Patent Application
20190020049
The present invention relates to the field of bioelectricity. More particularly, the present invention provides energy generating systems, methods, and devices that are capable of converting chemical energy stored in a variety of renewable sugars into useful electricity. In specific embodiments, the present invention relates to novel synthetic enzymatic pathways for converting chemical energy from six-carbon and five-carbon sugars to electricity using enzymatic fuel cells, and several key enzymes with engineered and/or newly-discovered functions.
Batteries are electricity storage devices. Rechargeable batteries are currently available. But, the energy storage densities of such rechargeable batteries are much lower than those the energy storage densities of hydrogen or liquid fuels (
Enzymatic fuel cells (EFCs) are a type of biological fuel cells that employ enzymes to convert the chemical energy in the fuels into electricity. EFCs are superior to batteries mainly because they: 1) have approximately 10-100 times higher energy storage densities than chemical batteries; and 2) are more environmentally friendly due to the biodegradability and elimination of heavy metals and costly rare metals. EFCs (
In EFCs, electrons are generated when fuels arc oxidized at an anode. The electrons then flow from the anode through an external load to a cathode. Protons are generated simultaneously (with the electrons) in the anodic reactions and pass through the polymer separator to the cathode to compensate for the electron flow. One of the largest challenges with EFCs is the extraction of most or all of the chemical energy from the low-cost and most abundant sugars for electricity generation. The methods to utilize the all energy of the sugars have not been developed so far, except for the methods that utilize sugars as a heat energy source by combustion in air or as a chemical energy source for the production of ATP through NAD(P)H generated by redox enzymes in living organisms (such as microorganisms, animals). There is no method that is capable of effectively utilizing most of the chemical energy of sugars directly as electric (electrical) energy.
In one embodiment, the present invention provides a process for generating electrons from hexose sugars comprising: generating glucose 6-phosphate (G6P) from a chemical reaction of 6-carbon sugar monomers or one or more 6 carbon sugars from oligohexoses or polyhexoses reacted with polyphosphate or ATP or phosphate, wherein: (i) when using polyphosphate, the chemical reaction is performed in the presence of polyphosphate-glucose phosphotransferase; or (ii) when using ATP, the chemical reaction is performed in the presence of hexokinase, and wherein the ATP is generated by reacting ADP and polyphosphate in the presence of polyphosphate kinase; (iii) when using free phosphate, the chemical reaction is performed in the presence of glucan phosphorylases (e.g., starch phosphorylase, maltose phosphorylase, sucrose phosphorylase, cellobiose phosphorylase, cellodextrin phosphorylase) and phosphoglucomutase; reacting the G6P and 6PG with NAD or its analogues (called biomimics in the oxidized form) in water to obtain NADH or the reduced biomimics; and oxidizing the NADH or the reduced biomimics on an anode at its surface to generate electrons. In one embodiment, the product of ribulose 5-phosphate is converted to G6P via a hybrid pathway of the non-oxidative pentose phosphate pathway, glycolysis and gluconeogenesis.
In another embodiment, the process further comprises NAD biomimetics (
In some embodiments, the process further comprises engineered glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase that can utilize NAD biomimics.
In some embodiments, the process further comprises glucose as the sugar. In one embodiment, glucose is converted to glucose 6-phosphate via polyphosphate glucokinase or hexokinase. In some embodiments, the process further comprises generating the glucose from: (i) converting fructose to glucose using glucose (xylose) isomerase; or (ii) converting fructose to glucose with sorbitol dehydrogenase and aldehyde reductase.
In other embodiments, the process further comprises generating fructose or fructose 6-phosphate by: (i) converting mannose to fructose using mannose isomerase and then, in one embodiment, enter the above fructose-utilization pathway; (ii) converting mannose to fructose 6-phosphate (F6P) using polyphosphate-glucose mannose phosphotransferase and phosphomannose isomerase and, in one embodiment, the product F6P enters a modified pentose phosphate pathway; or (iii) converting mannose to fructose-6-phopshate by using polyphosphate kinase, hexokinase, and phosphomannose isomerase and, in one embodiment,then the product F6P enters a modified pentose phosphate pathway.
In some embodiments, the process further comprises starch or maltodextrin as the sugars. In other embodiments, the process further comprises phosphate and phosphoglucomutase and starch or maltodextrin phosphorylase for G6P generation.
In some embodiments, the present invention provides a sugar battery comprising: a solution capable of generating glucose 6-phosphate (G6P) from 6-carbon sugar monomers or one or more 6 carbon sugars from oligohexoses or polyhexoses reacted with polyphosphate or ATP or phosphate, and for reacting the G6P with NAD or its analogues in water to obtain NADH or its analogues (biomimetics), wherein: (i) when using polyphosphate, the chemical reaction is performed in the presence of polyphosphate-glucose phosphotransferase; or (ii) when using ATP, the chemical reaction is performed in the presence of hexokinase, and wherein the ATP is generated by reacting ADP and polyphosphate in the presence of polyphosphate kinase; (iii) when using free phosphate, the chemical reaction is performed in the presence of glucan phosphorylases and phosphoglucomutase; an enzyme-modified anode; an enzyme-modified or standard platinum reference cathode; and an electrolyte, wherein the enzyme-modified anode or anode is in contact with the solution comprising the six carbon sugars and some enzymes in the synthetic pathways and both the cathode and the enzyme-modified anode are in contact with the electrolyte.
In some embodiments, an electrolyte is made of the pH-control buffer (e.g., HEPES) containing metal ions such as (Mg++, Mn++), NAD+ or NADH or its biomimics, and thiamine pyrophosphate: In some embodiments, the battery is operably configured for allowing the oxidation of the NADH or its biomimetics on the anode at its surface to generate electrons.
In some embodiments, the present invention provides a system for energy generation comprising: a fuel comprising a solution for generating glucose 6-phosphate (G6P) from 6-carbon sugar monomers or one or more 6 carbon sugars from oligohexoses or polyhexoses reacted with polyphosphate or ATP or phosphate, and for reacting the G6P with NAD+ and oxidized biomimetics in water to obtain NADH and reduced biomimics, wherein: (i) when using polyphosphate, the chemical reaction is performed in the presence of polyphosphate-glucose phosphotransferase; or (ii) when using ATP, the chemical reaction is performed in the presence of hexokinase, and wherein the ATP is generated by reacting ADP and polyphosphate in the presence of polyphosphate kinase; (iii) when using free phosphate, the chemical reaction is performed in the presence of glucan phosphorylases and phosphoglucomutase; a fuel cell operably configured for oxidizing the NADH and its biomimics at an anode to generate electrons and for delivering the electrons, e.g., via an outside circuit to a cathode. In one embodiment,; a catalyst on cathode converts protons and oxygen to water.
