AND/OR ATP, AND/OR ELECTRICITY GENERATION

The present invention relates to a modular system for the biosynthesis of hydrogen, and/or ATP and/or electricity generation .

As is well known, the production of hydrogen is commonly carried out by hydrocarbons and fossil fuels through a chemical process. Currently, about 97% of the hydrogen produced is obtained from fossil fuels, while only 3% is obtained through water electrolysis. This process, if carried out using fossil fuels, leads to the emission of high amounts of C02, which end up increasing the greenhouse effect. Alternatively, alternative energy sources can be used as input energy to the water electrolysis process. Hydrogen can be obtained by many methods, but the cheapest means are represented by hydrocarbon extraction. Hydrogen can be generated from natural gas with an efficiency of approximately 80%, or from other hydrocarbons with different degrees of efficiency.

Hydrogen can also be extracted from water through organic production in algae bioreactors, either by electrolysis or by thermolysis. However, these methods are less efficient in terms of the amount produced than the chemical process derived from hydrocarbons.

A known system for the biosynthesis of hydrogen is described in US2018/3452 63A1 which describes a hybrid nanoparticle semiconductor for the photocatalytic development of hydrogen, in different quantities with respect to environmental and light conditions.

Another known system is described in EP2131437A1. Even if known system have some useful application, their main limit and disadvantage is related to the direct enzyme immobilization on the electrodes or the insertion of a molecule inside a phospholipid bilayer.

In fact, enzymes and proton pumps, such as bacteriorhodopsin, have active sites and critical domains (portions of the molecules) whose role is central in their functioning. The direct "immobilization" bond of the molecules on the electrodes can lead to the inactivation of the same that come into contact and establish a bond with the "electrode" substrate by binding to it either with the active site or with a critical domain. The procedure is more critical for proton pumps (including bacteriorhodopsin) and a further limit is related to the direction of the molecule in the bilayer. In fact, the insertion of bacteriorhodopsin in a double layer or its immobilization on a support without taking into account the directionality, there is a risk of obtaining no or extremely low useful work. In fact, if a hypothetical number of bacteriorhodopsins are inserted into a system, and half of these are arranged in one direction and half in the opposite direction, the useful work carried out by the system will be zero since the work carried out by the first portion of bacteriorhodopsin will be thwarted by the activity performed by the other, that means that no electrochemical gradient is generated.

The main limitation of immobilization on the support of enzymes and / or proton pumps lies in the impossibility of controlling the binding site between the molecule and the support, much less directing the functioning of the molecule, everything translates into these main disadvantages :

- High level of inactivity of enzymes and / or proton pumps introduced into the system deriving from the link between the support and the molecule which, being random and not controllable, can occur at the level of active sites and / or critical domains; - Inability to direct the activity of the molecules, which is crucial for sodium potassium pumps, with the risk of having a system in which the yield is unpredictable, low (compared to the number of enzymes and / or proton pumps introduced) and even nothing.

The object of the present invention is to provide a modular system for the biosynthesis of hydrogen, and/or ATP and/or electricity generation that is less expensive than known systems, efficient and flexible.

According to the present invention, a modular system for the biosynthesis of hydrogen, and/or ATP, and/or electricity generation is made, as defined in claim 1.

For a better understanding of the present invention, four preferred embodiments are now described, by way of non-limiting example only, with reference to the accompanying drawings, in which: figure 1 shows a schematic view of a first embodiment of a system for the biosynthesis of hydrogen, and/or electricity generation, according to the invention; figure 2 shows a schematic view of a second embodiment of a system for ATP biosynthesis, according to the invention; figure 3 shows a schematic view of a third embodiment of a system for the biosynthesis of hydrogen, ATP and/or electricity generation, according to the invention; - figure 4 shows a schematic view of a fourth embodiment of a system for the biosynthesis of hydrogen, ATP and/or electricity generation, according to the invention .

With reference to such figure 1, there is shown a modular system for the biosynthesis of hydrogen, and/or

ATP, and/or electricity generation according to a first embodiment of the invention.

