Systems and methods for electro-fermentation

Abstract

The present disclosure provides an electro-fermentation bioreactor containing a culture of Clostridium species, which optimizes metabolic pathways to enhance carbon fuel production, demonstrating a sustainable and efficient method for converting waste into valuable biofuels with minimal energy consumption. The present disclosure also provides methods of using the microbial electro-fermentation bioreactor to generate biofuels from food waste products and / or algae.

Description

[0001] Docket No. 103362-092WO1 SYSTEMS AND METHODS FOR ELECTRO-FERMENTATION CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 755,586, filed February 7, 2025, entitled “ELECTRO-FERMENTATION TO PRODUCE ACETONE-BUTANOL-ETHANOL FROM FOOD WASTE,” which is incorporated by reference herein in its entirety.

[0003] FIELD

[0004] The present disclosure relates to systems and methods of using electro-fermentation to produce acetone-butanol -ethanol products from food waste.

[0005] BACKGROUND

[0006] Global food waste is about 2.5 billion tons annually, and the United States discards 60 million tons, which is almost 40% of its food supply. Wasted food contributes 11% of global greenhouse gas emissions (GHGEs). Current food waste management practices include landfilling, incineration, and composting. These techniques raise environmental concerns such as land use, air pollution, and GHGEs. Other health issues related to food waste include food insecurity, loss of nutrients, and occupational health risks to waste management workers. Due to the negative impacts of traditional food waste management methods and the need to recover valuable organic materials, finding sustainable and eco-friendly solutions to increase the value and utilization of wasted food is crucial. Various technologies are used to convert food waste into energy or fertilizer. Anaerobic digestion generates both biogas and nutrient-rich digestate, while gasification transforms waste into syngas for energy production. Traditional composting, through aerobic decomposition, produces nutrient-rich soil amendments. Additionally, emerging processes like hydrothermal liquefaction can convert food waste into biocrude oil under high temperature and pressure. However, these processes consume a significant amount of energy, and only a small portion of food waste is valorized, as they require ten times the amount of added water. Thus, what is needed is a cost-efficient fermentation system that maximizes output of carbon products while also minimizing energy consumption.

[0007] The systems and methods disclosed herein address these and other needs.Docket No. 103362-092WO1 SUMMARY

[0008] The present disclosure provides a microbial electrochemical system (MES) for converting food waste and / or algae into biofuel. The present disclosure also provides for methods of using the MES to ferment a food waste and / or algae. The present disclosure also provides for methods of using the MES to generate a biofuel.

[0009] In some aspects, disclosed herein is a microbial electrochemical system comprising two chambers connected by a bridge into an H-type configuration, wherein a first chamber is a cathode chamber comprising at least two Clostridium bacterial species and a second chamber is an anode chamber.

[0010] In some embodiments, the at least two Clostridium bacterial species are co-cultured in the cathode chamber. In some embodiments, the at least two Clostridium bacterial species comprise Clostridium carboxidivorans and Clostridium beijerinckii . In some embodiments, the first chamber comprises a food waste, algae, or a combination thereof.

[0011] In some embodiments, the food waste comprises an organic composition. In some embodiments, the organic composition comprises carbohydrates, fatty acids, and / or a plurality of essential elements selected from potassium (K), magnesium (Mg), sulfur (S), calcium (Ca), phosphorus (P), aluminum (Al), iron (Fe), manganese (Mn), sodium (Na), copper (Cu), boron (B), zinc (Zn), silicon (Si), strontium (Sr), selenium (Se), and boron (B). In some embodiments, the food waste comprises a coffee, an ice cream, a sour cream, or infant formula.

[0012] In some embodiments, the second chamber comprises an inorganic salt. In some embodiments, the inorganic salt is sodium sulfate (Na2SC>4). In some embodiments, the first chamber and the second chamber are separated by a proton exchange membrane.

[0013] In some embodiments, the at least two Clostridium bacterial species convert the food waste and / or algae into one or more biofuels. In some embodiments, the one or more biofuels comprise butanol, ethanol, acetone, or a combination thereof.

[0014] In some aspects, disclosed herein is a method of producing one or more biofuels in a microbial electrochemical system (MES), the method comprising adding an inorganic salt to an anode chamber, adding at least two Clostridium bacterial species and a food waste, algae, or a combination thereof, to a cathode chamber, assembling a MES by connecting the anode chamber to the cathode chamber using a connecting bridge, and applying a low voltage across the MES, wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane, and wherein the at least two Clostridium bacterial species convert the food waste, algae, or a combination thereof, into the one or more biofuels.Docket No. 103362-092WO1 In some aspects, disclosed herein is a method of fermenting a food waste using a microbial electrochemical system (MES), the method comprising adding an inorganic salt to an anode chamber, adding at least two Clostridium bacterial species and the food waste to a cathode chamber, assembling a MES by connecting the anode chamber to the cathode chamber using a connecting bridge, and applying a low voltage across the MES, wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane, and wherein the at least two Clostridium bacterial species convert the food waste into one or more biofuels.

[0015] In some aspects, disclosed herein is a method of fermenting algae using a microbial electrochemical system (MES), the method comprising adding an inorganic salt to an anode chamber, adding at least two Clostridium bacterial species and the algae to a cathode chamber, assembling a MES by connecting the anode chamber to the cathode chamber using a connecting bridge, and applying a low voltage across the MES, wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane, and wherein the at least two Clostridium bacterial species convert the algae into one or more biofuels.

[0016] In some embodiments, the at least two Clostridium bacterial species are co-cultured in the cathode chamber. In some embodiments, the at least two Clostridium bacterial species comprise Clostridium carboxidivorans and Clostridium beijerinckii . In some embodiments, the Clostridium beijerinckii metabolizes the food waste and / or algae. In some embodiments, the Clostridium carboxidivorans produces the one or more biofuels.

[0017] In some embodiments, the food waste comprises a coffee, an ice cream, a sour cream, or infant formula. In some embodiments, the one or more biofuels comprise butanol, ethanol, acetone, or a combination thereof. In some embodiments, the inorganic salt comprises sodium sulfate (Na2SC>4). In some embodiments, the low voltage ranges from 0V to -0.5 V.

[0018] BRIEF DESCRIPTION OF FIGURES

[0019] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0020] Figure 1 shows a graphical abstract for the production of butanol from electrofermentation of coffee waste.

[0021] Figures 2A and 2B show (Figure 2A) the configuration and (Figure 2B) the photograph of the MES bioreactor with glucose and coffee waste electro-fermentation.

[0022] Figures 3A and 3B show the coffee waste and glucose control co-culture electrofermentation. Figure 3 A shows the optical density. Figure 3B shows the pH profile.Docket No. 103362-092WO1 FIGS. 4A and 4B show the electrochemical performance of the carbon cloth in coculture electro-fermentation. Figure 4A shows the chronoamperogram of cathode, glucose control, and coffee waste. Figure 4B shows the cyclic voltammetry (CV) curve of glucose control and coffee waste.

[0023] Figures 5A, 5B, 5C, 5D, 5E, and 5F show the electro-fermentation products in coculture electro-fermentation of coffee waste and glucose control at -0.2V applied voltage. Figure 5A shows the acetone product. Figure 5B shows the ethanol product. Figure 5C shows the butanol product. Figure 5D shows the ABE product. Figure 5E shows the acetic acid product. Figure 5F shows the butyric acid product.

[0024] Figure 6 shows the graphical abstract of biomanufacturing of platform chemicals from industrial processing food waste.

[0025] Figures 7A and 7B show (Figure 7A) the configuration and (Figure 7B) the photograph of MES bioreactor with ice cream waste electro-fermentation.

[0026] Figure 8 shows the glucose concentration in ice cream and sour cream industry waste samples.

[0027] Figures 9A, 9B, and 9C show the pH profile during sour and ice cream waste fermentation. Figure 9A shows the conventional mono-fermentation of food waste. Figure 9B shows the conventional co-fermentation of food waste. Figure 9C shows the electrofermentation of ice cream waste. G = glucose control, IC = ice cream waste, SC = sour cream waste, -0.1 Volt (V), -0.2 Volt (V).

[0028] Figures 10A, 10B, and 10C show the optical density profile of sour cream and ice cream waste. Figure 10A shows the conventional mono-fermentation of food waste. Figure 10B shows the conventional co-fermentation of food waste. Figure 10C shows the electrofermentation of ice cream waste at -0.1V and -0.2V

[0029] Figures HA, 11B, 11C, HD, HE, and HF show the conventional fermentation products of ice cream and sour cream industrial waste employing Clostridium species. Figure 11A shows acetone product. Figure 11B shows ethanol product. Figure 11C shows butanol product. Figure 1 ID shows acetic acid product. Figure 1 IE shows butyric acid product. Figure 1 IF shows total ABE. IC= Ice cream; SC = Sour cream. * Solid line: Clostridium beijerinckii;** Dotted line: Clostridium beijerinckii + Clostridium carboxidivorans.