In some embodiments, the present invention provides a process for generating electrons from the pentose sugars comprising: generating xylulose 5-phosphate (X6P) from a chemical reaction of 5-carbon sugar monomers reacted with polyphosphate or ATP, wherein: (i) when using polyphosphate, the chemical reaction is performed in the presence of xylose isomerase and polyphosphate xylulokinase; or (ii) when using ATP, the chemical reaction is performed in the presence of xylose isomerase and ATP-based xylulokinase, and wherein the ATP is generated by reacting ADP and polyphosphate in the presence of polyphosphate kinase or a combination of polyphosphate:AMP phosphotransferase and polyphosphate-independent adenylate kinase; entering the pentose phosphate pathway for generation of G6P; reacting the G6P with NAD+ and the oxidized biomimics in water to obtain NADH and the reduced biomimics; oxidizing the NADH and the reduced biomimics on an anode at its surface to generate electrons.
In some embodiments, the present invention provides a sugar battery comprising:
a solution capable of generating xylulose 5-phosphate from 5-carbon sugar monomers reacted with polyphosphate or ATP, and for producing glucose 6-phosphate through non-oxidative pentose phosphate pathway, and reacting the G6P with NAD+ or its analogues in water to obtain NADH or its analogues (biomimetics), wherein: a. when using polyphosphate, the chemical reaction is performed in the presence of polyphosphate-xylulose kinase; or b. when using ATP, the chemical reaction is performed in the presence of xylulokinase, and wherein the ATP is generated by reacting ADP and polyphosphate in the presence of polyphosphate kinase; an enzyme-modified anode or anode; an enzyme-modified or standard platinum reference cathode; and an electrolyte, wherein the enzyme-modified anode or anode is in contact with the solution comprising the five carbon sugars and some enzymes in the synthetic pathways and both the cathode and the enzyme-modified anode are in contact with the electrolyte.
In some embodiments, an electrolyte is made of the pH-control buffer (e.g., HEPES) containing metal ions such as (Mg++ or Mn++), NAD+ or NADH or its biomimics, and thiamine pyrophosphate.
In some embodiments, the present invention provides a system for energy generation comprising: a fuel comprising a solution for generating xylulose 5-phosphate from five-carbon sugar monomers reacted with polyphosphate or ATP, and for generation of G6P through the non-oxidative pentose phosphate pathway, for reacting the G6P with NAD+ and its biomimetics in water to obtain NADH and reduced biomimics, wherein: (i) when using polyphosphate, the chemical reaction is performed in the presence of polyphosphate-xylulokinase; or (ii) when using ATP, the chemical reaction is performed in the presence of xylulokinase, and wherein the ATP is generated by reacting ADP and polyphosphate in the presence of polyphosphate kinase; a fuel cell operably configured for oxidizing the NADH and its biomimics at an anode to generate electrons and for delivering the electrons to a cathode; an electrical generator for converting the electrons from the cathode into electricity.
In some embodiments, the present invention provides a process for generating electrons from a sugar comprising: generating NADH or a reduced biomimic thereof from the sugar; and oxidizing the NADH or a reduced biomimic thereof to generate the electrons at an anode, wherein the NADH or a reduced biomimic thereof is generated using a group of enzymes comprising glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase, transaldolase, triose phosphate isomerase, aldolase, fructose 1,6-bisphosphatase, and phosphoglucose isomerase.
In some embodiments, the process comprises a hexose or a pentose sugar as the sugar.
In one embodiment of the process, the NADH or a reduced biomimic thereof is oxidized by diaphorase. In another embodiment of the process, the NADH or a reduced biomimic thereof is oxidized in the presence of one or more of vitamin K3, benzyl viologen, or a biomimetic thereof. In some embodiments, one or more of vitamin K3, benzyl viologen, or a biomimetic thereof is immobilized on the anode surface. In other embodiments, one or more of vitamin K3, benzyl viologen, or a biomimetic thereof is free in the anode compartment.
In some embodiments, the present invention provides a sugar battery comprising: a solution comprising a sugar, enzymes, and an electrolyte; an anode; and a cathode, wherein the anode and the cathode are in contact with the solution; wherein the electrolyte comprises a pH-control buffer containing metal ions, NAD or NADH or a biomimic thereof, and thiamine pyrophosphate, and wherein the enzymes comprise glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase, transaldolase, triose phosphate isomerase, aldolase, fructose 1,6-bisphosphatase, phosphoglucose isomerase, and an enzyme capable of oxidizing NADH or a biomimic thereof.
In some embodiments, the sugar battery comprises a hexose or a pentose sugar as the sugar. In some embodiments of the sugar battery, the enzyme capable of oxidizing NADH or a reduced biomimic thereof is diaphorase.
In some embodiments, the sugar battery further comprises one or more of vitamin K3, benzyl viologen, or a biomimetic thereof. In some embodiments, one or more of vitamin K3, benzyl viologen, or a biomimetic thereof is immobilized on the anode surface. In other embodiments, one or more of vitamin K3, benzyl viologen, or a biomimetic thereof is free in the anode compartment.
In some embodiments, the present invention provides a system for electricity generation comprising: a solution comprising a sugar, enzymes, and an electrolyte; a fuel cell comprising an anode and a cathode; and an electrical generator, wherein the solution and the electrical generator are in contact with the fuel cell; wherein the electrolyte comprises a pH-control buffer containing metal ions, NAD+ or NADH or a biomimic thereof, and thiamine pyrophosphate; and wherein the enzymes comprise glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase, transaldolase, triose phosphate isomerase, aldolase, fructose 1,6-bisphosphatase, phosphoglucose isomerase, and an enzyme capable of oxidizing NADH or a biomimic thereof.
In some embodiments, the system comprises a hexose or a pentose sugar or a mixture thereof as the sugar. In some embodiments of the system, the enzyme capable of oxidizing NADH or a reduced biomimic thereof is diaphorase.
In some embodiments, the system further comprises one or more of vitamin K3, benzyl viologen, or a biomimetic thereof. In some embodiments, one or more of vitamin K3, benzyl viologen, or a biomimetic thereof is immobilized on the anode surface. In other embodiments, one or more of vitamin K3, benzyl viologen, or a biomimetic thereof is free in the anode compartment.
These drawings illustrate certain aspects of some of the embodiments of the present invention, and should not be used to limit or define the invention.