The modular system 100, 200, 300, 400 for hydrogen biosynthesis, and/or ATP and/or electricity generation according to the invention, comprises and provides for the use of:

- at least one phospholipid membrane 101, 201, 301,

401 consisting of a phospholipid bilayer;

A plurality of monoclonal antibodies 102, 202, 302, 402 configured to each bind a first predefined molecule 103a, 203a, 303a, 403a or a second predefined molecule 103b, 203b, 303b, 403b; an inert support 104, 204, 304, 404 on which the plurality of monoclonal antibodies 102, 202, 302, 402 are immobilized; a source of light radiation 105, 205, 305, 405; wherein a first molecule 103a, 203a, 303a, 403a bound to at least a part of the plurality of monoclonal antibodies 102, 202, 302, 402 at the trans-membrane level is the molecule of bacteriorhodopsin.

The bond between the monoclonal antibodies, immobilized on the inert support, and the molecules allows a plurality of molecules to be inserted into the phospholipid bilayer in such a way that they are oriented uniformly, advantageously allowing greater control over the reactions and maintaining a standard of efficiency and effectiveness of the products obtained from industrial processes.

This uniform orientation of the molecules is particularly important in cases where said molecules are ion pumps. According to one aspect of the invention, the second molecule 103b, 203b, 303b, 403b bound to at least a part of the plurality of monoclonal antibodies 102, 202,

302, 402 at the trans-membrane level is an halorodopsin molecule. According to one aspect of the invention, the second molecule (103b, 203b, 303b, 403b) bound to at least another part of the plurality of monoclonal antibodies (102, 202,

302, 402) at the trans-membrane level is an ATP-synthase molecule.

According to one aspect of the invention, the modular system 100, 200, 300, 400 comprises: a plurality of phospholipid membranes 101, 201, 301, 401 consisting of a phospholipid bilayer stabilized laterally by means of supports; within which a plurality of monoclonal antibodies will be able to bind each predefined molecule inserted at the trans-membrane level, since the monoclonal antibodies are immobilized on one of the supports.

According to one aspect of the invention, the inert support 104, 204, 304, 404 is microperforated and is made of a material chosen from the group consisting of: Cellulose Nitrate, PVC (Poly Vinyl Chloride),

Polycarbonate .

According to one aspect of the invention, the modular system 100, is configured for the production of electricity and/or hydrogen, and comprises at the trans-membrane level a molecule of bacteriorhodopsin and a molecule of halorhodopsin which act as light-dependent ion pumps, specific for chloride and hydrogen ions. The modular system (100) comprises two chambers separated from each other by a septum, a first chamber in which there is an accumulation of H+ ions and a second chamber in which there is a high concentration of Cl- ions in aqueous solution, and two electrodes 110 respectively connected to the first chamber and the second chamber.

According to one aspect of the invention, the modular system 200 is configured for the production of ATP and comprises at the trans-membrane level a first molecule (203a) of bacteriorhodopsin and a second molecule (203b) of

ATP-synthase .

According to one aspect of the invention, the modular system (300), is configured for the production of ATP and/or electricity and/or hydrogen, said modular system (300) comprising at the trans-membrane level first molecules (303a) of: bacteriorhodopsin, and second molecules (303b) of ATP-synthase, and third molecules of halorhodopsin . The modular system 300 comprises two chambers separated from each other by a septum and a pair of electrodes 310. respectively connected to the first chamber and the second chamber.

The phospholipid membranes included in the system 100, 200, 300, 400 are laterally stabilized by means of supports in a material such as to directly or indirectly immobilize a plurality of molecules on the membrane.

According to one aspect of the invention, the inert support is preferably made of a material chosen from the group consisting of: Cellulose nitrate, PVC (Poly Vinyl

Chloride), Polycarbonate. The material of the inert support may in any case be any material that directly or indirectly immobilizes a plurality of molecules on the membrane.