[0030] Figures 12A, 12B, 12C, 12D, 12E, and 12F show the electro-fermentation products. Figures 12A and 12B show the fermentation products employing Clostridium beijerinckii at 0.1V in ice cream waste. Figures 12C and 12D show the glucose control (solid line indicates -Docket No. 103362-092WO1 0.1V; dotted line indicates -0.2V). Figures 12E and 12F show the ice cream industry waste employing Clostridium beijerinckii + Clostridium carboxidivorans . -0.1V and -0.2V are applied externally.

[0031] Figures 13A, 13B, and 13C show the chronoamperogram (Figure 13 A), the cyclic voltammetry of co-electrofermentation in the glucose control (Figure 13B) and ice cream waste (Figure 13C).

[0032] Figures 14A, 14B, 14C, 14D, 14E, and 14F show the scanning electron microscopy photomicrographs. Figure 14A shows the clean electrode. Figure 14B shows the mono-electro-fermentation with glucose. Figure 14C shows the mono-electro-fermentation with ice cream waste at -0.1V. Figure 14D shows the mono-electro-fermentation with ice cream waste at -0.2V. Figure 14E shows the co-electro-fermentation of glucose at -0.1V. Figure 14F shows the co-electro-fermentation of ice cream waste at -0.1V.

[0033] Figure 15 shows a chronoamperogram of microelectrofermentation of algae waste at two applied voltages

[0034] Figures 16A and 16B show the cyclic voltammetry of algae electrofermentation experiment. Figure 16A shows the voltage at -0.1 V. Figure 16B shows the voltage at -0.3V.

[0035] Figures 17A, 17B, and 17C show the profile of algae waste in electrofermentation at two applied voltages. Figure 17A shows the cathode pH. Figure 17B shows the optical density at 600 nanometers (nm) (OD600). Figure 17C shows the anode pH.

[0036] Figures 18A and 18B show the profile of algae waste in co-culture and monoculture conventional fermentation. Figure 18A shows the pH. Figure 18B shows the OD600.

[0037] Figure 19 shows the gas production in algae waste in co-culture conventional fermentation.

[0038] Figure 20 shows the chronoamperogram of algae 100% and Alage and Coffee waste (1 : 1) at two -0.1 V using microbial electrofermentation.

[0039] Figures 21A and 21B show the cyclic voltammetry of algae and coffee waste at -0.1 V using microbial electrofermentation. Figure 21 A shows coffee (100%) alone. Figure 21B shows the algae and coffee at 50% each.

[0040] Figures 22A and 22B show the profile of coffee waste and coffee+algae waste in coculture electrofermentation. Figure 22A shows the pH of coffee waste and coffee+algae waste. Figure 22B shows the OD600 of coffee waste and coffee+algae waste.

[0041] Figures 23A and 23B show the profile of co-culture and mono-culture coffee+algae waste in conventional fermentation. Figure 23A shows the pH of the co-culture and mono-Docket No. 103362-092WO1 culture of coffee+algae waste. Figure 23B shows the OD600 of the co-culture and monoculture of coffee+algae waste.

[0042] Figures 24A and 24B show the gas production in coffee+algae waste conventional fermentation. Figure 24A shows the gas production in the co-culture. Figure 24B shows the gas production in the mono-culture.

[0043] Figure 25 shows the chronoamperogram of infant formula waste at two applied voltages using microbial electrofermentation.

[0044] Figure 26 shows the cyclic voltammetry of infant formula using microbial el ectrofermentati on .

[0045] Figures 27A and 27B show the profile of infant formula waste in electrofermentation at two applied voltages using microbial electrofermentation. Figure 27A shows the pH of infant formula waste. Figure 27B shows the OD600 of infant formula waste.

[0046] Figures 28A and 28B show the profile of infant formula waste in electrofermentation at four applied voltages using microbial electrofermentation. Figure 28 A shows the pH of infant formula waste. Figure 28B shows the OD600 of infant formula waste.

[0047] DETAILED DESCRIPTION

[0048] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

[0049] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.Docket No. 103362-092WO1 Terminology

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0051] The following definitions are provided for the full understanding of terms used in this specification.

[0052] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0053] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0054] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.Docket No. 103362-092WO1 "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0055] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant.

[0056] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more decrease so long as the decrease is statistically significant.

[0057] As used herein, the term “lipid” or “lipid-like” refers to a macromolecule that is soluble in nonpolar solvents. These molecules are usually hydrophobic or amphiphilic molecules; the amphiphilic nature of some lipids allows formation of structures such as vesicles, liposomes, membranes, and nanoparticles. Lipids can be categorized into fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids, and prenol lipids.

[0058] As used herein, a “carbohydrate” refers to a large family of organic compounds including, but not limited to sugars, starch, and cellulose, containing hydrogen and oxygenDocket No. 103362-092WO1 in similar ratios to water (2:1) and used as structural materials in numerous biomolecules, such as DNA or RNA, and for energy storage with living tissues. Carbohydrates can be categorized into four groups including monosaccharides, disaccharides, oligosaccharides, and polysaccharides, wherein monosaccharides and disaccharides are the smallest forms of carbohydrates, commonly referred to as sugars, whereas oligosaccharides and polysaccharides are larger, complex structures used for energy storage of the structural foundation of nucleic acids and nucleotides.

[0059] “Culture” or “cell culture” is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. These conditions vary for each cell type but generally consist of a suitable vessel with a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH, buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. "Cell culture" also refers to the culturing of cells derived from multicellular eukaryotes, especially animal cells, in contrast with other types of culture that also grow cells, such as plant tissue culture, fungal culture, and microbiological culture (of microbes).

[0060] As used herein, a “co-culture” refers to a biological system in which two or more different strains of microorganisms, such as bacteria, are grown together. A “co-culture” also refers to any culture processes disclosed herein.

[0061] As used herein, “biofuel” refers to any renewable solid, liquid, or gaseous fuel produced biologically, for example fuels produced from biomass. The biofuels disclosed herein are primarily generated from the biological process including but not limited to fermentation and / or anaerobic digestion.

[0062] As used herein, an “anode” refers to an electrode of a polarized electrical device, such as a microbial electrochemical device, which is positively charged and which electrons leave said device.

[0063] As used herein, a “cathode” refers to an electrode of a polarized electrical device, such as a microbial electrochemical device, which is negatively charged and which electrons enter said device.Docket No. 103362-092WO1 As used herein, “fermentation” or “fermenting” refers to an anaerobic metabolic process, occurring in the absence of oxygen, where microorganisms, such as bacteria, convert carbohydrates into alcohols, organic acids, and gases to produce energy.

[0064] Microbial electrochemical systems (MES)

[0065] The present disclosure provides a microbial electrochemical system (MES) for converting food waste and / or algae into biofuel.

[0066] MES is a technology that uses applied voltage to accelerate the metabolism of microorganisms to produce byproducts. Such devices rely on the integration of microbial cells, biochemistry, material science, and electrochemical technologies. In general, MESs use processes that involve: (1) extracellular electron transfer in which an electrode potential lies within the physiological range of the microorganisms and rely primarily on Faraday processes and, (2) indirect interactions in which the microbial environment (including, but not limited to metabolites, pH, oxygen pressure, and carbon dioxide levels) is controlled by electrochemical processes. For these processes to occur, the microbial system should be in close vicinity to the electrochemical system. Said systems should also be integrated into a reactor that ideally requires low operation and maintenance costs. While several developments have been attempted to implement these systems, several challenges exist that prevented the successful integration of these systems in real world applications. The present disclosure demonstrates integration of a microbial co-culture system that convert chemical energy from food waste and / or algae into biofuels.

[0067] The MES of the present disclosure generates biofuels from food waste, algae, or a combination thereof, which is driven by the synergistic metabolisms of a first microbe and a second microbe.

[0068] In some aspects, disclosed herein is a MES comprising two chambers connected by a bridge into an H-type configuration, wherein a first chamber is a cathode chamber comprising at least two Clostridium bacterial species and a second chamber is an anode chamber.

[0069] In the MES, the Clostridium bacteria perform distinct functions resulting in the production of biofuels. In some embodiments, the at least two Clostridium bacterial species are co-cultured in the first chamber / cathode chamber. In some embodiments, the at least two Clostridium bacterial species convert the food waste and / or algae into one or more biofuels. In some embodiments, the first chamber / cathode chamber contains the food waste and / or algae.Docket No. 103362-092WO1 In some embodiments, a first Clostridium species facilitates fermentation of food waste and / or algae. In some embodiments, a second Clostridium species produces a carbon-based fuel, such as short chain fatty acids. In some embodiments, the at least two Clostridium bacterial species comprise Clostridium carboxidivorans and Clostridium beijerinckii . In some embodiments, the first Clostridium species comprises Clostridium beijerinckii. In some embodiments, the second Clostridium species comprises Clostridium carboxidivorans.