The present invention provides a method for converting chemical energy stored in sugars to electricity. In certain embodiments, the present invention provides synthetic (artificial) enzymatic pathways for converting chemical energy from six-carbon sugars and five-carbon sugars to electricity using enzymatic fuel cells. In a preferred embodiment of the present invention, sugar batteries are used to convert the chemical energy to electricity. One of the many advantages of the present invention is that the sugar batteries disclosed herein have an energy density that may be more than about 10-fold higher than the energy densities of the current enzymatic fuel cells and rechargeable batteries. Other advantages of the sugar batteries of the present invention include, but are not limited to, the utilization of low-cost feedstock, modest reaction conditions and low-cost catalysts (e.g., enzymes), high energy storage density, low safety concern (e.g., neither explosion nor flammability), abundant supply of all materials and catalysts, fast refilling, zero carbon emissions, environmentally friendly, and usability in a wide variety of applications.
Another advantage of the present invention is that, unlike primary and secondary batteries that suffer from low energy storage densities, the fuel cells described herein have much higher densities. Enzymatic fuel cells are a type of fuel cells that can utilize enzymes to convert chemical energy stored in chemical compounds to electricity. Six-carbon sugar monomers (e.g., glucose, fructose, mannose) and their derivatives (e.g., maltose, cellodextrins, sucrose, lactose, cellobiose, cellodextrin, cellulose, starch) and xylose and its derivatives (e.g., hemicellulose, xylan) are the most abundant carbohydrates and therefore a great source of energy. The present invention provides a number of novel non-natural synthetic enzymatic pathways that can convert these six-carbon sugars and five-carbon sugars to electricity through partial or complete oxidation mediated by a number of cascades of enzymes. Because the sugar batteries have a high energy storage density, are biodegradable and quickly refillable, and have a low explosion risk, they can successfully replace most primary batteries, secondary batteries and direct methanol fuel cells.
The preferred fuels for sugar batteries include six-carbon sugars from monosaccharides (e.g., glucose, mannose, fructose, and galactose), oligosaccharides (e.g., maltose, maltodextrins, sucrose, cellobiose, cellodextrins, lactose), and polysaccharides (e.g., cellulose, starch, and glycogen), D-xylose, L-arabinose, hemicellulose, xylan, and any combinations thereof. The output is electricity. A typical scheme of an exemplary sugar battery according to embodiments of the present invention is shown in
Starch is the most widely used energy storage compound in nature. The catabolism of starch allows for a slow and nearly constant release of chemical energy in living cells that is different from its monomer glucose. Maltodextrin, a partially hydrolyzed starch fragment, is a superior fuel to glucose in EFCs, because maltodextrin has 11% higher energy density than glucose. Maltodextrin is also less costly because glucose is the main product of its enzymatic hydrolysis, and low-cost linear maltodextrin can be made from cellulose. An equivalent weight of maltodextrin has a much lower osmotic pressure than glucose. Moreover, it can provide slowly-metabolized glucose 1-phosphate for more stable electricity generation in closed EFCs. Maltodextrin has been used as a fuel for EFCs, but only two electrons could be generated per glucose unit before this invention.
Pathways
The inventive pathways include specific enzymes for converting the stored energy of specific sugars into useful electricity. A complete utilization of the sugar fuel is possible by oxidizing G6P (glucose 6-phosphate) in the presence of NAD+ or a biomimetic analogue (or biomimic) thereof. The advantages of the embodiments of the present invention include the ability to use a sugar as a starting material, and not G6P and X5P (xylulose 5-phosphate) directly. This provides for a low-cost source of G6P and X5P. The starting material (e.g., sugar as a fuel) is efficiently used, which was not achieved previously, by using a modified pentose phosphate pathway along with enzymes from the glycolysis and gluconeogenesis pathways coupled with a complete conversion of NAD+ or a biomimic thereof into NADH or a biomimic thereof.
In some embodiments, the present invention provides a novel combination of enzymatic pathways for sugar batteries. In these embodiments, the novel synthetic enzymatic pathways contain three parts, referred to herein as modules. Module one generates low-cost glucose 6-phosphate from any six-carbon sugar. Module two allows NADH or a biomimic thereof to be generated from a modified pentose phosphate pathway together with enzymes from the glycolysis and gluconeogenesis pathways (G6P+7 H2O+12 NAD+→12 NADH+12 H++6 CO2). Module 3 allows reduced NADH or a biomimic thereof to be oxidized on the surface of an anode for electron generation.
In one embodiment, Module I contains a single pathway as shown below. In another embodiment, Module I may contain a combination of pathways in an enzymatic fuel cell. The present invention provides pathways that can generate G6P from any monomer hexose without consumption of costly ATP by using polyphosphate as a phosphate donor, followed by its regeneration.
Pathway 1
in one embodiment, the pathway is provided as Pathway 1. Pathway 1 starts with glucose, which can be generated by simple hydrolysis or phosphorolysis of oligosaccharides and polysaccharides. G6P can be generated as “Glucose+(Pi)n→G6P+(Pi)n−1” by polyphosphate-glucokinase (PPGK, EC 2.7.1.6, polyphosphate-glucose phosphotransferase) or “Glucose+ATP→G6P+ADP” by hexokinase (HK, EC2.7.1.1), where ATP can be generated at the cost of polyphosphate as ADP+(Pi)n→ATP+(Pi)n−1 by polyphosphate kinase (PPK, EC 2.7.4.1) or a combination of polyphosphate:AMP phosphotransferase (PPT, EC 2.7.4.B2) and polyphosphate-independent adenylate kinase (ADK, EC 2.7.4.3). After the complete oxidation of G6P, free phosphate can be regenerated to produce polyphosphate by chemical or biological means.
Pathway 2
In another embodiment, the pathway is provided as Pathway 2. Pathway 2 starts with fructose. The fructose can be converted into glucose by glucose (xylose) isomerase (EC 5.3.1.5), and then glucose is processed through the above pathway (Pathway 1). In another embodiment, glucose may be generated by coupled enzymes sorbitol dehydrogenase (EC 1.1.1.14) and aldehyde reductase (EC 1.1.1.21).
Pathway 3
In another embodiment, the pathway is provided as Pathway 3. Pathway 3 starts with mannose. In one embodiment, mannose can be converted to fructose by mannose isomerase (EC 5.3.1.7), and then fructose is processed through Pathway 2. In another embodiment, mannose can be converted to fructose 6-phosphate by two enzymes (polyphosphate-glucose mannose phosphotransferase, EC 2.7.1.63 and phosphomannose isomerase, EC 5.3.1.8), and then fructose 6-phosphate is processed through a modified pentose phosphate pathway (modified PPP) together with enzymes from the glycolysis and gluconeogenesis pathways. In another embodiment, fructose 6-phopshate is generated by using three enzymes—polyphosphate kinase (EC 2.7.4.1), hexokinase (EC 2.7.1.1) and phosphomannose isomerase (EC 5.3.1.8), resulting in the overall reaction of mannose+(Pi)n→fructose 6-phosphate+(Pi)n−1.
