Method for doping graphene with nitrogen and sulfur by reducing graphene oxide with microorganisms, nitrogen- and sulfur-doped graphene obtained thereby, and uses thereof

Abstract

The present invention relates to a method for producing graphene dual-doped with nitrogen and sulfur atoms through the microbial reduction of graphene oxide. Additionally, the present invention relates to the graphene dual-doped with nitrogen and sulfur atoms obtained by this method, and to the use of such doped graphene, for example for producing electronic components or water purification equipment. In particular, the method has the further advantages of being eco-sustainable and economical, and providing products with significantly improved performance compared to known products.

Description

Technical Field

[0001] The present invention relates to a method for producing graphene doped with nitrogen atoms and sulfur atoms through the reduction of graphene oxide by microorganisms. In addition, the present invention relates to graphene doped with nitrogen and sulfur obtained by this method, and to the use of such doped graphene for manufacturing, for example, electronic components or water purification devices. In particular, the method has the further advantage of providing an eco-sustainable and economical product with significantly improved performance compared to known products.

Background Art

[0002] Graphene is known to be a single atomic layer of carbon atoms. Graphene has the mechanical strength of diamond and the flexibility of plastic. Its discovery, actually attributed to two physicists, Andrej Gejm and Konstantin Novoselov, has opened new paths in nanotechnology. Specifically, graphene has been experimentally demonstrated to be extremely stable and to provide very excellent mechanical, thermal, optical and chemical properties, as well as improved electrical properties, as shown through many studies conducted in the last decade.

[0003] As suggested by the name (ending with "ene"), the carbon atoms are sp 2Graphene is hybridized in its morphology, and therefore arranged to form hexagons with 120° angles, resulting in a pattern with high crystalline properties. Thus, as a two-dimensional crystalline material, graphene offers unique and adaptable physical properties, namely its controllability, enabling the manufacture of a wide variety of devices with different functions, including sensors, transistors, memories, and filtering systems, which can be used in many fields such as electronics, biology, biotechnology, and energy. The promising properties of graphene in these fields necessitate the definition of large-scale production methods in anticipation of increasing demand. Furthermore, due to its easily modifiable structure, its highly interesting physical-chemical properties, and its natural abundance, this material is associated with "malleable clay."

[0004] As is well known in the latest technology, in order to enable the use of this material for different devices in many fields, it is necessary to subject graphene to doping methods suitable for improving its properties relative to unmodified graphene. In fact, doping graphene with heteroatoms is an effective method for improving its properties, preferably its electrical properties. Therefore, most researchers in this field are currently focused on modifying the properties of graphene through doping. In detail, the insertion of different atoms such as nitrogen (N) and sulfur (S) into graphene oxide is sp 2 This causes disruption of the carbon atom pattern, affecting the chemical and physical properties of graphene and allowing the properties to be adapted according to the degree and type of doping. Theoretical studies have shown that doping graphene with nitrogen (N) and sulfur (S) favorably modifies its properties, preferably its electrochemical properties, producing so-called "N,S dual-doped graphene" (N,S-DDG), which can be further modified, for example, in catalysts for oxygen reduction reactions, as will be demonstrated. Currently, the most effective method typically used for doping graphene is based on the deposition of evaporated chemical substances on the graphene surface (chemical vapor deposition, CVD technique) in a reactor heated to 800°C. Specifically, to produce N,S dual-doped graphene oxide (N,S-DDG), graphene oxide (GO) is used as the starting material and doped with chemicals such as methane (CH4), ammonia (NH3), and sulfuric acid (H2SO4). Without being bound by any theory, at high temperatures (800°C), some covalent bonds are broken, for example, nitrogen atoms in ammonia, sulfur atoms in sulfuric acid, and hydrogen atoms associated with each of the doping substances. Then, under reactor conditions, the resulting unstable molecules, such as amide ions (NH2) - ) and sulfate ions (SO4 2- ) interacts with the covalent bonds of graphene, which are also weakened by high temperatures, modifying its chemical composition and transforming it into N,S dual-doped graphene (N,S-DDG).

[0005] The methods described above utilize polluting chemicals for the environment and toxic substances for humans, namely methane (CH4), ammonia (NH3), and sulfuric acid (H2SO4). The temperatures of the methods are extremely high, requiring the consideration of a suitable plant adapted to maintain such conditions. Furthermore, the flammability of methane in the presence of oxygen (potentially released by graphene oxide or accidentally introduced into the plant) poses a very high security risk to operators. The final purification process for N,S dual-doped graphene (N,S-DDG) involves removing the toxic residue of the material used in the previous step, namely ammonium ions (NH3). + ) and sulfate ions (SO4 2-This includes the removal of ). Typically, this purification step for N,S dual-doped graphene (N,S-DDG) involves dissolving the residue by rinsing with hydrochloric acid (HCl). Thus, the method involves further contamination and toxic substances, namely hydrochloric acid (HCl), which, in addition to the enumeration of substances listed above necessary to carry out the method, may also affect the properties of graphene, such as robustness. As an alternative to the previously described procedure, the use of microorganisms has been proposed to allow for a reduction in critical experimental conditions in certain phases compared to those described above.