[0070] In some embodiments, the food waste comprises a dairy product, a produce, a beverage product, or any food product thereof. Non-limiting examples of food waste used herein includes a coffee, an ice cream, a sour cream, cheese, milk, fruits (such as for example apples, bananas, oranges, lemons, limes, tomatoes, berries, and melons), vegetables (such as for example leafy greens, broccoli, carrots, celery, onions, potatoes, peppers, and beans), grain products (such as, for example breads, pasta, oats, wheat, and rice), dairy products, baby food waste (such as, for example purees), infant formula, and any combinations thereof. In some embodiments, the MES system of any aspect disclosed herein is capable of fermenting and / or digesting any food waste known in the art.

[0071] In some embodiments, the MES system of any aspect disclosed herein is capable of fermenting and / or digesting any combination of food waste products. In some embodiments, the MES system of any aspect disclosed herein is capable of fermenting and / or digesting 1 or more food waste products. In some embodiments, the MES system of any preceding aspect disclosed herein is capable of fermenting and / or digesting 2 or more, 3 or more, 4 or more, or 5 or more food waste products.

[0072] In some embodiments, the food waste comprises an organic composition. In some embodiments, the organic composition comprises carbohydrates, fatty acids, and / or a plurality of essential elements selected from potassium (K), magnesium (Mg), sulfur (S), calcium (Ca), phosphorus (P), aluminum (Al), iron (Fe), manganese (Mn), sodium (Na), copper (Cu), boron (B), zinc (Zn), silicon (Si), strontium (Sr), selenium (Se), and boron (B).

[0073] Algae are a diverse, largely aquatic group of photosynthetic organisms ranging from microscopic unicellular diatoms to massive, multicellular seaweeds, such as kelp. In some embodiments, the algae comprises a unicellular (or single cell) organism, also known as microalgae. In some embodiments, the algae comprises a multicellular organism, also known as marcoalgae or seaweed. In some embodiments, the algae is derived from a aquatic habitat. In some embodiments, the algae is derived from a terrestrial habitat. In some embodiments, the algae is from any one of the following classifications: green algae (Chlorophyta), brown algaeDocket No. 103362-092WO1 (Phaeophyta), red algae (Rhodophyta), blue-green algae (Cyanobacteria), golden-brown algae and diatoms (Chrysophyta).

[0074] In some embodiments, the algae of any aspect disclosed herein can be combined with a food waste product including but not limited to includes a coffee, an ice cream, a sour cream, cheese, milk, fruits (such as for example apples, bananas, oranges, lemons, limes, tomatoes, berries, and melons), vegetables (such as for example leafy greens, broccoli, carrots, celery, onions, potatoes, peppers, and beans), grain products (such as, for example breads, pasta, oats, wheat, and rice), dairy products, baby food waste (such as, for example purees), infant formula, and any combinations thereof. In some embodiments, the algae of any aspect disclosed herein can be combined with any food waste known in the art. In some embodiments, the algae of any aspect disclosed herein can be one with 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more food waste products.

[0075] In some embodiments, the second chamber comprises an inorganic salt. In some embodiments, the inorganic salt is sodium sulfate (Na2SC>4).

[0076] A non-limiting example of a MES, according to the present disclosure, is shown in Figure 7B. In some embodiments, the MES of any preceding aspect comprises a sample input port (100). In some embodiment, the sample input port is used to introduce the food waste and / or the at least two bacteria into the system. In some embodiments, the first chamber (cathode chamber) (102) and the second chamber (anode chamber) (101) are separated by a proton exchange membrane (106). As used herein, a proton exchange membrane refers to a polymer film that conducts protons (such as hydrogen ions, H+) while simultaneously blocking electrons and gases, acting as a solid electrolyte to enable clean energy conversion. Spacing between the anode chamber (101) and the cathode chamber (102) can vary, as is understood by those of skill in the art.

[0077] In some embodiments, the cathode chamber (102) comprises an effluent outlet (104).

[0078] In some embodiments, the anode chamber (101) comprises an effluent outlet (103). In some embodiments, the MES of any preceding aspect comprises a reference electrode (107). In some embodiments, the reference electrode (107) is located in the anode chamber (101).

[0079] In some embodiments, the MES comprises an anode electrode (108). In some embodiments, the MES comprises a cathode electrode (109). As used herein, the anode electrode (108) can also be referred to as the “counter electrode”, and the cathode electrode (109) can also be referred to as the “working electrode”. In some embodiments, the anode electrode (108) and cathode electrode (109) are selected from any known conductive material,Docket No. 103362-092WO1 including but not limited to carbon, precious or non-precious metals, metal organic compounds, stainless steel, conductive polymers, and the like, further including combinations thereof. In some embodiments, the anode electrode (108) and cathode electrode (109) can be made of the same conductive material. In some embodiments, the anode electrode (108) and cathode electrode (109) can be made of different conductive materials. In some embodiments, the cathode chamber (102) and the anode chamber (101) are in an “H-type” configuration (105), which refers to a structural or functional arrangement resembling the capital letter “H” used for stability and efficiency of the MES system.

[0080] In some embodiments, the one or more biofuels comprises 2-carbon (C2) molecules or 4-carbon (C4) molecules. In some embodiments, the MES of any aspect disclosed herein can produce 1, 2, 3, or more biofuels. In some embodiments, the one or more biofuels comprise butanol, ethanol, acetone, or a combination thereof. The MES of any preceding aspect provides production levels of biofuels in the following order: butanol > ethanol > acetone.

[0081] In some embodiments, the MES of any aspect disclosed herein is a gas releasing system. In some embodiments, the gas released includes hydrogen gas or carbon dioxide gas. In some embodiments, the MES of any aspect disclosed herein does not release a gas. In some embodiments, the MES of any aspect disclosed herein does not release hydrogen gas or does not release carbon dioxide gas.

[0082] Methods of using an MES

[0083] The present disclosure also provides for methods of using the MES to ferment a food waste and / or generating a biofuel.

[0084] In some aspects, disclosed herein is a method of producing one or more biofuels in a microbial electrochemical system (MES), the method comprising adding an inorganic salt to an anode chamber, adding at least two Clostridium bacterial species and a food waste, algae, or a combination thereof to a cathode chamber, assembling a MES by connecting the anode chamber to the cathode chamber using a connecting bridge, and applying a low voltage across the MES, wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane, and wherein the at least two Clostridium bacterial species convert the food waste, algae, or a combination thereof, into the one or more biofuels.

[0085] In some aspects, disclosed herein is a method of fermenting a food waste using a microbial electrochemical system (MES), the method comprising adding an inorganic salt to an anode chamber, adding at least two Clostridium bacterial species and the food waste to aDocket No. 103362-092WO1 cathode chamber, assembling a MES by connecting the anode chamber to the cathode chamber using a connecting bridge, and applying a low voltage across the MES, wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane, and wherein the at least two Clostridium bacterial species convert the food waste into one or more biofuels.

[0086] In some aspects, disclosed herein is a method of fermenting algae using a microbial electrochemical system (MES), the method comprising adding an inorganic salt to an anode chamber, adding at least two Clostridium bacterial species and the algae to a cathode chamber, assembling a MES by connecting the anode chamber to the cathode chamber using a connecting bridge, and applying a low voltage across the MES, wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane, and wherein the at least two Clostridium bacterial species convert the algae into one or more biofuels.

[0087] In some embodiments, the method comprises using the MES of any preceding aspect, including but not limited to the MES as shown in Figure 7B. In some embodiments, the method comprises Clostridium beijerinckii for metabolizing the food waste, algae, or a combination thereof. In some embodiments, the method comprises Clostridium carboxidivorans for producing the one or more biofuels. In some embodiments, the method comprises fermenting a food waste of any preceding aspect. In some embodiments, the method comprises fermenting an algae of any preceding aspect. In some embodiments, the method comprises fermenting a combination of food waste and algae of any preceding aspect, including but not limited to a combination of coffee and algae.

[0088] In some embodiments, the MES of any preceding aspect comprises a low voltage range from 0V to -0.5 V. In some embodiments, the MES of any preceding aspect comprises a low voltage of 0V, -0.05V, -0.1V, -0.15V, -0.2V, -0.25V, -0.3V, -0.35V, -0.4V, -0.45, -0.5V, and any voltage in between the range disclosed herein. In some embodiments, the low voltage range provides for optimal generation of biofuels. It should be noted that setting the MES to a voltage outside of 0V to -0.5V range can result in overloading the system and / or further limiting the amount of biofuels produced.

[0089] In some embodiments, the biofuels disclosed herein are carbon-based fuels. The carbon-based fuels generated by any method disclosed are advantageous because they bum clean with minimal greenhouse gas emissions. In some embodiments, the one or more biofuels comprises 2-carbon (C2) molecules or 4-carbon (C4) molecules. In some embodiments, the method of any aspect disclosed herein can produce 1, 2, 3, or more biofuels. In some embodiments, the one or more biofuels comprise butanol, ethanol, acetone, or a combinationDocket No. 103362-092WO1 thereof. The MES of any preceding aspect provides production levels of biofuels in the following order: butanol > ethanol > acetone.