Pathway 4
In another embodiment, the pathway is provided as Pathway 4. Pathway 4 starts with galactose. Five enzymes together convert galactose to glucose 6-phopshate in the overall reaction of: galactose+(Pi)n→glucose 6-phosphate+(Pi)n−1. These five enzymes are polyphosphate kinase (EC 2.7.4.1), galactokinase (EC 2.7.16), UDP-glucose-hexose-1-phosphate uridylyltransferase (EC 2.7.7.12), UDP-galactose-4-epimerase (EC 5.1.3.2), and phosphoglucomutase (EC 5.4.2.2).
Polyphosphate regeneration can be performed by chemical and/or biological approaches. Free phosphate ion can be precipitated by forming insoluble salts including, but not limited to, Mg3(PO4)2 and Ca3(PO4)2. Polyphosphate can be made by adding concentrated H2SO4, followed by heating. Alternatively, Microlunatus phosphovorus takes up free phosphate and accumulates polyphosphate intracellularly under glucose-limited conditions.
The present invention provides pathways that can generate G6P from oligosaccharide without consumption of costly ATP by using polyphosphate and its regeneration and substrate phophosphorylation by using respective phosphorylases.
Pathway 5
In another embodiment, the pathway is provided as Pathway 5. Pathway 5 starts with maltodextrin. Maltodextrin with DP (Degree of Polymerization)=n can be converted to (n−1) glucose-1-phosphate and glucose by maltodextrin phosphorylase (EC 2.4.1.1) and maltose phosphorylase (EC 2.4.1.8). Glucose 1-phosphate is produced by phosphoglucomutase (EC 5.4.2.2) followed by the PPP with enzymes from the glycolysis and gluconeogenesis pathways, and glucose is processed through Pathway 1.
Pathway 6
In another embodiment, the pathway is provided as Pathway 6. Pathway 6 starts with sucrose. Sucrose can be converted to glucose 1-phosphate and fructose by sucrose phosphorylase (EC2.4.1.7). The fructose and glucose 1-phosphate may be processed through Pathway 2 and by phosphoglucomutase (EC 5.4.2.2), respectively.
Pathway 7
In another embodiment, the pathway is provided as Pathway 7. Pathway 7 starts with water-soluble cellodextrins, including, but not limited to, cellobiose and cellodextrins. Cellodextrins with DP=n can be converted to (n−1) glucose 1-phosphate and glucose by cellobiose phosphorylase (EC 2.4.1.20) and cellodextrin phosphorylase (EC 2.4.1.49). The remaining pathway may be the same as Pathway 5.
The present invention provides various exemplary pathways that can generate monomeric hexoses from oligosaccharides or polysaccharides after hydrolysis, which are then processed by Pathways 1-7.
Pathway 8
In another embodiment, the pathway is provided as Pathway 8. Pathway 8 starts with sucrose. Sucrose can be hydrolyzed to glucose and fructose by sucrase (EC 3.2.1.10). The products may enter Pathways 1 and 2, respectively.
Pathway 9
In another embodiment, the pathway is provided as Pathway 9. Pathway 9 starts with lactose. In one embodiment, lactose can be hydrolyzed to glucose and galactose by lactase (EC 3.2.1.23). In another embodiment, lactose can be phosphorolyzed to galactose 1-phosphate and glucose by lactose phosphorylase. The products may enter Pathways 1 and 4.
Pathway 10
In another embodiment, the pathway is provided as Pathway 10. Pathway 10 starts with starch, glycogen, maltose or maltodextrins. The starch or glycogen can be partially hydrolyzed to maltodextrins and maltose by using alpha-amylase (EC 3.2.1.1) and other starch hydrolyzing enzymes, such as isoamylase (EC3.2.1.68), pullulanase (EC3.2.1.41). The starch, maltose or maltodextrin can be hydrolyzed to glucose by glucoamylase (EC 3.2.1.3), alpha-amylase (EC 3.2.1.1), and other starch hydrolyzing enzymes. Glucose may enter Pathway 1.
Pathway 11
In another embodiment, the pathway is provided as Pathway 11. Pathway 11 starts with insoluble cellulose or pretreated biomass. The insoluble cellulose or pretreated biomass can be hydrolyzed to soluble cellodextrins by individual endoglucanase (EC 3.2.1.4) and/or cellobiohydrolase (EC 3.2.1.74) or a combination thereof. The products of soluble cellodextrins and glucose may enter Pathways 1 and 7.
The present invention provides pathways that can generate glucose 1-phosphate from polysaccharides (starch, cellulose, or glycogen) and may be phosphorolyzed by their respective glucan phosphorylases and accessory enzymes.
Pathway 12
In another embodiment, the pathway is provided as Pathway 12. Pathway 12 starts with linear starch (amylose) or their short-fragments with DP=n. (n−1) glucose 1-phosphate and glucose may be generated from starch and free phosphate by starch phosphorylase (EC 2.4.1.1) and maltose phosphorylase (EC 2.4.18). After the conversion of glucose 1-phosphate to glucose 6-phosphate by phosphoglucomutase, G6P enters the PPP for electricity generation.
Pathway 13
In another embodiment, the pathway is provided as Pathway 13. Pathway 13 starts with branched starch (amylopectin) or glycogen. Most glucose units in the linear chains may be removed to generate glucose 1-phosphate by starch or glycogen phosphorylase (EC 2.4.1.1). At the end of branch points, starch debranching, enzymes or pullulanases (EC 3.2.1.41) can be used to enhance further conversion. The minor product, glucose, can be used to generate G6P through Pathway 1.
The present invention provides pathways that utilize xylose and ATP or polyphosphate to generate xylulose 5-phosphate, which will be converted to G6P through the pentose phosphate pathway and enzymes in glycolysis and gluconeogenesis. Then G6P is consumed to generate NADH or a biomimic thereof through Pathway 1.
Pathway 14
In another embodiment, the pathway is provided as Pathway 14. Pathway 14 starts with xylose. Xylose is converted to xylulose by xylose isomerase (XI, EC 5.3.1.5), and then to xylulose 5-phosphate by xylulokinase (XK, EC 2.7.1.17) by using ATP. ATP can be regenerated as ADP+(Pi)n→ATP+(Pi)n−1 by polyphosphate kinase (PPK, EC 2.7.4.1) or a combination of polyphosphate:AMP phosphotransferase (PPT, EC 2.7.4.B2) and polyphosphate-independent adenylate kinase (ADK, EC 2.7.4.3). The product xylulose 5-phosphate can be converted to G6P through the non-oxidative pentose phosphate pathway. Then G6P is consumed to generate NADH or a biomimic thereof through Pathway 1.