According to one aspect of the invention, the inert support is microperforated, for example by the use of lasers.

The use of monoclonal antibodies, in the embodiments of the modular system 100, 200, 300, 400 allows, unlike polyclonal antibodies, to recognize all an epitope of a given molecule, obtaining a predictable binding behavior. The fact that monoclonal antibodies bind to a single epitope makes it possible to determine a single binding site and to direct the molecule in the membrane insertion phase. Polyclonal antibodies, on the other hand, are a set of antibodies produced by different B cell clones that each recognize a different epitope of a given different molecule.

Advantageously, in the system 100,200,300 the monoclonal antibodies specifically and precisely bind a predetermined epitope thus allowing the molecule/ enzyme to carry out its activity, in fact if the antibody binds the enzyme in a critical portion such as in a portion defined as an active site, it would make it non-functional. The embodiment, shown in Figure 1, and referred to as system 100 comprises:

- at least one phospholipid membrane 101;

- at least one substrate, or inert support 104, on which a plurality of monoclonal antibodies 102 are bound, in turn bound to first and second predetermined molecules 103a, 103b;

- a septum of electrical and chemical separation of the system 100 into two chambers;

- a light source or light radiation 105; - a plurality of first molecules of bacteriorhodopsin

103a and a plurality of second molecules of halorhodopsin 103b bound to monoclonal antibodies;

- a pair of electrodes 110 each connected to one of the chambers separated by the septum. According to one aspect of the invention, the embodiment called the system 100 originates a proton gradient.

The system 100 predicts that the first molecules and second molecules to be bound by monoclonal antibodies to the phospholipid membrane 101— are bacteriorhodopsin and halorhodopsin . The bacteriorhodopsin and halorhodopsin molecules are inserted into the phospholipid bilayer. Advantageously, the molecule of bacteriorhodopsin is photoabsorbent and proton pump thus originating a proton gradient adapted to be used for the production of electricity and/or the synthesis of hydrogen.

Halorhodopsin, on the other hand, also photoabsorbent will advantageously accumulate Cl - ions. The molecule of halorhodopsin is a light-dependent ion pump, specific for

Cl - ions. In this way, the system 100 has two chambers, a first chamber in which there is an accumulation of H+ ions (on which the membrane that has molecules of bacteriorhodopsin acts) and a second chamber in which there is a high concentration of Cl- ions (on which the membrane that has molecules of halorhodopsin acts) . Thus, an electrolytic dissociation is carried out, that is, a separation of ions with opposite charge in aqueous solution. Cations are the positive ions of an electrolyte solution, while anions are the negative ions. This separation between ions with opposite charge generates electricity that can be used directly or used to produce hydrogen starting from the H+ ions present in the solution

(water). The second and third embodiments of the system 200, 300 provide for bond between monoclonal antibodies and ATP synthase molecules (the ATP synthase) for the production of ATP. If prepared, the ATP synthase (placed at the trans membrane level) can use all or part of the originated proton gradient, respectively in the case of the second embodiment of the system 200 and the third embodiment of the system 300, to catalyze the synthesis of adenosine triphosphate (ATP) using adenosine diphosphate (ADP) and inorganic phosphate (P i) - in this configuration, this molecule can also be placed at the trans-membrane level, so that it crosses the membrane facing both sides.

The second embodiment of the system 200, is shown in Figure 2 and comprises:

- A phospholipid membrane;

- At least one support or substrate to which monoclonal antibodies are bound;

- At least one light source; - A plurality of molecules of bacteriorhodopsin, bound to monoclonal antibodies;

- A plurality of ATP-synthase molecules bound to monoclonal antibodies. The second embodiment of the system 200 allows the production of ATP.

The molecule of bacteriorhodopsin is a light- dependent ion pump, specific for H+ ions. - ATP synthase will use the work carried out by bacteriorhodopsin (H+ ion shift) to synthesize ATP starting from ADP and P i.