[0090] As used herein, the biofuels produced by the methods of any preceding aspect can be used as a renewable alternative energy source. Non-limiting examples of renewable alternatives derived from the biofuels produced herein include transportation, electricity generation, heating and other specialty applications. Regarding transportation, said biofuels can be blended with conventional fuels (including, but not limited to the blending of ethanol with gasoline) for use in existing engines and infrastructures. Electricity generation implements the burning or conversion of biofuels to generate electricity for residential and / or commercial buildings. Said biofuels could also be used for heating, as referred to as “biohea ’, wherein the burning of biofuels generates heat used for residential and / or commercial buildings.

[0091] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0092] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0093] EXAMPLES

[0094] The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0095] Example 1: Production of butanol from electro-fermentation of coffee waste

[0096] Food waste occurs at every stage of the production chain, from farms to consumers. Managing food waste is one of the biggest challenges for municipalities, especially in urban centers. Reducing food waste is a clear pathway to cutting greenhouse gas emissions (GHGEs) and addressing climate change. In the U.S., up to 40% of food produced goes uneaten, and 95% of discarded food ends up in landfills, contributing 21% of solid waste. It accounts for 6.1% of total GHGEs in the United States. Food manufacturers waste a significant 20% of the initial food production that doesn't meet grading requirements in regions like North America, Europe,Docket No. 103362-092WO1 Oceania, and Latin America. This waste often includes produce that is perfectly edible but falls short of cosmetic or size standards, such as oddly shaped fruits and vegetables or products with slight imperfections. Food waste from industries and food manufacturers is a uniform source of waste and can be valorized using biological conversion techniques. Commercial fruit processing generates 16% of waste, contributing 6% of GHG emissions from landfills. Environmental concerns around coffee waste focus on the 2.5 billion coffee cups and 30,000 tons of spent coffee grounds generated annually in the UK alone. It is estimated that 65% of spent coffee waste is produced from each ton of green coffee beans. Coffee waste is a valuable source of biomass and can be repurposed for various applications, including bioenergy production. It can serve as additives for silages, and functional compounds can be extracted for use in food additives and other nutraceuticals.

[0097] These wastes can be transformed through thermal and biological processes into useful products. For waste with a high total solids content, it is possible to produce solid biofuels through processes like pyrolysis, which also generates biochar. However, pyrolysis requires a drying step to remove moisture from the waste, adding complexity and cost. Alternatively, hydrothermal carbonization is another thermal conversion process that doesn't require moisture removal. This process can handle wet biomass directly but operates at high temperatures, which leads to additional energy costs. Biological processes are alternative to thermal processes, and it includes composting, anaerobic digestion, and fermentation. Composting can produce valuable fertilizer, but the process needs approximately 180 days and land acreage to complete the process. Anaerobic digestion can produce biogas and digestate. Digestate needs additional waste management and methane production is 60%. Cellulose, hemicellulose, and lignin are the main components of plant cell walls and present significant challenges for microbial metabolism due to their recalcitrance. To overcome this, pretreatment and saccharification are necessary to convert the biomass into a fermentable form. Pretreatment enhances microbial access to cellulose by increasing the accessible surface area, reducing the degree of polymerization, and lowering the crystallinity of the cellulosic biomass. Literature reported enzymatic hydrolysis increases sugar yield that may be used as feedstock for fermentation processes primarily in biofuel production. Microbial Electrochemical Systems (MES) facilitate anodic oxidation and cathodic reduction reactions in separate chambers divided by an ionexchange membrane. In the cathodic chamber, microbial species act as biocatalysts, transforming biowaste into biochemicals by accelerating microbial metabolism. SpecificallyDocket No. 103362-092WO1 acclimatized microbial species can carry out the electro-fermentation of food waste, while coculturing different microbial species can diversify or enhance product yields.

[0098] As energy demand continues to rise, the need for alternative energy sources has become increasingly urgent. In the light of circular economic principles, the utilization of waste and by-products for energy production has emerged as a preferred solution. Consequently, research on food waste valorization has become a primary focus. This study investigates producing liquid fuels from spent coffee waste generated during industrial processing. The study's novelty lies in the innovative co-culturing of two Clostridium species, which optimizes metabolic pathways to enhance butanol production, demonstrating a sustainable and efficient method for converting industrial waste into valuable biofuels.

[0099] Materials and Methods

[0100] Coffee waste procurement and characterization. Coffee waste industry was contacted for samples procurement. Spent coffee waste was provided by J. M Smucker Co. Waste samples were stored at 4 °C for further analysis. Waste samples were characterized for physicochemical parameters. Total solids, moisture content, calorific content and pH were measured. Ash content of food waste samples was analyzed following the procedure described in TMECC’s method 03.02-A. Elemental composition of waste samples was analyzed by following TMECC’s 04.05, 04.06 and 04.07 methods using inductively coupled plasma optical emission spectroscopy (ICP-OES). Completely dried samples (1g) were each used for elemental analysis. The estimated elements are reported in mg per g sample (dry basis).

[0101] Pre-treatment and bacterial strain cultures. Spent coffee grounds (95 g) were pretreated in a Parr reactor at 180 °C for 1 hour with 393 mL of water (15% solid loading). After cooling to 20 °C, the slurry was neutralized to pH 5 using 1 N NH4OH, followed by centrifugation. The solid fraction was dried at 75 °C, while the supernatant was stored at -20 °C for subsequent enzyme hydrolysis. For hydrolysis, the supernatant was thawed, adjusted to pH 5, and treated with cellulase and viscozyme. The reaction was conducted at 50 °C with continuous shaking for 48 hours. Soluble reducing sugars were quantified using the DNS assay. Clostridium beijerinckii spores and Clostridium carboxidivorans vegetative cells were cultured in TGY medium under anaerobic conditions (82% N2, 15% CO2, 3% H2) at 35 ± 1°C. C. beijerinckii spores were heat-shocked (75 °C for 3 minutes) before inoculation. Once precultures reached specific optical densities (OD600: 0.9-1.1 for C. beijerinckii,' 0.67 for C. carboxidivorans),Docket No. 103362-092WO1 they were transferred to fresh medium and grown further to prepare inocula. For fermentation, cultures were added at 6% of the total volume, with co-culture experiments comprising 2.5% C. beijerinckii and 3.5% C. carboxidivorans.

[0102] Microbial electrosynthesis bioreactor configuration. The MES reactor is a glass H-type bioreactor with two IL chambers (anode and cathode), each sealed with external clamps and O-ring grooves. It has two influent ports on the lid for the reference electrode and electrode wire, secured with caps and stoppers. A proton exchange membrane (PFSA D125-U) separates the chambers, sealed with rubber gaskets. Carbon cloth electrodes (platinum-coated) are used, with platinum wire for electrical connections.

[0103] Hydrolysate electro-fermentation. The MES reactor was set up with 50 mM Na2SC>4 at pH 3.5 in the anode chamber and 1 L of fermentation medium (including food waste and P2 components) in the cathode chamber. The fermentation was inoculated with C. beijerinckii and C. carboxidivorans co-culture. The reactors were operated at room temperature with controlled cathode potentials (-0.1 V, -0.2 V) using a potentiostat. Potentials above -0.2 V were ineffective due to overload.

[0104] Analytical procedures. Cell growth was tracked by measuring optical density (OD600) using UV-visible spectrophotometer (DU®800, Beckman Coulter Inc., Brea, CA) at 12-hour intervals, with initial OD subtracted from final OD at each fermentation time point. pH was monitored with a pH meter. Solvents (acetone, butanol, ethanol, acetic acid, and butyric acid) were quantified using gas chromatography (Agilent 7890A). ABE yield was calculated as the total ABE produced per gram of glucose or biosolids, and productivity was determined by ABE concentration produced per hour.

[0105] Results and Discussion

[0106] Coffee waste characteristics

[0107] The characteristics of the coffee waste sample, as presented in Table 1, reveal that its pH is approximately 6, which is close to neutral. This shows the absence of significant acid fractions, making it unlikely to inhibit the acidogenesis phase of fermentation. The low ash content of 1.7% indicates a minimal presence of inorganic material, implying that the coffee waste is predominantly organic in nature. This organic composition is advantageous for biological transformation methods, which often require high organic matter content. AshDocket No. 103362-092WO1 contains inorganic species (K, P, Ca, Fe, Mg) and they can act as cofactors in enzymatic reactions of the biomass conversion. The total solids content of 72% and a moisture content of 27.8% indicate that the coffee waste retains substantial moisture while also containing a high level of solids. High solids and moisture content are known to support biological activity, as they provide a favorable environment for microbial growth and fermentation. These characteristics underscore coffee waste’s suitability for biological valorization processes, such as fermentation or other methods aimed at recovering value-added products.