[0006] The scientific publication “One-Pot Microbial Method to Synthesize Dual-Doped Graphene and Its Use as High-Performance Electrocatalyst, Guo et al., 16 December 2013” ​​(https: / / www.nature.com / articles / srep03499), incorporated herein by reference, describes a method for reducing graphene oxide (GO) by microbial respiration of sulfate-reducing bacteria (SRBs). In detail, this document describes the procedure for preparing N,S dual-doped graphene (N,S-DDG) by reduction of graphene oxide (GO) via microbial respiration of sulfate-reducing bacteria (SRB) at a temperature of 37°C to obtain the desired doping with N and S atoms. During anaerobic respiration, sulfate-reducing bacteria (SRBs) use sulfate (SO4) as their final electron acceptor. 2- It is well known that ) is primarily used. Since graphene oxide (GO) clearly exhibits electron acceptor properties, it is possible to achieve reduction of graphene oxide (GO) during the SRB respiration process.

[0007] It is noteworthy that the SRB bacteria used in this procedure originated from moist, oily soil in Shengli, China, and were grown in API-RP38 medium consisting of 4.0 mL of sodium lactate, 1.0 g of yeast extract, 0.2 g of magnesium sulfate (MgSO4·7H2O), 0.1 g of vitamin C (Vc), 0.01 g of potassium diphosphate (K2HPO4), and 10 g of sodium chloride (NaCl) (per liter of ultrapure water). The pH was adjusted to 7.0-7.2 with 1 M sodium hydroxide (NaOH). The final solution was sterilized in an autoclave at 121°C for 20 minutes, cooled to room temperature, and 0.2 g of ultraviolet-sterilized FeSO4·(NH4)2SO4·6H2O was added. 100 mL of GO solution (0.1 mg / mL) -1 The reduction of graphene oxide (GO) was achieved by mixing the mixture with 10 mL of SRB medium and 30 mL of fresh medium. The mixture was incubated in an incubator under anaerobic conditions at 37°C for several days. The resulting black dispersion was centrifuged (14,000 rpm), washed with an aqueous HCl solution to remove organic matter and cellular debris, and washed several times with ultrapure water. Finally, the resulting solid was dried under vacuum at 80°C. When the XPS spectrum for nitrogen was detected using high-resolution X-ray photoelectron spectroscopy, the percentages of doped nitrogen and sulfur atoms were approximately 6.11% and 1.1%, respectively.

[0008] The aforementioned procedure is certainly advantageous because, thanks to the use of sulfate-reducing bacteria (SRBs), the doping requirements are significantly reduced. In particular, the temperature is much lower, even outside of the final washing phase, and the reagents are completely environmentally friendly. Nevertheless, the performance of N,S-DDG obtained by the aforementioned microbiological methods has not been satisfactorily demonstrated. In fact, experiments have observed that the more similar the distribution of N and S on graphene, the more effective the doped graphene appears to be, i.e., it appears to be conductive with respect to electric current. In other words, without being bound by theory, it is thought that in order to obtain a substrate with superior conductivity, the percentage ratio of N to S must be as close as possible to a 1:1 value. Furthermore, SRBs are excessively sensitive to high doses of GO. This is thought to significantly limit their use in large-scale production, because the insertion of excessive doses of GO as a substitute for what is needed only for industrial methods is thought to cause the death of "doped" bacteriological media.

[0009] Finally, despite the detailed physicochemical properties of the microbially doped graphene oxide, the above publication does not describe N,S-DDG synthesis clearly enough for those skilled in the art. More specifically, it lacks any information regarding the isolation and acquisition of SRBs, as well as the conditions for their maintenance / culturing in the laboratory. Therefore, this publication may not provide the information necessary to reproduce the described process on any scale. It is speculative that the authors isolated the SRB bacteria from the Deltaproteobacteria class, the genus of sulfate-reducing bacteria used, without disclosing any species, and that this could therefore be highly variable, possibly belonging to the genera Desulfovibrio, Desulfobacter, Desulfococcus, or Desulfonema. As a result, there is a need to define a method for preparing N,S dual-doped graphene oxide (N,S-DDG) that overcomes the challenges described above. Specifically, the manufacturing method, preferably on a large scale, must employ conditions that involve a low risk of contamination, low hazard to operators, such as low temperatures, and minimize the use of contaminating and toxic products, while simultaneously ensuring that the final product possesses high performance in terms of conductivity. [Overview of the project]

[0010] Therefore, the technical challenge inherent in the present invention is to provide a method for producing nitrogen and sulfur-doped graphene (N,S-DDG) that does not involve the risk of large-scale use or release of environmentally polluting and human-toxic chemicals such as methane (CH4), ammonia (NH3), and sulfuric acid (H2SO4), and that involves a substantially low reaction temperature.

[0011] The above challenges are addressed by a method for producing dual-doped graphene (N,S-DDG) with nitrogen and sulfur atoms, which includes a reduction phase of graphene oxide (GO) by specially selected microbial means. Therefore, the first object of the present invention is a method for producing dual-doped graphene (N,S-DDG) with nitrogen and sulfur atoms, comprising the step of contacting a culture medium of selected microorganisms with a mixture of graphene oxide (GO). The second objective is a method for producing dual-doped graphene (N,S-DDG) with nitrogen and sulfur atoms, wherein the culture medium also provides the elements necessary for doping reduced graphene oxide (GO), such as nitrogen (N) and sulfur (S). A third objective is a method for producing nitrogen and sulfur-doped graphene (N,S-DDG) that does not require interruption when occurring in an isolated system, and does not involve any non-operational phase, i.e., an "in-loop" method or a continuous recirculation method. A further objective is to provide a method that enables the release of unsettling active molecules with a high insertion rate into graphene oxide, resulting in a product with extremely low toxicity and extremely low contamination grade.