Pathway 15
In another embodiment, the pathway is provided as Pathway 15. Pathway 15 starts with xylose. Xylose is converted to xylulose by xylose isomerase (XI, EC 5.3.1.5), and then to xylulose 5-phosphate by polyphosphate xylulokinase (PPXK, EC NA) by using polyphosphate. The product xylulose 5-phosphate can be converted to G6P through the non-oxidative pentose phosphate pathway. Then G6P is consumed to generate NADH or a biomimic thereof through Pathway 1.
Xylulokinase is an enzyme responsible for converting xylulose to xylulose-5-phophosphate with help of ATP. We discovered a wild-type T. maritima xylulokinase has a promiscuous activity by utilizing polyphosphate rather than ATP as a phosphate donor. As a result, Pathway 15 can work directly.
In some embodiments, the synthetic pathway is comprised of four functional modules: glucose 6-phosphate (G6P) generation from maltodextrin mediated by alpha-glucan phosphorylase and phosphoglucomutase (Equation 1); 2 NADH generated from G6P mediated by two NAD-dependent G6PDH and 6-phosphogluconate dehydrogenase (6PGDH) (Equation 2); NADH electro-oxidation through DI to VK3 that generates 2 electrons per NADH (Equation 3); and 5/6 moles of G6P regeneration from one mole of ribulose 5-phosphate via a hybrid pathway comprising enzymes in the pentose phosphate, glycolysis, and gluconeogenesis pathways (Equation 4). The overall anode reaction for the combination of Equations 1-4 approximately results in Equation 5. Clearly, each glucose unit from maltodextrin can generate 24 electrons on the anode via this de novo pathway (Equation 5).
(C6H10O5)n+Pi→G6P+(C6H10O5)n−1 [1]
G6P+H2O+2NAD+→ribulose-5-phosphate+CO2+2NADH+2H+ [2]
NADH+H+→2H++2e− [3]
6 ribulose-5-phosphate+H2O→5 G6P+phosphate [4]
C6H10O5+7H2O→24e−+6CO2+24H+ (anode compartment) [5]
The pathway utilizes two NAD-dependent G6PDH and 6PGDH to generate NADH differently from natural NADP-dependent enzymes in the pentose phosphate pathway used for anabolism. The above pathway does not require either ATP or CoA, which are very costly and unstable in EFCs. Moreover, phosphate ions can be recycled to maintain constant pH and ion concentrations. This cyclic pathway design is different from the linear pathways typically used in EFCs.
Enzymes
In some embodiments, enzymes are immobilized using a variety of methods, including gel entrapment, physical adsorption, chemical covalent linking, and immobilization with nanoparticics and nanotubes. These methods originated from biosensors that focus on achieving reproducible signals by immobilizing commercially available mesophilic enzymes to enhance their stability without concern for slow reaction rates.
However, enzymes immobilized on the surface of solid electrodes generally exhibit much lower activities (e.g., 1%) due to enzyme deactivation and poor fuel transfer from the bulk solutions to the immobilized enzymes. To achieve constant high-power EFCs, we considered an alternative strategy for mediating electron transfer in the EFCs without immobilizing the enzymes. Our strategy retains the enzymatic activity and facilitates mass transfer by immobilizing the electron mediator (that is, vitamin K3 (VK3)) on the surface of the electrode (
Two typical enzyme immobilization approaches for EFCs are polymer matrix entrapment in a quaternary tetrabutylammonium bromide (TBAB)-modified Nafion and covalent binding on carbon nanotubes (CNTs) (
The stability of enzymes can be addressed by the use of thermoenzymes. The thermoenzymes may be produced in E. coli and purified by three methods: heat precipitation, His-tag/nickel charged resin, and adsorption of cellulose-binding module tagged proteins on a cellulosic adsorbent (
The half-life time of the non-immobilized enzymes may be increased by adding bovine serum albumin and 0.1% Triton X-100. (
Electron Mediators
In some embodiments, an electron mediator is immobilized on the surface of the anode.
In a particular embodiment, the electron mediator is vitamin K3.
In some embodiments, an electron mediator is not immobilized on the surface of the anode. In other embodiments, an electron mediator is free in the anode compartment.
In a particular embodiment, the electron mediator is benzyl viologen.
Sugar batteries or sugar EFCs
In some embodiments, the EFC is a cuvette-based EFC (
In some embodiments, the biobatteries equipped with non-immobilized enzyme cascades might be refilled by the addition of the sugar solution because the sole gaseous product (CO2) can be easily released from the anode compartment and the non-immobilized enzymes are not washed out of the EFCs. In other embodiments, the EFCs are refilled by the addition of the substrate and enzyme mixture.
Alternative Uses:
Materials and Methods
Chemicals
All chemicals, including maltodextrin (dextrose equivalent of 4.0-7.0, i.e., a measured degree of polymerization of 19), vitamin K3 (VK3), nicotinamide adenine dinucleotide (NAD, including both the oxidized form (NAD+) and the reduced form (NADH)), poly-L-lysine (PLL, MW ˜70-150 kDa), dithiothreitol (DTT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were reagent grade or higher and purchased from Sigma-Aldrich (St. Louis, Mo., USA) or Fisher Scientific (Pittsburgh, Pa., USA), unless otherwise noted. Restriction enzymes, T4 ligase, and Phusion DNA polymerase were purchased from New England Biolabs (Ipswich, Mass., USA). Oligonucleotides were synthesized either by Integrated DNA Technologies (Coraville, Iowa, USA) or Fisher Scientific. The carbon paper (AvCarb MGL200) used in the anodes was purchased from Fuel Cell Earth (Stoneham, Mass., USA). Membrane electrode assemblies (MEAs) consisting of Nafion 212 membranes and a carbon cloth cathode modified with 0.5 mg cm−2 Pt were purchased from Fuel Cell Store (San Diego, Calif., USA). COOH-functionalized multi-wall carbon nanotubes (CNTs) with an outer diameter of <8 nm and a length of 10-30 μm were purchased from CheapTubes.com (Brattleboro, Va., USA). Regenerated amorphous cellulose used in enzyme purification was prepared from Avicel PH105 (FMC, Philadelphia, Pa., USA) through its dissolution and regeneration, as described elsewhere. Escherichia coli Top10 was used as a host cell for DNA manipulation and E. coli BL21 Star (DE3) (Invitrogen, Carlsbad, Calif., USA) was used as a host cell for recombinant protein expression. Luria-Bertani (LB) medium including either 100 mg L−1 ampicillin or 50 mg L−1 kanamycin was used for E. coli cell growth and recombinant protein expression.