[0108] The calorific content of coffee waste is 22 MJ / kg, which is comparable to that of sub-bituminous coal, reported at 24.4 MJ / kg. This similarity highlights coffee waste as a renewable energy source with properties akin to conventional fossil fuels. Additionally, studies on food waste conversion to biochar report a calorific value of 23.6 MJ / kg, further reinforcing the suitability of coffee waste for energy recovery applications.

[0109] The major and minor element analysis of coffee waste reveals significant concentrations of essential elements, with potassium (K) being the highest, followed by magnesium (Mg), sulfur (S), calcium (Ca), phosphorus (P), aluminum (Al), iron (Fe), manganese (Mn), sodium (Na), copper (Cu), boron (B), and zinc (Zn). Trace elements include silicon (Si), strontium (Sr), selenium (Se), boron (B), and others (Table 2). These trace elements play a crucial role as cofactors in enzymatic reactions, particularly in maintaining the redox balance of the butanol dehydrogenase enzyme, which is critical for fermentation and biofuel production. The elemental composition of coffee waste not only supports energy applications but also highlights its utility in biological valorization processes.

[0110] Growth and pH profile in electro-fermentation

[0111] A gradual increase in cell density indicated the exponential growth phase of the cells (Figure 3 A). The optical density (OD 600 nm) peaked on day 3 in the control and day 2 in the coffee waste bioreactors. The growth profile in the pure glucose medium was 41% higher than in the coffee waste medium. Growth stabilized for 24 hours before beginning to decline. The subsequent 24-hour decline in growth was 32% in the coffee waste bioreactor and 33% in the control bioreactor.

[0112] A classical pH trend was observed in both the control and coffee waste samples (Figure 3B). The pH profile corresponded to the OD profile, with similar pH drops of 5.3 and 5.4 observed on day 3 and day 2, respectively. The pH decrease indicates rapid bacterial growth during the acidogenic phase, where sugars are oxidized into pyruvate and subsequentlyDocket No. 103362-092WO1 converted into acetic or butyric acid. After day 5, a microbial acidogenic phase shift towards the solventogenic phase was observed.

[0113] Electrochemical characteristics and electrosynthesis efficiency

[0114] Chronoamperogram profile of cathode reflects stability and performance of the two systems (Figure 4A). Coffee waste shows more steady profile than control. The two primary mechanisms of electron transfer currently recognized are: (i) extracellular electron shuttles or bacterial secretion of reduced secondary metabolites, and (ii) direct electron transfer by membrane-bound c-type cytochromes or conductive pili (nanowires). Extracellular pili were not observed under scanning electron microscope (another study) most likely electron transfer mechanism is direct transfer or mediated transfer. Cyclic voltammetry (CV) analysis was performed using a three-electrode system within a potential range of -900 to +400 mV and scan rates varying from 1 to 200 mV / s. In the MES, the biocathode functioned as the working electrode for the CV analysis (Figure 4B). CV was performed three times over the six-day batch operation. The maximum current reduction response was -0.28 mA for glucose and -0.3 mA for coffee waste. The reduction peaks are overlapping and a significant difference in oxidation peaks is observed. The electrochemical behavior of control and coffee waste shows similar pattern which reflects biofilm formation of co-culture Clostridium species is accepting electrons and reducing the substrate. VFAs production also reflects the successful acclimatization of co-culture Clostridium species.

[0115] Butanol and other products formation

[0116] Electro-fermentation with co-culture of Clostridium species showed synergistic relationship and produced acetone-butanol-ethanol. The acetic acid and butyric acid are precursors of solvent formation. The acetic acid concentration showed from day 0 as the buffer contains some amount of ammonium acetate that is converted to acetic acid in the medium (Figure 5A). The assimilation of acetic acid and conversion into acetone started after almost 50% of acetic acid was reduced from initial concentration. Acetic acid concentration from control declined faster than coffee waste and reaches 0 g / L on day 5 and day 6 and the maximum concentration of acetone was achieved on day 6, 2.4 and 1.9 g / L for control and coffee waste respectively (Figure 5C). As the pH profile reflects lowest at day 3 maximum butyric acid (2.8 and 2.2 g / L) production was observed (Figure 5B). After maximum butyric acid production butanol production started at day 3 and keeps increasing until day 8. Only 3.7 % difference between butanol production was observed in coffee waste and control (FigureDocket No. 103362-092WO1 5D). Ethanol production was higher in coffee waste than control and reached 3.2 g / L on day 6 and it is 43% higher than control (Figure 5E). Co-culturing of Clostridium species in electrofermentation were able to produce 17.5 g / L of ABE and it is 42% higher than coffee waste enzyme treated and 65% higher than sulphuric acid pretreated conventional fermentation. At day 6 the difference between control and coffee waste ABE concentration was only 20% (Figure 5F). Conventional fermentation has shown 4.4 g / L butanol and 6.7 g / L ABE, and 7 g / L butanol and ABE 10 g / L using C. acetobutylicum and C. beijerinckii . Electro-fermentation with the application of -0.2V and co-culture of two Clostridium species can produce higher butanol and ABE than other conventional fermentation studies.

[0117] Example 2: Biomanufacturing of platform chemicals from industrial processing food waste

[0118] Biomanufacturing platform chemicals from food waste through electro-fermentation holds significant commercial potential. This study examined the effects of mono- and coculture fermentation on the production of platform chemicals in both conventional and electrofermentation systems. In co-culture conventional fermentation, butyric acid and butanol were the primary products, with production levels following the order: butanol > ethanol > acetone. A constant cathode potential was maintained using an external voltage, and lower applied voltages were found to enhance the yields of platform chemicals. Mono-culture electrofermentation mainly generated C2 to C4 products, such as acetone, butanol, and butyric acid, while co-culture electro-fermentation predominantly produced C2 compounds like acetone and acetic acid. Use of lower voltages may allow the potentiostat to be replaced with a simpler fixed DC power supply, improving the scalability and cost-efficiency of the process.

[0119] Improved conversion efficiency can be achieved through a microbial electrochemical system (MES), which facilitates anodic oxidation and cathodic reduction reactions in separate chambers divided by an ion-exchange membrane. This innovative approach is effective in breaking down organic components in food waste while synthesizing valuable products. Electrical energy powers the metabolic pathways in the cathodic chamber, where electron transfer mechanisms regulate the formation of these byproducts.

[0120] Moreover, MES systems can utilize CO2 at the cathode, reducing greenhouse gas emissions and integrating waste treatment with resource recovery — offering significant advantages over conventional biological methods. Co-culturing microbial species further enhances product diversity from biomass and syngas. Specifically, Clostridium beijerinckiiDocket No. 103362-092WO1 metabolizes biomass hydrolysates, while Clostridium carboxidivorans, a gas fermenter, utilizes CO2 and H2 for growth and product formation.

[0121] Research on liquid product synthesis in MES is limited and low product yield from mixed microbial consortia is a significant obstacleA 6 L tubular MES reactor was recently explored and a maximum volatile fatty acids (VFA) titer of 1.76 g / L from biowaste substrate was reported. MES integrated with microalgae has been demonstrated and it was further reported 7.6 g / L VFAs from CO2. There is still a limited understanding of critical factors, such as system configuration, materials, dimensions, biocatalyst, and substrate influence on the performance of both the anode and cathode in MES. As a result, further research is urgently needed to close the knowledge gap in MES and to tackle the challenges related to scaling up this technology.

[0122] In this study, an H-type, two-chambered microbial electrolysis system (MES) was developed with a 2 L working volume to electro-ferment food waste into valuable products such as volatile fatty acids (VFAs) and acetone-butanol -ethanol (ABE). The system employed co-culturing of Clostridium species to optimize resource utilization. Specifically, Clostridium beijerinckii facilitated fermentation, producing CO2 as a byproduct, which was then consumed by Clostridium carboxidivorans to produce short-chain fatty acids. A series of fermentation experiments were conducted by varying key parameters, including types of feedstocks, organic loading rates, and applied voltages. These efforts aimed to optimize conversion efficiency and evaluate the scalability of this innovative technology.

[0123] 1. Dairy industry food waste is a promising feedstock for production of biochemicals. 2. Co-fermentation of Clostridium species produce platform chemicals in higher yield.

[0124] 3. Electro-fermentation of food waste performs better at lower applied voltage.

[0125] 4. Electro-fermentation improves microbial metabolic reactions over conventional methods.

[0126] Materials and Methods

[0127] Food Waste Collection. Ice cream and sour cream waste samples were used as model wastes in the experiment. Food waste samples were provided by Smith Foods. The ice cream waste samples were a mix of ice cream, and flavoring agents while sour cream samples had traces of spices, onion and emulsifiers.