[0012] A further objective is to produce graphene dual-doped with nitrogen and sulfur atoms using special microorganisms. A further objective is to obtain graphene (N,S-DDG) dual-doped with nitrogen and sulfur atoms using this method, which is therefore low-cost and poses minimal risks to the environment and human health. A further objective is a simplified manufacturing plant specifically designed in a simple way to reduce the cost of producing dual-doped graphene (N,S-DDG) with nitrogen and sulfur atoms.

[0013] The ultimate goal is the use of doped graphene according to the present invention for the manufacture of electronic and electrochemical components (e.g., fuel cells), analytical systems, purification systems, and eco-sustainable macromaterials as nanocomponents for pharmaceuticals, telephones, aerospace, and robotics, as well as automotive machinery components, aircraft components, aerospace components, and robotics components. The problems and objectives described above, as well as other problems and objectives that will appear more clearly later herein, are solved and achieved by the methods, plants, specific doped microorganisms, graphene and their uses as defined in the separate claims attached herein.

[0014] Further characteristics and advantages of the method for producing dual-doped graphene (N,S-DDG) with nitrogen and sulfur atoms according to the present invention will become apparent in the following description of several preferred embodiments, which are given as non-limiting examples, and with reference to the following figures. [Brief explanation of the drawing]

[0015] [Figure 1]This diagram shows the phylogenetic tree position of six new Haloarchaea species belonging to the class Halobacteria, the Stenosarchaea branch, and the phylum Euryarchaeota. [Figure 2] This figure shows a general scheme of an industrial method for producing nitrogen and sulfur-doped graphene according to the present invention. [Figure 3] This figure shows a graph comparing the results of Raman spectroscopy analysis of graphene oxide (GO) and graphene oxide reduced in N,S-DDG, using different haloalkae grown on a formate (electron donor) when thiosulfate (DDGO-T) and polysulfide (DDGO-S) were supplied as electron acceptors in place of elemental sulfur. [Figure 4] This figure shows two comparative graphs of results between cyclic voltammetry of graphene oxide (control) obtained using different haloalkae species grown on different carbon sources, and graphene oxide samples doped with N and S, where elemental sulfur is donated as the final electron acceptor and grown on formate (Graph A), and where thiosulfate and polysulfide are donated as electron acceptors (Graph B). [Figure 5] The figure shows three graphs illustrating the XPS spectra of three doped graphene samples against undoped graphene oxide, each using three different species of microorganisms (HSR) grown on three different substrates (PYR, AC, FORM): a) 1s hybridization of C, b) 1s hybridization of N, and c) 2p hybridization of S. [Figure 6] The figures represent two graphs, respectively, showing the analysis of the chemical forms of nitrogen in doped graphene samples after microbial treatment of the AARC-S strain cultured with thiosulfate (Graph A) and polysulfide (Graph B). [Figure 7]Figure showing the XPS spectrum of the chemical form of sulfur in a sample of graphene modified with the microorganism (AARC-S) of the present invention grown with thiosulfate.

BRIEF DESCRIPTION OF THE INVENTION

[0016] The present invention essentially refers to a biochemical method of doping graphene oxide (GO) in view of the industrial applications of nanomaterials. The so-called "doping" is a method made possible exclusively by the physical proximity between graphene oxide, specific microorganisms, and their culture media. In the following description, the terms "doping" and related words mean a method of changing the chemical composition of a molecule by inserting atoms that were initially absent in its original structure. This method modifies the physicochemical properties of the material to which it is applied. In a specific case of the present invention, "N,S-DDG" means graphene doped with nitrogen (N) atoms and sulfur (S) atoms, that is, literally, N[nitrogen], S[sulfur]-D[dual]D[doped]G[graphene].

[0017] It should be noted that no interruption is required to perform the doping, except for the final purification step of the product. In addition, generally, the respiratory function of the selected microorganism allows the release of unstable molecules having a very high insertion rate. Then, the proximity between the unstable molecules and GO enables the insertion of sulfur (S) atoms and nitrogen (N) atoms into the two-dimensional structure of graphene oxide (C n H i O j ) in a surprisingly effective way. As already explained, the physicochemical properties of such doped and reduced graphene oxide have recently been recognized for their versatility in a variety of applications, from the replacement of silicon semiconductors to the operation of innovative water decontamination systems. Therefore, there is a great deal of interest in a simple, effective, and economical method.

[0018] Therefore, according to the present invention, a method for producing graphene (N,S-DDG) doped with nitrogen and sulfur atoms comprises the following steps: - Strictly anaerobic, sulfite-reducing, and 200 g·L -1 A step to prepare microorganisms of the class Halobacteria that can survive at temperatures between 20°C and 50°C under salinity conditions exceeding a certain level and a pH between 7.0 and 10.0, - A step of culturing the microorganism in a medium containing, as an electron donor, hydrogen (H2), acetate (C2H4O2), formate (CH2O2), glycerol (C3H8O5), glucose (C6H12O6), sucrose (C12H22O11) and other similar sugars, lactate (C3H6O3), short-chain fatty acids (C4-C9) and / or pyruvate (C3H4O3) in an amount of up to 100 mmol, and as an electron acceptor, any one of the sulfur forms oxidized more than S2-, including elemental sulfur (S8°), polysulfide (-S-S6-S-), thiosulfate (S2O32-), dimethyl sulfoxide (CH3)2SO, and tetrathionate (S4O62-), in an amount of up to 50 mmol; - A step of contacting a graphene oxide (GO) solution with the culture medium containing the microorganism for a sufficient time to obtain doping with nitrogen and sulfur, - A step of washing the graphene to remove both the organic phase and the nitrogen and sulfur molecules that have not reacted with the oxidized graphene.