Production and Purification of Recombinant Enzymes
The E. coli BL21 Star (DE3) strain harboring a protein expression plasmid was incubated in a 1-L Erlenmeyer flask with 250 mL of the LB medium containing either 100 mg L−1 ampicillin or 50 mg L−1 kanamycin. Cells were grown at 37° C. with rotary shaking at 250 rpm until the absorbance of the cell culture at 600 nm reached 0.6-0.8. Protein expression was induced by adding 100 μM of isopropyl-β-D-thiogalactopyranoside (IPTG) during an 18° C. overnight incubation. The cells were harvested by centrifugation at 4° C. and washed once with 20 mM HEPES (pH 7.5) containing 0.3 M NaCl. The cell pellets were resuspended in the same buffer and lysed by ultra-sonication (Fisher Scientific Sonic Dismembrator Model 500; 5-s pulse on and off, total 300 s at 50% amplitude). After centrifugation, the target proteins in the supernatants were purified.
Three approaches shown in (Table 2) were used to purify the various recombinant proteins. His-tagged proteins were purified by the Profinity IMAC Ni-Charged Resin (Bio-Rad, Hercules, Calif., USA). Fusion proteins containing a cellulose-binding-module (CBM) and self-cleavage intein were purified through high-affinity adsorption on a large surface-area regenerated amorphous cellulose. Heat precipitation at 80° C. for 20 min was used to purify RPI, Ru5PE, TIM, and ALD. The purity of the recombinant proteins was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, (
Measurement of Enzyme Activity
Clostridium thermocellum alpha-glucan phosphorylase (αGP) activity was assayed in 100 mM HEPES buffer (pH 7.5) containing 1 mM MgCl2, 5 mM DTT, 30 mM maltodextrin, and 10 mM sodium phosphate at 23° C. for 5 min. The reaction was stopped by adding HClO4 followed by neutralization with KOH. Glucose 1-phosphate (G1P) was measured using a glucose hexokinase/G6PDH assay kit (Pointe Scientific, Canton, Mich., USA) supplemented with phosphoglucomutase (PGM).
C. thermocellum phophoglucomutase (PGM) activity was measured in 100 mM HEPES buffer (pH 7.5) containing 5 mM MgCl2, 0.5 mM MnCl2, and 5 mM GIP at 23° C. for 5 min. The glucose 6-phosphate (G6P) product was determined using a hexokinase/G6PDH assay kit.
Geobacillus stearothermophilus glucose-6-phosphate dehydrogenase (G6PDH) activity was assayed in 100 mM HEPES buffer (pH 7.5) containing 100 mM NaCl, 2 mM G6P, 2 mM NAD+, 5 mM MgCl2, and 0.5 mM MnCl2 at 23° C. An increase in the absorbance due to the formation of NADH was measured at 340 nm.
Morella thermoacetica 6-phosphogluconate dehydrogenase (6PGDH) activity was measured in a 100 mM HEPES buffer (pH 7.5) containing 2 mM 6-phosphogluconate, 2 mM NAD+, 5 mM MgCl2, and 0.5 mM MnCl2 at 23° C. for 5 min.
Thermotoga maritima ribose-5-phosphate isomerase (RPI) activity was assayed using a modified Dische's cysteine-carbazole method.
T. maritima ribulose-5-phosphate epimerase (Ru5PE) activity was determined on a substrate of D-ribulose 5-phosphate as described previously.
Thermus thermophilus transketolase (TK) activity was measured in a 50 mM Tris/HCl (pH 7.5) buffer containing 0.8 mM D-xylulose 5-phosphate, 0.8 mM D-ribose 5-phosphate, 5 mM MgCl2, 0.5 mM thiamine pyrophosphate, 0.15 mM NADH, 60 U mL−1 of TIM and, 20 U mL−1 of glycerol 3-phosphate dehydrogenase. The reaction was started with the addition of TK at 23° C. The D-glyceraldehyde 3-phosphate product was quantified through the consumption of NADH measured at 340 nm for 5 min.
T. maritima transaldolase (TAL) activity was assayed as reported previously in Huang et al., A thermostable recombinant transaldolase with high activity over a broad pH range. Appl. Microbiol. Biotechnol. 93: 2403-2410 (2012).
T. thermophilus triosephosphate isomerase (TIM) activity was determined in 50 mM Tris/HCl (pH 7.5) containing 5 mM MgCl2, 0.5 mM MnCl2, 0.5 mg mL−1 BSA, 20 U mL−1 of glycerol 3-phosphate dehydrogenase, and 0.25 mM NADH.
T. thermophilus fructose 1,6-bisphosphate aldolase (ALD) was assayed in a 50 mM Tris/HCl Buffer (pH 7.5) at 23° C. with 1.9 mM fructose 1,6-biphosphate as a substrate. The glyceraldehyde 3-phosphate product was quantified with 0.15 mM NADH, 60 U mL−1 of TIM, and 20 U mL−1 of glycerol 3-phosphate dehydrogenase at 340 nm.
T. maritima fructose 1,6-bisphosphatase (FSP) activity was determined based on the release of phosphate.
C. thermocellum phosphoglucose isomerase (PGI) activity was assayed at 23° C. In 100 mM HEPES (pH 7.5) containing 10 mM MgCl2, 0.5 mM MnCl2, and 5 mM fructose 6-phosphate. After 3 minutes, the reaction was stopped with the addition of HClO4 and neutralized with KOH. The G6P product was analyzed at 37° C. with a hexokinase/G6PDH assay kit.
G. stearothermophilus diaphorase (DI) activity was assayed in 10 mM, phosphate buffered saline solution containing 0.16 mM NADH and 0.1 mM dichlorophenolindophenol (DCPIP) at 23° C. A decrease in the absorbance at 600 nm due to the consumption of DCPIP was measured using a spectrometer.
Activities of the G6PDH and DI immobilized on the carbon paper electrodes were assayed under the same conditions as for the free enzymes. The reactions were started by immersing the electrodes in the substrate solution at 23° C. After removing the electrodes from the reactions, the changes in the absorbance in the reaction solutions were measured as described for the G6PDH and DI assays.
Anode Preparation
Two enzyme immobilization methods were used to prepare the anodes equipped with the immobilized enzymes.