The third embodiment of the system 300 comprises:

Phospholipidic membranes, consisting of a phospholipid bilayer, stabilized on the sides by supports of various types of material that directly or indirectly advantageously immobilize molecules, and that present at the trans-membrane level (so that it crosses the membrane facing both sides) the molecule of halorhodopsin, ATP synthase and bacteriorhodopsin.

The molecule of halorhodopsin is a light-dependent ion pump, specific for Cl - ions. In this way, the system 100 has two chambers, a first chamber in which there is an accumulation of H+ ions (on which the membrane that has molecules of bacteriorhodopsin ) acts) and a second chamber in which there is a high concentration of Cl- ions (on which the membrane that has molecules of halorhodopsin acts). As in system 100, an electrolytic dissociation is carried out, that is, a separation of ions with opposite charge in aqueous solution. Cations are the positive ions of an electrolyte solution, while anions are the negative ions. This separation between ions with opposite charge generates electricity that can be used directly or used to produce hydrogen starting from the H+ ions present in the solution (water).

The system 300 differs, therefore, from the system 100 for the presence at the level of one of the two membranes in addition to the bacteriorhodopsin also the ATP synthase (in particular at the level of the membrane that faces the compartment in which there is an accumulation of H+ ions). In the opposite compartment in which Cl- ions accumulate, the membrane presents exclusively halorhodopsin. With this configuration, ATP, hydrogen and/or electricity can be obtained.

A fourth embodiment of the modular system 400 presents ferredoxin free to move (in the modular compartment), nitrogenase and/or hydrogenase immobilized on the support. It is specified that in this case that in the absence of molecular nitrogen, nitrogenases can catalyze the production of molecular hydrogen through the reduction of protons, using ferredoxin as an electron donor. In the fourth embodiment of the modular system 400, in addition to the aforementioned molecules

(nitrogenase/hydrogenase, ferredoxin, bacteriorhodopsin, ADP, Pi, ATP synthase) the following molecules must be used:

- Hexokinase

- phosphoglucose isomerase

- phosphofructokinase

- Aldolase

- triosephosphate isomerase

- glyceraldehyde-3-phosphate dehydrogenase

- phosphoglycerate kinase

- phosphoglycerate mutase

- Enolase

- Pyruvate kinase (PK)

Ferredoxin oxidoreductase (PFOR) / formate lyase

(PFL)

- ferredoxin,

- bacteriorhodopsin,

- ADP adenosine diphosphate,

- Pi (inorganic phosphate),

- ATP synthase

- Nitrogenase and/or hydrogenase Thus configured, the modular system 400 according to the fourth embodiment provides for a series of reactions (in which the molecules just listed are involved) through which glucose (6-carbon monosaccharide) is oxidized to 2 pyruvate molecules with a net production of two ATP molecules and 2 NADH molecules (starting from NAD molecules) . The pyruvate is converted to acetyl-CoA.

Ferredoxin oxidoreductase (PFOR) from pyruvate converts ferredoxin to its reduced form (Fd). Reduced Fd is an electron donor for hydrogenases. Hydrogenases and/or nitrogenase use reduced Fd, NADH, proton gradient, ATP will synthesize^ (ATP will partly be derived from reactions catalyzed by glycolysis enzymes partly resulting from the work of ATP synthase - ATP is employed only by nitrogenase) . The series of reactions takes place within bioreactors or containers capable of providing an adequate environment in which the biochemical reaction carried out by the selected molecules and active from the biochemical point of view is carried out. In particular, the reaction involving [Fe-Fe] hydrogenases will provide an environment free of 02 considering the inactivation to which it goes against after contact with oxygen. In these containers/ bioreactors the temperature and pH will ensure the best yield of biochemical reactions. Monoclonal antibodies will be used which will advantageously bind enzymes at the level of non- critical epitopes ensuring their enzymatic functionality (impossible to guarantee this aspect using polygonal antibodies as they would bind enzymes in different epitopes, inactivating some of them). The separation of the enzymes in different bioreactors will allow, if there are decreases in efficiency for a physiological degradation of the enzymes themselves, the replacement not of the whole pool of enzymes but only of the enzymes catalyzing the intermediate reaction in which the decrease in performance has been recorded - this will allow a saving to economic - in terms of time since we identify the problem immediately and immediately remedy it by replacing the support with bound antibodies and enzymes. By using monoclonal advances, we have an immediate count of the number of enzymes that catalyze each intermediate reaction, managing to better control the global biochemical reaction. In the bioreactor where the reaction is catalyzed by Glyceraldehyde-3- phosphate Dehydrogenase there are NAD molecules that will be converted to NADH. Acetyl-CoA is the main waste product.