[0019] The process of preparing the microorganisms is particularly important because prior art information regarding the use of graphene to carry out the biological doping of microorganisms is insufficient and unclear to provide data on the true efficacy of such methods. As described above, the microorganisms that have been experimentally used to dope graphene to date belong to the Eubacteria kingdom, the "classic" bacterial stem, the sulfate-reducing bacteria (SRB) group, which are chemosynthetic bacterial oxidizers of sulfur, sulfur in sulfites and sulfuric acids, and thiosulfates in sulfates.

[0020] Even when classification systems are continuously restructured based on the discovery of new microbial species, it is unanimously agreed that in any given case, Eubacteria are fundamentally different from Archaea, to which the class Halobacteria (or Haloarchaea) belongs.

[0021] Preferably, the halobacteria of the present invention are selected from the genera Halalkaliarchaeum, Halanaeroarchaeum, Halodesulfurarchaeum, Halarchaeoglobus, Natranaeroarchaeum, and Natronolimnobius (Figure 1), and more preferably, among the genus Halalkaliarchaeum, the species Halalkaliarchaeum desulfuricum, among the genus Halanaeroarchaeum, the species Halanaeroarchaeum sulfurireducens, and among the genus Halodesulfurarchaeum, the species Halodesulfurarchaeum formicicum The electron donors are selected from species such as Halarchaeoglobus desulfuricus (from the Halarchaeoglobus genus), Natranaeroarchaeum sulfidigenum (from the Natranaeroarchaeum genus), and Natronolimnobius sulfurireducens (from the Natronolimnobius genus). 12 O6), sucrose (C 12 H 22 O 11 ) and / or simple organic substances such as pirubate (C3H4O3) are used, and these physiologically exclusive organic substances contain the element sulfur (S8°), polysulfide ( - S-S6-S - ), thiosulfate (S2O3 2- ), dimethyl sulfoxide (CH3)2SO, tetrathionate (S4O6 2-It reduces both of these and produces H2S as the final product of their sulfur-dependent respiration.

[0022] In particular, the species *Halalcarialcaeum desulfuricum* and *Natronorimnovius sulfurreducens* mentioned above are described and characterized with the codes AArc-S and AArc1, respectively, in "Sulfur respiration in a group of facultatively anaerobic natronoarchaea ubiquitous in hypersaline soda lakes," Frontiers in Microbiology, Volume 9, Article 2359, 2 October 2018, Sorokin et al. The species *Halodesulfuracaeum formicicum* is described and characterized with the codes HTSR1 and HSR6, respectively, in "Discovery of anaerobic lithoheterotrophic haloarchaea, ubiquitous in hypersaline habitats," The ISME Journal, volume 11, pages 1245-1260 (2017), Sorokin et al. The species *Haranaeroarchaeum sulfireducens* is described and characterized under the code HSR12 in "Elemental sulfur and acetate can support life of a novel strictly anaerobic haloarchaeon," The ISME Journal, volume 10, pages 240-252 (2016), Sorokin et al. The species *Haralcaeoglobus desulfricus* is also described and characterized under the code HSR12 and is held in the UNIQEM (Vinogratsky Institute of Microbiology Culture Collection) collection center of the Russian Academy of Sciences in Moscow under the identification number U1000. TIt is deposited there. The species Natranaeroarcaeum sulfidigenum has been described and characterized with the code AArc-S, and is in the UNIQEM (Vinogratsky Institute of Microbiology Culture Collection) Collection Center of the Russian Academy of Sciences in Moscow, with identification number U999. T It is being held in deposit.

[0023] In addition, the Halodesulfuracaeum formicicum species HTSR1, mentioned in the paper, was deposited at the UNIQEM Collection Center of the Russian Academy of Sciences in Moscow. Therefore, its genome is available in the GenBank database under access number CP016070. The Natronorimnovius sulfureleducens species AArc1 was also deposited at the UNIQEM Collection Center of the Russian Academy of Sciences in Moscow under identification number U932. T Furthermore, access number JCM30663 for the Japan Microbial Collection Center. T It is deposited at the same Russian center (UNIQEM) with number U983. T And, the Japanese center JMC has number 30662 T It is deposited at: Haralkaliarcaeum desulfuricum species AArc-S1, number U999 at UNIQEM. T And also, the JCM Center has number 30664 T They were deposited at [location]. As stated in the aforementioned publications, all these haloalcaea possess some form of sulfur respiration based on an unknown sulfur. They are, in any case, eccentrically distributed in some high-salinity habitats. Using the growing media described above, alcaea strains belonging to the genera *Haranaeroalcaeum*, *Halodesulfuracaeum*, *Hararcaeoglobus*, *Natranaeroalcaeum*, and *Natronorimnovius* were isolated from brine and high-salinity sediment samples collected on the island of Stromboli (Aeolian Archipelago, Mediterranean Sea, Italy). These strains were analyzed and exhibited identical chemical / morphological / genetic characteristics to the strains described in the publications mentioned above, and were therefore indicated by the same codes.