Method 1 was based on the entrapment of enzymes into a quaternary ammonium bromide salt modified Nafion. The casting solution mixture was prepared by adding 39 mg of tetrabutylammonium bromide (TBAB) with 1 mL of 5% Nafion 1100 EW suspension (ion Power, Inc., New Castle, Del., USA). After drying overnight, the mixture was washed with 3.5 mL of 18 MΩ deionized water and re-suspended in 1 mL of isopropanol. The enzyme solution mixture consisted of 1 unit of G6PDH, 40 units of DI, 1 mM NAD+, and 0.29 M VK3. The carbon paper anode was covered by a mixture of 100 μL of the casting solution and 100 μL of the enzyme solution and dried at room temperature.
Method 2 was based on covalent bond linkage between the enzymes and the carbon nanotubes (CNTs). A 10 μL volume of a 2% wt/v PLL solution was used to coat the carbon paper, followed by addition of 20 μL of 25 mM EDC. Meanwhile, 2.5% wt/v COOH-functionalized CNTs were suspended in a 50% ethanol solution and sonicated for 30 min. The carbon paper was then treated with 40 μL of the CNT-containing solution and dried at room temperature. Another 10 μL of 400 mM EDC and 10 μL of 100 mM NHS were then added, followed by the addition of 1 unit of G6PDH, 40 units of DI, 1 mM NAD+, and 10 μL of 0.29 M VK3 acetone solution.
Both types of anodes with immobilized enzymes were stored in 100 mM HEPES buffer containing 2 mM NAD+ and 100 mM NaNO3 at 4° C. before use.
For preparation of the non-immobilized enzyme anodes, 1 or 3 mg of CNTs were added to the surface of a 1 cm2 carbon paper (AvCarb MGL200) from Fuel Cell Earth using poly-L-lysine (PLL, MW ˜70-150 kDa) as described previously in Zhu et al., Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases, Biosens. Bioelectron. 36: 110-115 (2012). A 10 or 30 μL solution of 0.29 M vitamin K3 dissolved in acetone was deposited on the dry CNT-containing anode under a hood. After two hours of acetone evaporation, the water-insoluble vitamin K3 was deposited onto the anode through physical adsorption.
Electrochemical Characterization of EFCs
All electrochemical tests were performed using a 1000B Multi-Potentiostat (CH Instruments Inc., Austin, Tex., USA) interfaced to a PC. Experimental data pertaining to current and power outputs were normalized to a 1 cm2 of anode area because the reaction occurring at the anode was the rate-limiting step and the oxidation of protons mediated by Pt in MEAs was not rate-limiting. The measurements of open circuit potential and linear sweep voltammetry were performed at a scan rate of 1 mV s−1.
For the comparison of the power generation from the immobilized and non-immobilized enzyme EFCs (
When maltodextrin was used as a substrate (
The one-dehydrogenase EFC contained the first three enzymes (i.e., alpha-glucan phosphorylase, phosphoglucomutase, and glucose 6-phosphate dehydrogenase) plus DI. The two-dehydrogenase EFC contained the first four enzymes (Le., alpha-glucan phosphorylase, phosphoglucomutase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) plus DI. The EFC used for the complete oxidation of maltodextrin contained all thirteen enzymes. The enzyme loading conditions are shown in Table 2.
The complete oxidation of maltodextrin (0.1 mM) was measured (
F
MD−current
=C
total/(Δcglucose unit×V×24×F)
To further increase the power density of the EFCs, several factors were optimized (
The effect of CNTs loading on one 1 cm2 carbon paper as the anode was measured under the following conditions: 20 mM G6P, 1 U of G6PDH, 80 U of DI, 100 mM HEPES (pH 7.5), 10 mM MgCl2, 0.5 mM MnCl2, 8 mM NAD+, 4 mM sodium phosphate, 0.5 mM thiamine pyrophosphate, 1 electrode, 1 mg, 3 mg, or 5 mg CNTs per electrode and 23° C. The effect of the number of the stacked anodes made by 1 cm2 carbon paper deposited with 3 mg CNTs was measured under the following conditions: 20 mM G6P, I U of G6PDH, 80 U of DI, 100 mM HEPES (pH 7.5), 10 mM MgCl2, 0.5 mM MnCl2, 8 mM 4 mM sodium phosphate, 0.5 mM thiamine pyrophosphate, 1, 3, or 6 electrodes and 23° C. The effect of G6PDH loading from 1 to 30 U in the EFC containing a stack of 6 electrodes, each of which contained 3 mg CNTs was measured under the following conditions: 20 mM G6P, 1, 10, or 30 U of G6PDH, 80 U of DI, 100 mM HEPES (pH 7.5), 10 mM MgCl2, 0.5 mM MnCl2, 8 mM NAD+, 4 mM sodium phosphate, 0.5 mM thiamine pyrophosphate and 23° C. The effect of reaction temperature at 23, 50, or 65° C. In the EFC containing a stack of 6 electrodes, each of which contained 3 mg CNTs was measured under the following conditions: 20 mM G6P, 30 U of G6PDH, 80 U of DI, 100 mM HEPES (pH 7.5), 10 mM MgCl2, 0.5 mM MnCl2, 8 mM NAD+, 4 mM sodium phosphate, and 0.5 mM thiamine pyrophosphate. In
The effect of scanning rate was also measured in the buffer (
To demonstrate the capability of the “cuvette-like” EFC that can power up a digital clock or a LED light (
The sugar-refilling experiment for the non-immobilized-enzyme EFC (
The preliminary diagnostic experiment for the aged EFC (
To compare the stability of the immobilized and non-immobilized enzyme systems (
The best EFC condition was 100 mM HEPES buffer (pH 7.5), 10 mM MgCl2, 0.5 mM MnCl2, 4 mM NAD+, 0.5 mM thiamine pyrophosphate, 5 mM DTT, 15% (wt/v) maltodextrin, 40 mM sodium phosphate as substrates, and enzyme loading conditions of 30 units of #1-#4 enzymes, 10 units of #5-#12 enzymes, 80 units of DI, 50 mg L−1 kanamycin, 40 mg L−1 tetracycline, 40 mg L−1 cycloheximide, and 0.5 g L−1 sodium azide. For long-term non-disruptive operation, an external resistance of 150Ω was applied. The power density was measured for 60 hours at 23° C. (
Comparison of Non-Immobilized and Immobilized Enzymes
To compare the EFCs equipped with non-immobilized enzymes to the two EFCs equipped with immobilized enzymes, equivalent amounts of glucose 6-phosphate dehydrogenase (G6PDH) and diaphorase (DI) were used to test the polarization and the power outputs of the EFCs for glucose-6-phosphate fuel (
The mass transport region for the non-immobilized EFCs occurred at higher current densities compared to the covalent binding-based EFCs (
The EFC based on non-immobilized G6PDH exhibited the highest power density of 0.13 mW cm−2, three times higher than that of the covalent binding method. The EFC based on the TBAB-modified Nafion polymer entrapment method had the lowest maximum power density of 0.0013 mW cm−2, which was only 4% of the density for the covalent binding method. The G6PDH immobilized by Nafion polymer entrapment and the covalent binding retained 0.2% and 6° A of its non-immobilized activity, respectively. The DI immobilized by Nafion polymer entrapment and the covalent binding retained 0.4% and 7.5% of its non-immobilized activity, respectively (Table 3). These data for enzyme activity clearly suggest that a dramatic activity loss occurs due to enzyme immobilization. The power density data validate the feasibility of using non-immobilized enzyme(s) to achieve high-power output in EFCs.