The system according to embodiments 100,200,300 according to the invention thus advantageously allows to produce: - ATP(Figure 2);

- Electricity (Figure 1);

- Electricity and Hydrogen (figure 1);

- ATP, Electricity and Hydrogen (figure 3); The production of hydrogen is therefore configured in a particular scenario, when the demand for electricity is zero (in this case, hydrogen is constituted as the possibility of "storing the unsolicited energy" - in this way, an Energy Harvesting process is configured). It should be noted that the production of hydrogen has no environmental impact as it involves reactions/processes catalyzed by enzymes/molecules

Figure 1 shows a modular system 100 suitable for the production of electricity or electricity and hydrogen. Figure 2 shows a modular system 200 adapted to the production of ATP.

Figure 3 shows a modular system 300 suitable for the production of ATP, electricity and/or hydrogen.

A modular system 400 according to a fourth embodiment is shown in Figure 4.

Thus, the system for the biosynthesis of hydrogen, ATP and/or electricity generation according to the invention makes it possible to establish in advance the number and type of enzymes or molecules and therefore to standardize the production of energy having a total control of the reaction stoichiometry.

According to the invention, the use of monoclonal antibodies allows to bind all the enzymes and / or proton pumps at the level of a single and single point selectable a priori for example a TAG (in molecular biology this term indicates a protein label - a short sequence of amino acids). Also, the use of monoclonal antibodies offers the possibility of directing the entire activity of the enzymes and / or proton pumps and obtaining a predictable, constant, optimized yield of the system.

The use of monoclonal antibodies according to the invention, also allows to have the functionality of all the enzymes and / or proton pumps, the bond between monoclonal antibodies immobilized on the support and the molecules can be established precisely excluding critical domains and active sites (virtually all enzymes and / or proton pumps that enter the system will be functional and will carry out their activity in unison in order to optimize the determination of any electrochemical potential difference).

A further advantage according to the invention, and related to the use of monoclonal antibodies in the event that a planar phospholipid bilayer is used is the possibility of having greater physical stability of the membrane. In fact, the monoclonal antibodies will be covalently immobilized to the support enzymes and / or proton pumps will be firmly linked by the antibody and the phospholipid protein interactions will stabilize the planar phospholipid bilayer.

Another advantage related to the use of monoclonal antibodies according to the invention, is to finely control the amount of enzymes and / or proton pumps active in the system, this possibility allows a fine control of the reactions catalyzed by these molecules.

The fine control of the reactions in the system according to the invention, derives from a series of facts:

- The number of antibodies immobilized on the support can be precisely established in the activation phase of the support itself (the number of COOH or NH groups present on the support can be precisely established);

- Each antibody will bind a precise number (1-2) of molecules; - The molecules bound by monoclonal antibodies will all be active and directed in functionality ensuring a precise and definable catalyzing of the reactions within the system. Another advantage of the modular system for the biosynthesis of hydrogen, and /or ATP and/or electricity generation according to the invention is to be advantageously industrializable by being able to determine and reproduce the series the amount of energy that can be produced based on the number and type of molecules bound and/or inserted in the phospholipid bilayer.

Finally, it is clear that modifications and variations can be made to the modular system for the biosynthesis of hydrogen, and/or ATP and/or electricity generation according to the invention described and illustrated herein without departing from the protective scope of the present invention, as defined in the attached claims.