[0024] The activity of all the microorganisms listed above has been observed to end in the final phase of the respiratory process with the production of up to 10-15 mmol of H2S. Furthermore, these microorganisms also possess a favorable resistance to the toxicity of GO. In fact, it has been shown that the presence of GO negatively affects the growth and survival conditions of SRBs, but this is not the case for the halobacteria listed above. In particular, to promote GO doping, as will be explained below, 1.0-2.0 mg / mL -1 The amount used is 10 to 20 times greater than that used by Guo et al. (2013). Therefore, using these specific and selected microorganisms makes it possible to perform far more doping than the biological methods described earlier.

[0025] The culture medium also, in one respect, provides the necessary sources of nitrogen and sulfur for enabling microbial growth and simultaneously doping with graphene oxide (GO). Preferably, the culture medium is 240 g / L -1 NaCl, 3g·L -1 K2HPO4, 0.5g·L -1 It contains NH4Cl, 1-5 mM MgCl2×6H2O, sterilized, and then 20-50 mg·L -1 Yeast extract, 1 ml / L -1 Acidic trace metal solution, 1 mL·L -1 A mixture of Se / W alkaline solution and vitamins is added. The final pH is controlled to 7. More preferably, with Halodesulfuracaeum formicicum and Haranaeroalcaeum sulfireducens, the medium is also 10 g / L -1This also includes HEPES. In addition, 1 mL of acidic solution of trace metals preferably contains the following substances (in 1 liter of culture medium): HCl 0.01N (i.e., 10 mmol), 0.6 g of CoCl2 × 6H2O, 30 mg of CuCl2, 0.3 g of FeCl2 × 4H2O, 1.14 g of H3BO3, 4 g of MnCl2 × 4H2O, 0.5 g of Na2MoO4 × 2H2O, 0.3 g of NiCl2 × 6H2O, and finally 0.42 g of ZnCl2.

[0026] Preferably, the vitamin mixture contains 1 mg of vitamin B per liter of distilled water. 12 It contains 20 mg of biotin, 20 mg of folic acid, 50 mg of nicotinic acid, 50 mg of p-aminobenzoic acid, 50 mg of calcium pantothenate, 100 mg of pyridoxine × HCl, 50 mg of riboflavin, 50 mg of thiamine, and 50 mg of thionic acid. The Se / W alkaline solution is preferably made from the following substances (per liter of 0.01 N [i.e., 10 mmol] NaOH): 2 mg of Na2SeO3 and 4 mg of Na2WO4 × 1.5 H2O. The pH of the medium can also be adjusted to a specific requirement, for example, 7.0, by adding 1 M KOH.

[0027] According to embodiments of the present invention, the culture medium is 240 g·L -1 NaCl, 5g·L -1 KCl, 2g·L -1 K2HPO4, 0.5g·L -1 First medium containing NH4Cl, 190 g·L -1 Na2CO3, 30g·L -1 NaHCO3, 16g·L -1 NaCl, 5.0g·L -1 KCl, 8 mM NH4Cl, 1.0 g·L -1 The second medium consists of a mixture of two media, containing K2HPO4. Both media are supplemented with 1 mM MgCl2 × 6H2O. As before, after sterilization, 20-50 mg of L-1 yeast extract and 1 ml of L - 1. The above-mentioned acid trace metal solution, 1 mL / L -1The above-mentioned Se / W alkaline solution and the above-mentioned vitamin mixture are added. The final pH is adjusted to 7. More preferably, along with Halodesulfuracaeum formicicum and Haranaeroalcaeum sulfireducens, the above medium is also 10 g / L -1 This also includes HEPES.

[0028] In a further embodiment, the medium used to grow Halalkaliarcaeum desulfrycum is preferably obtained by mixing a first medium and a second medium in a 1:1 ratio to obtain a final pH of 9.6, while the medium for Natronolimnovius sulfireducens is formed by mixing a first medium and a second medium in a 3:1 ratio to obtain a final pH of 9.3. Generally, the microorganisms of the present invention are maintained in their culture medium under static conditions during growth, i.e., without vigorous agitation.

[0029] The step of contacting graphene oxide (GO) with the cell culture medium is preferably performed in a quantity of 2 mg / mL. -1 This is carried out by directly adding graphene oxide (in the solid phase as a powder) to the cell growth medium with or without agitation at a concentration of less than or equal to This phase is carried out in an isolated doping chamber or container in which the empty space, i.e., the space not filled with cell culture medium containing graphene oxide, is saturated with an inert gas such as nitrogen or argon. At the end of the doping phase, which involves contacting microorganisms with graphene oxide, the washing phase preferably includes separating organic matter from the doped graphene oxide, for example, by centrifugation and / or filtration. More preferably, this phase separates the doped graphene by centrifugation at 2,000 to 6,000 × g for 2 to 10 minutes, followed by the graphene being dissolved in an isotonic solution (240 g / L). -1The process includes washing with NaCl, followed by two consecutive washing steps with tap water or distilled water, and then filtering through Whatman qualitative filter paper grade 1 having a porosity between 5 and 20 μm to retain the doped graphene. After the filtration phase, a further washing step may be applied, which involves rinsing the material retained on the filter with, for example, Milli-Q® water. Rinsing may be repeated two or more times, preferably under vigorous agitation in a container or washing chamber. Finally, the treated material is dried in a conventional oven at 40-80°C for 2-6 hours. Conveniently, neither organic solvents nor acidic substances are required in any of the above-mentioned processing or cleaning steps for N,S-DDG products.