[0128] Physico-chemical Characterization. Food waste samples were physico-chemically characterized. Total solids, moisture content, calorific content and pH were measured. AshDocket No. 103362-092WO1 content of food waste samples was analyzed following the procedure described in TMECC’s method 03.02-A. Elemental composition of waste samples was analyzed using inductively coupled plasma optical emission spectroscopy (ICP-OES) as described by TMECC’s 04.05, 04.06 and 04.07 methods. Completely dried samples (1g each) were used for elemental analysis. The elemental content was expressed in mg / g sample (dry basis). Total reducing sugar concentration with reference to glucose was measured using a 3, 5 -dinitrosalicylic acid-based assay. The absorbance of the standard solution and samples to which the DNS reagent was added was determined in a UV-Visible spectrophotometer at 546 nm. The obtained absorbance of standard solutions was plotted against glucose concentration and used to calculate the glucose concentration in the ice cream and sour cream waste.

[0129] Reactor Design and Configuration. The MES reactor design and configuration is illustrated in Figures 7A and 7B. This bioreactor, crafted from glass by experts at the Ohio State University glassblowing laboratory, features an H-type configuration consisting of two chambers: the anode chamber and the cathode chamber, each with a working volume of IL made in identical design.

[0130] The lid of the bioreactor includes two influent ports. One port, with a 5.5 mm internal diameter, was fitted with a screw cap to secure the reference electrode, while the other port, with a 6.5 mm internal diameter, was equipped with a rubber stopper and an aluminum cap, held in place by a crimper for the electrode wire. Both the lid and the flask have an internal diameter of 60 mm, designed with grooves for O-rings, and are secured using external clamps. Each chamber also features an external effluent port, secured with a rubber stopper and sealed with an aluminum cap. The bridge connecting the two chambers has an internal diameter of 60 mm on both sides, complete with grooves for O-rings. A PFSA D125-U proton exchange membrane is positioned between the chambers, secured with abrasion-resistant natural rubber gaskets on both sides, and sealed with external clamps to ensure a tight fit. Carbon cloth (0.5 mg / cm260% Platinum on Vulcan) used as electrodes was purchased from a fuel cell store and electrical connections were made with platinum wire (0.5mm diameter, 99.99% metal basis) from Fisher Scientific.

[0131] Bacterial Strain cultures. The Clostridium carboxidivorans vegetative cells (500 pL) cultures were inoculated into 10 mL tryptone glucose yeast (TGY) media and incubated anaerobically for 12-14 hours at 35 ± 1 °C. C. beijerinckii spores (400 pL) were reactivated by heat-shocking at 75 °C for 3 minutes, followed by cooling on ice for 3 minutes and inoculated in TGY media. After incubation, they were sub-cultured in TGY broth until the optical density (OD600nm) ofDocket No. 103362-092WO1 C. beijerinckii reached 0.9-1.1, and that of C. carboxidivorans reached 0.65, prior to use in fermentation.

[0132] Conventional Fermentation Experiments. Pyrex screw-capped bottles (150 mL) containing 100 mL of fermentation medium were used for fermentation. The fermentation medium was composed of 91% food waste, and 1 mL each of filter-sterilized P2 vitamin stock, (0.1 g / L para-amino-benzoic acid; 0.1 g / L thiamine; 0.001 g / L biotin), P2 buffer (50g / L KH2PO4; 50g / L K2HPO4; 220g / L ammonium acetate), and P2 mineral stock (20 g / L MgSO 7H2O; 1 g / L MnSO4 H2O; 1

[0133] g / L FeSO4'7H2O; 1 g / L NaCl). Food wastes ice cream and sour cream were sterilized in an autoclave for 15 min at 121 °C then allowed to cool to 40 °C before transferring into the anaerobic chamber for 14-16 h prior to fermentation. The anaerobic chamber (Coy Laboratory Products Inc., Ann Arbor, MI) was maintained at a modified atmosphere of 82% N2, 15% CO2, and 3% H2. Actively growing precultures in TGY medium were used as inocula, and each bottle was inoculated with 6% (v / v) C. beijerinckii for mono-culture fermentation and with 2.5% C. beijerinckii and 3.5% C. carboxidivorans for cocultures. Fermentation was performed for 84 h and broth was sampled every 12 h to quantify growth, and solvent and organic acid production.

[0134] Electro-Fermentation Experiments. For electro-fermentation in MES the anode chamber was filled with 50 mM Na2SC>4 at pH 3.5 and the cathode chamber was filled with 1 L fermentation medium. About 910 mL of sterile food waste and 10 mL each of filter-sterilized P2 vitamin, P2 buffer, and P2 mineral were added. As used for the conventional fermentation, the MES fermentation medium was inoculated with either 6% (v / v) C. beijerinckii alone, or with 2.5% C. beijerinckii and 3.5% C. carboxidivorans. Clostridium precultures were not added to the control experiment.

[0135] The MES reactors containing ice cream waste were operated at room temperature (around 22 °C) with a constant cathode potential managed by a potentiostat (VMP3, BioLogic) in a three-electrode configuration. In this arrangement, the cathode functioned as the working electrode, while the anode served as the counter electrode, and a Ag / AgCl reference electrode (3 M KC1) was placed in the cathode chamber. To explore the effects of cathode potential on MES performance, tests were conducted at cathode potentials of -0 V, -0.1 V and -0.2 V versus Ag / AgCl with 3 M KC1. Potentials above -0.2 V were ineffective with the food waste substrates due to excessive overload potential.Docket No. 103362-092WO1 Analytical Procedures. Optical culture densities (ODeoonm; pathlength, 1 cm) were used to estimate cell growth using a UV-visible spectrophotometer (DU®800, Beckman Coulter Inc., Brea, CA) at intervals of 12 h. The pH profile was monitored with an AB 15 pH meter (Fischer Scientific accumet®). The quantitative evaluation of solvent (acetone, butanol, ethanol, acetic acid and butyric acid) was determined using the Agilent Technologies 7890A, Gas Chromatography system (Agilent Technologies Inc., Wilmington, DE, USA) equipped with a flame ionization detector (FID) and 30 m (length) x 320 pm (internal diameter) x 0.50 pm (HP-Innowax film) J x W 19091 N-213 capillary column. Nitrogen was the carrier gas, while the inlet and detector were maintained at 250 and 300 °C, respectively. The oven temperature was programmed to increase from 60 to 200 °C in 20 °C / min increments, with a 5-min hold at 200 °C. The injection volume of samples was 1 pL, with a split ratio of 10:1.

[0136] The ABE produced per gram of substrate was used to calculate yield while productivity was calculated as the concentration (g / L) of ABE produced per hour.

[0137] The MES cathode materials and the attached microbes were examined after prolonged (6 days) fermentation using scanning electron microscopy (Hitachi S-3500N scanning electron microscope). The electrodes from the cathode chamber were first fixed for 3 h in 2% glutaraldehyde solution in a 0.1 M sodium phosphate buffer and dried under vacuum. Afterward, the cathodes were rinsed with 0.1 M phosphate buffer for 1 hour and subjected to a stepwise dehydration process in ethanol (25%, 50%, 75%, 90%, 100%). Before characterization with SEM, the electrodes were coated with gold using a Leica EM ACE600 sputter coater.

[0138] Results and Discussion

[0139] Food Waste Characteristics

[0140] Food waste sample characteristics (Table 3) indicated that the pH of the ice cream samples was close to neutral, whilst the sour cream sample was acidic at pH 4.42. The moisture content of the sour cream sample was nearly twice that of the ice cream, and its ash content was three times higher. High moisture content makes it an excellent feedstock for fermentation and hydrothermal carbonization. The high ash content could be attributed to the presence of spices and vegetables. Also, the calorific content of ice cream was lower than sour cream, making sour cream the better candidate for bioenergy generation processes, such as anaerobic digestion and fermentation. Low total solids were similar to those previously reported. Lactic acid is a key component of sour cream, produced during fermentation by lactic acid bacteriaDocket No. 103362-092WO1 inoculated to achieve the desired acidity. The acidic pH of sour cream waste could also be influenced by the onion content, which contributes additional organic acids.

[0141] Sour cream waste was rich in calcium, potassium, sulfur, sodium, and phosphorus (Table 4). The sodium concentration in ice cream waste was six times higher than in sour cream waste, as NaCl is added as a preservative and taste enhancer. Ice cream waste was also rich in calcium, iron, potassium, and phosphorus. Potassium plays a vital role in plant stomatai conductance, and its higher concentration than other elements in both samples reflects the presence of plant sources of onion and green herbs in sour cream and fruits in ice cream. The iron concentration in ice cream samples was over five times higher than in sour cream and probably caused by the fruit content in the ice cream, because blueberries, cranberries, apples, grapes, and oranges are good sources of iron. Silicon was more than 11 times higher in sour cream than in ice cream. Silicon, the second most abundant element in the Earth's crust, is present in plants and plays an important role in managing both abiotic and biotic stress in plants. It is possible that the vegetables and spices added to sour cream were grown with silicon supplementation, as it increases plant resistance against insects, pests, and nematodes. Sugar concentration in ice cream waste samples (94 g glucose / L) was higher than in sour cream, because it is a sweet desert, but sour cream still had 64 g glucose / L (Figure 8) added as a taste enhancer. Thus, both wastes are good feedstocks for bacterial fermentation and probably also for yeast fermentation which requires high amounts of sugars and can produce enzymes.