Complete Oxidation of Maltodextrin
To release the maximum electron potential from each glucose unit (i.e., 24 per glucose), we designed a non-natural enzymatic pathway containing 13 enzymes (
(C6H10O5)n+Pi→G6P+(C6H10O5)n−1 [1]
G6P+H2O+2NAD+→ribulose 5-phosphate+CO2+2NADH+2H+ [2]
NADH+H+→2H++2e− [3]
6 ribulose 5-phosphate+H2O→5 G6P+phosphate [4]
C6H10O5+7H2O→24e−+6CO2+24H+ (anode compartment) [5]
The pathway utilizes two NAD-dependent G6PDH and 6PGDH to generate NADH differently from natural NADP-dependent enzymes in the pentose phosphate pathway. The above pathway does not require either ATP or CoA, which are very costly and unstable in EFCs. Moreover, phosphate ions can be recycled to maintain constant pH and ion concentrations. This cyclic pathway design is different from the linear pathways typically used in EFCs.
The power densities from maltodextrin fuel (i.e., 2 mM glucose units) were compared for three EFCs that used one dehydrogenase (i.e., G6PDH), two dehydrogenases (i.e., G6PDH and 6PGDH), or the entire pathway (
To quantitatively validate the complete oxidation of the glucose units of maltodextrin, we measured the Faraday efficiency from NADH to electrons through the diaphorase and vitamin K3 in the air-breathing EFC (
F
NADH−current
=ΔC/(Δc×V×2×F)
where FNADH−current is Faraday efficiency of electro-enzymatic oxidation of NADH, ΔC is the slope of total charge increase (C), Δc is the slope of NADH concentration decrease (M), V is reaction volume (L), 2 respresents 2 electrons generated per NADH consumed, F is Faraday constant.
Under oxygen-free conditions for the anode compartment, the Faraday efficiency of the EFC was 97.6±3.0% (
In a 15-mL EFC containing 13 enzymes and a low concentration of maltodextrin at room temperature (
It was noted that the negative control (i.e., the same EFC without the substrate) did not generate significant current outputs (
High-Energy-Density High-Power EFCs
Power density is another important consideration in EFCs. To increase the power density, we optimized a number of factors, including the EFC configuration, the enzyme loading, and the experimental conditions under which the non-immobilized G6PDH acts on the G6P.
The optimal CNT loading was 3 mg per cm2 of carbon paper (
The EFC comprised of the 13 non-immobilized enzymes based on 15% (wt/v) maltodextrin generated the maximum power density of 0.4 mW cm−2 at a scanning rate of 1 mV s−1 at room temperature. The EFC generated a nearly constant power output of approximately 0.32 mW cm−2 for 60 hours in a closed system (
The complete oxidation of the glucose units of the 15% maltodextrin solution means that the energy storage density of this sugar-powered EFC can be as high as 596 Ah kg−1, which is more than one order of magnitude higher than the energy storage densities for lithium-ion batteries and primary batteries (
10
7
Although the voltages of the EFCs (e.g., 0.5 V) are much lower than the voltages of lithium-ion batteries (3.6 V), the energy density of the 15% sugar-powered EFC can reach up to 298 Wh kg−1, several times that of common rechargeable batteries (e.g., Pd-acid, NiMH, and lithium-ion batteries) and higher than that of common primary batteries (e.g., zinc-carbon, alkaline, and Li—MnO2 batteries) (
In addition to the one order of magnitude improvement in the energy density of the sugar biobatteries via this synthetic pathway, relative to the system with one redox enzyme (
These sugar biobatteries represent a new type of rechargeable battery. One of the greatest advantages of fuel cells compared to closed primary and secondary batteries is that they are open systems that use high-energy density fuels (e.g., H2, methanol, glucose, and maltodextrin) that can be fed into the fuel cell device continuously (e.g., proton exchange membrane fuel cells) or sporadically (for example, direct methanol fuel cells and sugar batteries). When the weight ratio of the fuel to the fuel cell system is large enough (i.e., 5-10) or if the fuel cell is refilled a number of times, the energy density of the entire system including the fuel, a fuel tank, and a fuel cell system can be close to the theoretical energy density of the fuel that is used. Clearly, the use of water-free chemicals as fuels is more attractive in terms of energy storage density (
Maltodextrin is a better EFC fuel than alcohols (e.g., methanol) or glucose. Maltodextrin is slowly-utilized via the synthetic pathway to generate a nearly constant power output (
One of the most important issues for sugar biobatteries is extending their lifetime. This involves improving the stability of enzymes, cofactors, and mediators. A preliminary diagnostic experiment was conducted to study the decreased performance of the non-immobilized EFCs (
Thus, a synthetic ATP- and CoA-free catabolic pathway comprised of 13 enzymes in an air-breathing EFC is constructed to completely oxidize the glucose units of maltodextrin, yielding nearly 24 electrons per glucose: We found that the EFC based on non-immobilized enzymes exhibited a maximum power output far higher than those of the immobilized enzymes. These sugar-powered biobatteries feature high energy storage densities and high safety. Thus, these batteries represent next-generation micro-power sources that could be especially useful for portable electronics.
Engineered GsG6PDH
Wild-type Geobacillus stearothermophilus G6PDH (GsG6PDH) prefers NAD to NADP but does not work on biomimics (
The above disclosure provides particular embodiments of the present invention. It is intended that the above disclosure be considered as exemplary in nature and that variations that do not depart from the essence of the invention are intended to be within the scope of the invention. The particular embodiments disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein.
It is evident that the particular illustrative embodiments disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the present invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the above disclosure in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that these features may be used singularly or in any combination based on the requirements and specifications of a given application or design.
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This application is a divisional application of U.S. application Ser. No. 14/773,510 filed on Sep. 8, 2015, which is a national stage application of PCT/US2014/041103 filed on Jun. 5, 2014, which claims the benefit of U.S. Provisional Application No. 61/831,346 filed on Jun. 5, 2013, respectively, the contents of which are incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61831346 | Jun 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14773510 | Sep 2015 | US |
| Child | 16135796 | US |