[0030] A second object of the present invention is the use of strictly anaerobic, sulfur-reducing halobacteria for reduction and, at the same time, doping graphene oxide with sulfur and nitrogen. Such microorganisms are 200 g·L -1 They can survive under extremely saline conditions and a pH between 6.5 and 10.0 at temperatures between 20°C and 50°C. Preferably, the microorganisms are those described above.

[0031] According to a third object of the present invention, as shown in Figure 2, the method described above may be carried out in a plant for the production of nitrogen and sulfur dual-doped graphene, the plant comprising a first storage and maintenance / growth container 1 for the graphene oxide-reducing microorganisms described above, at least a second container (graphene oxide doping chamber) 2 for mixing the microorganisms with graphene oxide (GO), the second container being hydrodynamically connected to the first container, a control means 3 for the first container, a pH control and adjustment means 4 connected to the first container, a temperature control and adjustment means 5 for the second container, a separation / washing device 6, 7 for doped graphene oxide, and a drying means 8 for doped and washed graphene.

[0032] Preferably, the first container 1 is equipped with a stirring means 9 (not shown in Figure 2) for the microbial culture medium, which consists of a paddle agitator driven in rotation by a suitable motor that has an adaptable speed and is controllable by a prior art device. Such a container is sold by Eppendorf under the trade names New Brunswick® BioFlo Fermenters & CelliGen® Bioreactors. The first container adjustment means 3 preferably includes a thermometer capable of detecting the temperature inside the container and sending a signal to a conventional control unit, which detects and processes this signal to control the circulation of the heating fluid on the outer wall of the container to a set temperature for maintaining the microorganisms. Instead of a heating fluid, an electrical element such as a coil can be used. In either case, the heating device is entirely conventional and is used, for example, in the Eppendorf products mentioned above. The pH control and adjustment means 4 comprises a completely conventional sensor or pH meter connected to a control unit, which receives a representative signal of the pH inside the container 1 and sends the signal to an optional agitation pump (not shown) for the release of an acid (e.g., HCl) or base (e.g., KOH) substance to maintain the pH under desired conditions for the growth of microorganisms.

[0033] The means 5 for temperature control and adjustment in the second container may also be the same as the adjustment means described with reference to the first container 3. The second container may also be equipped with the same stirring device 10 as described above. Separation / washing means 6, 7 for removing the organic phase of doped graphene comprises a centrifuge and / or Whatman qualitative filter paper grade 1 having a porosity between 5 and 20 μm. A bench centrifuge is proposed, e.g., an Eppendorf 5804R centrifuge. Preferably, the separation / washing of the medium is a vacuum filter with a funnel equipped with a filter and placed over the mouth of a flask, the flask being connected to a vacuum pump. Such systems are sold, for example, by Membrane Solution LLC under the trade names BIO-PURE® Vacuum Filters, SIGMA-ALDRICH®.

[0034] Further separation / washing means may include, for example, a glass solvent system, i.e., a hard glass compound filtration system designed to separate the main body (microorganisms, precipitates, and similar particles) from the liquid suspension. Therefore, these means can be identified either as a system for simple separation, such as a system for centrifugation, or as a washing system, which also includes separation through filtration. The drying means 8 preferably comprises a static oven operated under vacuum, for example, one sold by ZZKD Instrument Equipment under the trade name DZF-6010 Vacuum Drying Oven.

[0035] It will be noted that a programmable supply pump 11, such as a peristaltic pump, is installed between the first container 1 and the second container (doping chamber) 2 to supply at least a suitable amount to the second container 2 or doping chamber, necessary to carry out the desired doping according to the selected desired parameters. These adjustments are any example for those skilled in the art, once the conditions of the doping process described above are known. Furthermore, a typical plant will be equipped with a fully prior art hydraulic coupling and associated valves (shown without reference numerals in Figure 2), which is to ensure proper operation for the reuse of the culture medium separated from the doped graphene and other possible contaminants for emergency or safe release, or for emptying for periodic cleaning of the liquid contained in the tank.

[0036] Once graphene oxide is doped in the second container, the second container is preferably separated from recirculation in the system and opened to collect the culture medium, microorganisms, and mixture of NS dual-doped graphene. After separation, the container is repositioned under the required conditions and GO may be supplied again (as shown from the reference GO in Figure 2) for further doping processes. If necessary, it may then be reconnected to circulation. These operations may be carried out with a fluid pressure circuit such as that shown in Figure 2, where conduits 20, 60, 70, 80 and their respective valves 21, 61 enable the circulation.