[0142] Growth and pH Profile in Conventional and Electro-Fermentation

[0143] In both mono- and co-culture conventional fermentation the pH decrease (Figures 9A and 9B) signifies the rapid growth of bacterial species during the acidogenic phase, where sugars are oxidized to produce pyruvate and subsequently acetic or butyric acid. After the pH drop, the co-culture fermentation pH increased (Figure 9B) indicating the culture’s transition into the solventogenic phase, during which acids are reabsorbed and converted into acetone, our results substantiate previous reports. This pH increase was less pronounced in the monoculture fermentation (Figure 9A). During electro-fermentation, the pH drop was slower than in conventional fermentation, but pH declined much faster at -0.2V than at -0.1V (Figure 9C) and did not subsequently increase. This is because higher voltage accelerates the metabolic phase, and bacterial species remain in their acetogenic phase. The pH of the ABE fermentation broth is largely affected by the balance between protonated and unprotonated forms of acetic and butyric acids. The elevated acid levels in -0.2V MES show that acid reassimilation mayDocket No. 103362-092WO1 have been impaired, and modifications to the medium may be needed to absorb excess protons. Also, bacterial species may require additional time to acclimate to the new environment.

[0144] Optical density (OD) in conventional fermentation of glucose peaked at 36 h (Figures 10A and 10B). In mono-fermentation, OD was lower in the waste samples than in the glucose control until 72 h when the ice cream and glucose were similar (Figure 10A). However, the sour cream monoculture OD remained much lower than the other two treatments throughout the 84 hours fermentation. In co-fermentation, the OD of ice cream waste was similar to that of the glucose control, while the OD of sour cream waste was only slightly lower. Maximum cell growth in electro-fermentation of ice cream waste (Figure 10C) occurred at -0.1 V, followed by -0.2V at day 3 (72 hours), although bacterial growth that -0.1V exhibited a prolonged lag phase (Figure 10C). The high voltage differential of -0.2V suppressed bacterial growth.

[0145] Platform Chemical Production in Conventional Fermentation

[0146] Co-fermentation produced more acetone from sour cream than ice cream waste, reaching a peak at 36 h, whereas mono-fermentation of ice cream started around 48 h and reached up to 5 g / L higher than the control (Figure 11 A). Ethanol was produced earlier during fermentation than acetone and the other analytes. More ethanol was produced from sour cream than either ice cream or glucose in both mono- and co-fermentation (Figure 1 IB). Fermentation of both wastes and control samples produced more butanol than acetone or ethanol, although most was produced from glucose reaching 12 g / L within 72 h (Figure 11C). Butanol was produced from ice cream and sour cream at 60% and 44% of the control yield, respectively, showing that waste samples can be valorized with 60% efficiency compared to the control. The addition of trace amounts of inhibitors as a pH control strategy, such as phenolic compounds, can enhance butanol production. This approach should be further evaluated in complex waste samples.

[0147] Acetic acid levels were initially high at 8-9 g / L in co-fermentation of ice cream and sour cream waste, then decreased by 50% in sour cream samples at 72 hours, indicating a transition from the acidogenic to the solventogenic phase (Figure 1 ID). The buffer contains some amount of ammonium acetate that translate to the acetic acid in the medium. In contrast, mono-fermentation and control samples showed little to no phase transition. Lower pH levels were reached than in co-fermentation, which also reduced ABE yield. A rise in pH signals the transition from the acidogenic phase to the solventogenic phase.Docket No. 103362-092WO1 Butyric acid production was high in co-fermentation of both ice cream and sour cream samples, consistent with butanol production, as more product remained in the form of butyric acid rather than being converted to butanol (Figure 1 IE). The presence of protonated acid forms restricted acid assimilation and alcohol production. The balance between dissociated and associated acid species, closely linked to pH, is essential in the fermentation process.

[0148] Total ABE (acetone-butanol-ethanol) production was highest in the glucose control (16.8 g / L), followed by ice cream then sour cream in both mono- and co-fermentation. Several factors contribute to the lower product concentration and yield from fermentation of food waste samples than from glucose. First, glucose is easier to ferment than the complex forms of reducing sugars present in the waste samples. The presence of lactose in cream-based wastes creates a less favorable metabolic condition for Clostridium species, as they preferentially metabolize glucose. Additionally, the physicochemical characteristics of the waste samples negatively impacted growth and ABE production, as cultures showed a prolonged lag phase.

[0149] The comparatively higher yields in co-culture fermentation indicate a synergistic interaction between Clostridium species, allowing them to adapt more effectively to the complex nature of the waste samples. However, medium modification and pH control strategies are needed to increase sugar consumption rates and produce even higher ABE yields.

[0150] Platform Chemical Production in Electro-Fermentation

[0151] In MES mono-fermentation with ice cream waste at -0.1 V, alcohol and acid production were monitored over 11 days to capture the full production profile. Ethanol production began on day 1, but butanol production didn’t start until day 3 (Figure 12A). The highest yield of both was achieved around day 6, as Clostridium required time to adapt to the applied voltage conditions (Figure 12B). Acetic acid levels decreased within the first three days, ethanol production is much less than the acetic acid loss, while butyric acid levels remained stable, consistent with a low pH profile (Figure 9C). Excessive acid production and low pH can lead to an acid crash, where the buildup of undissociated acids inhibits glucose consumption and butanol production, ultimately resulting in ABE production failure.

[0152] Under similar conditions, glucose control was evaluated in MES at -0.1 and -0.2 V, producing a wider variety of products. Butanol production was 12 times higher at -0.1 V than -0.2 V (Figure 12C). Negative cathode potential led to the increased acetic acid production rate and positive potential lacks the reducing force needed to trigger electron consumption. Co-fermentation of ice cream waste at both voltages yielded only acetic acid and ethanol (FiguresDocket No. 103362-092WO1 12E and 12F). Product formation in the waste samples surpassed that in the glucose control, with lower voltages resulting in higher production of VFAs compared to higher voltages.

[0153] In MES, VFA production accelerated after 48 h, aligning with the timing observed in conventional fermentation. Ethanol and acetic acid production levels in conventional fermentation were similar to those in electro-fermentation at 24 and 48 hours. However, co-electro-fermentation produced a narrower range of products than mono-electro-fermentation, with no synthesis of four-carbon compounds. Thus, electro-fermentation appears more suitable for producing short-chain fatty acids than conventional fermentation. These experiments used batch fermentation with a fresh bacterial consortium, and it was contemplated that fed-batch experiments with pre-acclimatized consortia would yield better results. In similar studies using artificial growth medium, acetate was the only VFA detected in the cathode chamber. The highest VFA production was achieved at -0.89 V, though these studies were primarily conducted with artificial media and mixed microbial consortia. In this study, which used real food waste samples, voltages greater than -0.1 V caused overload potential, showing that the system's internal resistance at higher applied voltage impeded electrochemical reactions. Acetic acid can be produced electrochemically under biologically relevant conditions at a potential of -0.28 V. However, in practice, more negative potentials are applied to overcome overpotential losses and enhance the production rate.

[0154] MES Electrochemical and Microscopic Characteristics

[0155] Current density is the most critical parameter in a bioelectrochemical system, as it reflects both performance and stability. The current density profile during the co-electro-fermentation of glucose was quite steady at near 0 mA / cm2over more than five days at -0.1V (Figure 13 A). In contrast, co-electro-fermentation of ice cream initially exhibited a pronounced negative current density that gradually normalized to near 0 mA / cm2. The indentations in the profile indicate the points where the system transitioned to cyclic voltammetry analysis. Cyclic voltammetry (CV) analysis was performed using a three-electrode system within a potential range of -900 to +400 mV and scan rates varying from 1 to 200 mV / s. In the MES, the biocathode functioned as the working electrode for the CV analysis. CV was performed three times over the six-day batch operation (Figure 13). The maximum current reduction response was 3 mA for glucose (Figure 13B) and 24 mA for ice cream waste (Figure 13C) (MES at -0.1 V vs Ag / AgCl). Redox peaks at -500 mV were because of flavoproteins. The onset potential for glucose and ice cream waste was -0.1 V and -0.4 V, respectively, indicating thatDocket No. 103362-092WO1 microbial electrochemical activity in the cathodic biofilm was higher in glucose control than ice cream waste MES. The lower onset potential observed for the ice cream shows that less energy is required to overcome the thermodynamic barrier for microbial conversion reactions than when glucose was the substrate. CV analysis confirmed electrochemical interaction between the microorganisms and the electrode in the broth. The combined electrochemical characteristics and current consumption data show that electro-fermentation operates by adjusting the redox potential of the external medium rather than acting as a direct electron donor.