[0037] The matching of doping conditions is controlled by a suitable computer, where the program receives signals from conventional sensors, probes, and thermometers that detect all operating conditions (temperature, pressure, chemical-physical values, e.g., salinity and pH) and runs to send command signals to ensure the correct control of the doping process in the best possible way. According to a further object of the present invention, the nitrogen and sulfur dual-doped graphene oxide obtainable by the above method is characterized by a nitrogen content between 1% and 9%, preferably between 1% and 5%, and a sulfur content between 0.3% and 15%, preferably between 1% and 15%, more preferably between 1% and 10%, relative to the total percentage of atoms inserted in the graphene oxide crystal. These values ​​were obtained as follows. The chemical analysis employed is a destructive technique for measuring the carbon, sulfur, nitrogen, and hydrogen (CHNS) composition in the sample. The analysis is based on the complete combustion of the sample at approximately 1000°C in an oxygen-rich atmosphere (according to the method described in Analytical Methods Committee (2006) Evaluation of analytical instrumentation. Part XIX. CHNS elemental analyzers. Accreditation and Quality Assurance 11(11), 569-576. Doi:10.1007 / s00769-006-0185-x), with the recovery of gases (CO2, H2O, N2, and SO2) produced during combustion, and assigning the original composition as elemental percentages. The instrument used for elemental analysis was a LECO CHNS-932 (model no: 601-800-500), and approximately 2 mg of material was used for each measurement.

[0038] In the case of sulfur, it should be noted that the chemical form in which it is used for doping affects the percentage of molecules incorporated. Therefore, the percentages of nitrogen and sulfur in N,S-DDG are highly variable and depend on the species used, the chemical form of the precursor used for doping (in the case of sulfur), and the growth conditions (in the case of nitrogen). Experimental tests conducted show that the use of elemental sulfur significantly increases the percentage of its content in graphene oxide.

[0039] Therefore, a particular advantage of the method according to the present invention (other than cost, ecological impact, and practicality) is that the insertion ratio of N atoms to S atoms can be adjusted as needed by diversifying the conditions specified above. In the present invention, deviations from the optimal S:N ratio (1:1) preferred for sulfur do not adversely affect the catalytic efficacy of the product, presumably because they do not exclusively exclude the sulfur components integrated into the new material. In fact, the addition of doped graphene during the washing phase with an organic solvent removes surface sulfur deposits and significantly reduces their percentage. The results obtained according to the method of the present invention demonstrate that biologically doped N,S-DDG possesses excellent oxygen reduction catalytic (ORR) properties (see the example below).

[0040] Specific X-ray photoelectron spectroscopy (XPS) analyses were also performed to characterize two samples (AARC-S in the presence of thiosulfates and polysulfides) in relation to their chemical form and elemental bonding. The techniques used allowed for elemental, structural, and quantitative analysis of the sample surface (to a depth of a certain nm, typically 2-4 nm). The analysis was performed on solid samples under a vacuum pressure of approximately 10⁻⁸ Pa. The samples were subjected to X-ray photons (aluminum or, instead, magnesium K). α The line, in this case, is aluminum K α X-rays were used. X-ray photons excite elements present in the sample, and the result can be the direct emission of electrons from a specific energy level (photoionization). The analysis consists of filtering the energy to detect these photoelectrons. The dynamic energy of the photoelectrons is given by the equation: E 動態 (Photoelectron)=E0(X-ray)-E 結合 (photoelectron) This is a function of the energy of photon X according to the formula. Aluminum X-ray source is K α12= 1486.7 eV. Elemental identification (qualitative analysis) is performed by measuring the binding energy of photoelectrons. The analysis system filters the electrons according to their dynamic energy, and the resulting spectrum is presented on a binding energy scale (inverse scale). Once the photons are emitted, the element is in an excited state. Possible deenergized states correspond to the emission of Auger electrons, which bring the element to three different electron levels. The dynamic energy of Auger electrons is independent of the energy of the incident X-rays. While X-rays penetrate to a significant depth (1 μm) in the sample, photoelectrons cannot be extracted from extremely thin layers, some on the order of a few nanometers thick. XPS technology is both qualitative and quantitative because its sensitivity is on the order of 0.1% atoms. However, the main advantage lies in the potential to obtain information about the chemical environment of the elements. The precise location of the photoelectron peaks in energy allows for the determination of the nature of covalent bonds between the analyzed element and its neighbors. In the case of carbon-oxygen bonding, for example, the electronegativity of oxygen induces a partial transfer of electrons from carbon to oxygen. In this way, carbon protons meet in an electron-deficient environment, and the bond energy of these electrons increases.

[0041] In particular, the above analysis was performed using a monochromatic X-ray source (aluminum K α (Line), dual anode X-ray source (aluminum and magnesium K α The analysis was performed using a PHI Versaprobe 500 emission spectrometer equipped with an electron source (for pure Auger analysis with a lateral resolution of approximately 200 nm), a low-energy ion source (XPS or Auger lateral spectroscopy), and a sample cooling system from the insertion chamber to the analysis chamber. Using this instrument, it is possible to focus the X-ray source on the sample with a spot diameter ranging from 10 μm to 1 millimeter. Specifically, the analysis on the sample according to the present invention was performed with an X-ray tube power of 50 W for a circular spot diameter of 200 μm. Information was collected and the average across the entire surface was calculated.

[0042] Regarding the spectrophotometer, photoelectrons are collected at an emergency angle of 45°. The settings vary for each spectrum and window. The conditions are silver 3D 5 / 2 Determined by measuring the full width at half maximum (FWHM) of the level (purchase in the pure silver standard): Wraith: FWHM = 2.3eV Windows: FWHM=0.8eV The operation was performed using the MultiPak logic program. For quantification, a sensitivity factor method was used, and the area was measured as the peak formed by windows after subtracting the continuous background using the Shirley method. The results are shown in Graph A in Figure 6 and Table 1 below, referring to graphene samples doped with the AARC-S strain grown with thiosulfate, while Graph B in Figure 6 and Table 2 below refer to samples doped with the AARC-S strain grown with polysulfide.