[0156] After a complete cycle of electro-fermentation, microbes attached to the carbon cloth electrodes were visualized using SEM (Figure 14). A biofilm was visible, though not dense, indicating that biofilm formation is not a prerequisite for VFA production. This show that suspended or agglomerated bacterial consortia are actively fermenting (Figures 14B and 14C). Clostridium species tested in MES demonstrated electroactive compatibility. Biofilm formation depends on factors such as cathode potential, electrode material, and pH. At a higher voltage of -0.2V, biofilm agglomerates showed an increased area of microbial colonization (Figure 14D). In co-electro-fermentation, denser biofilms indicate synergistic growth of both Clostridium species (Figures 14E and 14F). Direct electron transfer or external mediators likely facilitated electron acceptance, as no external appendages were observed. The biofilm development demonstrates that carbon cloth is a promising material for biocathodes; however, biocompatible catalyst coatings could further enhance electron transfer.

[0157] Microbial Electrosynthesis Efficiency

[0158] Direct comparison of monoculture and co-culture inoculation in conventional and electro-fermentation for acetone, butanol, and ethanol production is an innovative approach that has not been previously evaluated. Most studies have used a nutrient catholyte but this disclosure provides the first exploration using real food wastes. Additionally, very few studies have used Clostridium species or mixed culture inoculum from anaerobic digestate. Herein, a synergistic relationship was developed between C. beijerinckii and C. carboxidivorans.

[0159] In a previous study, acetic acid was identified as the primary product and propionic acid as the secondary product when using a nutritional medium. Herein, however, the microbial culture successfully metabolized food waste to produce four-carbon products. A stillage has been previously used as the inoculum source and observed caproic acid (6.8 g / L) as the major product in conventional fermentation, which is less than half the yield achieved in the presentDocket No. 103362-092WO1 disclosure. In an H-type bioreactor, butyrate was the main product during electro-fermentation using anaerobic sludge inoculum. A similar experiment using a nutrient catholyte and Shewanella oneidensis at an applied voltage of -0.1 V, observing 160 mg / L of butanol. Herein, 6 g / L of butanol was observed using ice cream waste as feedstock. Table 5 summarizes the comparison of the present study with existing literature.

[0160] Clostridium species has been observed to produce caproic acid as the main product, though the yield was low at a high voltage of -1.0 V. A microbial electrosynthesis system (MES) with mixed cultures enriched under THCCh conditions was observed to produce low amounts of caproic acid. Observations from other studies indicate that adjusting the applied voltage or using specific fermentative bacterial species could enhance productivity. The results of the present disclosure are promising, and further increases possibilities of adjusting the operating conditions.

[0161] Conclusion

[0162] Mono-electro-fermentation of ice cream waste produced higher amounts of butanol while co-electro-fermentation produced higher amounts of acetic acid and ethanol. Low applied voltage favored higher yield. The complex nature of food waste was the critical reason for lower current density and VFAs production in electro-fermentation as compared to conventional fermentation. Medium modification, pH adjustment, MES configuration with higher contact area across the membrane could improve the electro-fermentation yields. The findings underscore the biocathode MESs in food waste valorization and environmental remediation.

[0163] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.Docket No. 103362-092WO1

[0164] TABLES

[0165] Table 1

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[0167] Table 2

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[0171] Docket No. 103362-092WO1 Table 3. Physio-chemical characteristics of waste samples

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[0173] Table 4. Major and minor elements in food waste samples

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[0177] Docket No. 103362-092WO1 Table 5. Platform chemical production in conventional and electro-fermentation reported in literature

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Claims

Docket No. 103362-092WO1 CLAIMSWhat is claimed is:

1. A microbial electrochemical system (MES) comprising two chambers connected by a bridge into an H-type configuration, wherein a first chamber is a cathode chamber comprising at least two Clostridium bacterial species and a second chamber is an anode chamber.

2. The MES of claim 1, wherein the at least two Clostridium bacterial species comprise Clostridium carboxidivorans and Clostridium beijerinckii .

3. The MES of claim 1 or 2, wherein the first chamber comprises a food waste, algae, or a combination thereof.

4. The MES of claim 3, wherein the food waste comprises an organic composition.

5. The MES of claim 4, wherein the organic composition comprises carbohydrates, fatty acids, and / or a plurality of essential elements selected from potassium (K), magnesium (Mg), sulfur (S), calcium (Ca), phosphorus (P), aluminum (Al), iron (Fe), manganese (Mn), sodium (Na), copper (Cu), boron (B), zinc (Zn), silicon (Si), strontium (Sr), selenium (Se), and boron (B).

6. The MES of claim 3 or 4, wherein the food waste comprises a coffee, an ice cream, a sour cream, or infant formula.

7. The MES of claim of any one of claims 1-6, wherein the second chamber comprises an inorganic salt.

8. The MES of claim 7, wherein the inorganic salt is sodium sulfate (Na2SC>4).

9. The MES of any one of claims 1-8, wherein the first chamber and the second chamber are separated by a proton exchange membrane.Docket No. 103362-092WO1 10. The MES of any one of claims 1-9, wherein the at least two Clostridium bacterial species are co-cultured in the cathode chamber.

11. The MES of any one of claims 1-10, wherein the at least two Clostridium bacterial species convert the food waste, algae, or a combination thereof into one or more biofuels.

12. The MES of claim 11, wherein the one or more biofuels comprise butanol, ethanol, acetone, or a combination thereof.

13. A method of producing one or more biofuels in a microbial electrochemical system (MES), the method comprising:a. adding an inorganic salt to an anode chamber,b. adding at least two Clostridium bacterial species and a food waste, algae, or a combination thereof, to a cathode chamber,c. assembling a MES by connecting the anode chamber to the cathode chamber using a connecting bridge, andd. applying a low voltage across the MES,wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane, and wherein the at least two Clostridium bacterial species convert the food waste, algae, or a combination thereof, into the one or more biofuels.

14. A method of fermenting a food waste using a microbial electrochemical system (MES), the method comprising:a. adding an inorganic salt to an anode chamber,b. adding at least two Clostridium bacterial species and the food waste to a cathode chamber,c. assembling a MES by connecting the anode chamber to the cathode chamber using a connecting bridge, andd. applying a low voltage across the MES,wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane, and wherein the at least two Clostridium bacterial species convert the food waste into one or more biofuels.Docket No. 103362-092WO1 15. The method of claim 13 or 14, wherein the at least two Clostridium bacterial species comprise Clostridium carboxidivorans and Clostridium beijerinckii .

16. The method of any one of claims 13-15, wherein the at least two Clostridium bacterial species are co-cultured in the cathode chamber.

17. The method of any one of claims 13-16, wherein the Clostridium beijerinckii metabolizes the food waste.

18. The method of any one of claims 13-17, wherein the Clostridium carboxidivorans produces the one or more biofuels.

19. The method of any one of claims 13-18, wherein the food waste comprises a coffee, an ice cream, a sour cream, or infant formula.

20. The method of any one of claims 13-19, wherein the one or more biofuels comprise butanol, ethanol, acetone, or a combination thereof.

21. The method of any one of claims 13-20, wherein the inorganic salt comprises sodium sulfate (Na2SC>4).

22. The method of any one of claims 13-21, wherein the low voltage ranges from 0V to - 0.5V.

23. A method of fermenting algae using a microbial electrochemical system (MES), the method comprising:a. adding an inorganic salt to an anode chamber, b. adding at least two Clostridium bacterial species and the algae to a cathode chamber,c. assembling a MES by connecting the anode chamber to the cathode chamber using a connecting bridge, andd. applying a low voltage across the MES,Docket No. 103362-092WO1 wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane, and wherein the at least two Clostridium bacterial species convert the algae into one or more biofuels.

24. The method of claim 23, wherein the at least two Clostridium bacterial species comprise Clostridium carboxidivorans and Clostridium beijerinckii .

25. The method of claim 24 or 25, wherein the cathode chamber further comprises a food waste.

26. The method of claim 25, wherein the food waste comprises coffee.

27. The method of any one of claims 23-26, wherein the at least two Clostridium bacterial species are co-cultured in the cathode chamber.

28. The method of any one of claims 23-27, wherein the Clostridium beijerinckii metabolizes the algae.

29. The method of any one of claims 23-28, wherein the Clostridium carboxidivorans produces the one or more biofuels.

30. The method of any one of claims 23-29, wherein the one or more biofuels comprise butanol, ethanol, acetone, or a combination thereof.

31. The method of any one of claims 23-30, wherein the inorganic salt comprises sodium sulfate (Na2SC>4).

32. The method of any one of claims 23-31, wherein the low voltage ranges from 0V to - 0.5V.

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