[0043] [Table 1]

[0044] [Table 2] As shown in the data in the table above, the procedure according to the present invention led to doping with very reasonable values ​​of nitrogen and sulfur. Furthermore, the graph in Figure 6 shows that the doping occurs deeply, and therefore the graph shows that they are not simple deposition. Moreover, comparing the chemical form of sulfur before and after doping, it can be seen that N,S-DDG is not only doped with sulfur but also cleaned from surface imperfections (before this method, only the measured chemical form of sulfur corresponds to sulfate, which actually originates from the sulfuric acid residue used during the chemical process of separating graphene oxide; after this method, sulfur is clearly in the form of sulfate, and is mainly hybridized in the 2p form, i.e., inserted into carbon hexagons, and therefore completely integrated into the graphene oxide crystal).

[0045] In particular, referring to the graph in Figure 6, the inventors have obtained more precise information from the bond energy value corresponding to the peak of approximately 400.1 eV, indicating that the bond from nitrogen to the graphene structure is mainly pyrolysis nitrogen. In fact, compared to known techniques, nitrogen doping has a value of over 90% (peak area), and is almost exclusively pyrolysis among all forms of nitrogen that can be created by doping (pyrimidine, pyrolysis, and graphite). In contrast, known techniques show a percentage of approximately 40% pyrolysis nitrogen. Similarly, looking at the graph in Figure 7, we can see how the curve showing a peak of approximately 162.9 eV indicates the presence of sulfur in the thiophenol form at a percentage of over 40%. Doping in this form of sulfur does not appear in any known art.

[0046] Consequently, the above analysis clearly shows that this doping method makes it possible to obtain nitrogen and sulfur-doped graphene oxide characterized by having a percentage of over 90% pyrolysis nitrogen and / or over 40% thiophenol sulfur. Therefore, a further object of the present invention is the use of the previously described doped graphene for the manufacture of eco-sustainable macromaterials such as electronic and electrochemical components (e.g., fuel cells), analytical systems, purification systems, nanomaterials used in pharmaceuticals, telephones, aerospace, and robotics, automotive machinery components, and aerospace. The following are some embodiments of the present invention, provided as non-exclusive examples.

[0047] (Example 1) Reduction of graphene oxide to N,S-DDG using cell culture medium containing Natronolimnovius sulfireducens (AArc1 strain isolated from Stromboli, Italy).

[0048] A 1L "Schott" bottle was filled with 900mL of mineral medium containing a 3:1 mixture of the first and second media (final pH approximately 9.6), supplemented with 50 mmol of polysulfide and 50 mmol of formate. 100mL of cell suspension (10 7 cells mL -1 ) was added as the inoculum. The doping process was initiated by subsequently adding 1.5 g of graphene oxide powder. The headspace of the bottle was then washed five times with nitrogen and once with argon, and carefully sealed. The culture medium was kept in static mode (no shaking) at 40°C using a thermometer. The bottle was inverted daily to mix the settled GO / N,S-DDG. The GO doping period was set to one month. Polysulfides (electron donors in the respiration of the microorganism) are soluble under highly alkaline culture conditions. This also applies to sulfide ions, CO2 molecules (final respiration products), and formate molecules (electron donors in the respiration chain of the microorganism). Therefore, apart from the formed biomass, there are no insoluble products in the culture medium and dopant medium. After the above 1 month, centrifugation (4,000 × g, 5 min) was performed to separate N,S-DDG from the biomass, followed by the extraction of 240 g / L in a 50 mL isotonic solution. -1The precipitated N,S-DDG was washed twice with NaCl and then subjected to final filtration through a Whatman Grade 1 paper filter with a porosity of 8 μm. The resulting material was rinsed through a BIO-PURE® Vacuum filters module consisting of a vacuum pump, filtration lamp, and filter flask. A hard glass filtration system designed to separate particles from a liquid suspension was used. Washing was performed two / three times with Milli-Q® water, and the filtrate was then dried in a vacuum furnace, Type DZF-6010 Vacuum Drying Oven, at 60°C for 4 hours. It should be noted that all steps of the N,S-DDG purification process did not involve the use of any organic solvents or acid products.

[0049] The N,S-DDG obtained from the above procedure exhibited the following characteristics when analyzed by Raman spectroscopy. The information obtained from Raman scattering analysis is shown graphically as a DDG-S diagram (Raman spectrum) for N,S-DDG doped according to the present invention in Figure 3 (the first one in HTSR1 in the presence of thiosulfate is the DDGO-T curve, and the second one in HTSR1 in the presence of polysulfide is the DDGO-S curve), where the horizontal axis is cm. -1 This reports the Raman shift corresponding to the jump energy between the fundamental vibration levels v=0 and v=1, represented by (wave number:

[0050] [ka] This is reminiscent of the direct proportional relationship between energy and the inverse of the wavelength of electromagnetic radiation E=hv=hc·1 / λ). The Raman intensity, which is proportional to the number of Stokes photons collected by the instrument's detector, is reported on the vertical axis. Therefore, the previously processed N,S-DDG shows the spectrum reported in Figure 3, obtained with a Renishaw Raman microscope. Monochromatic light is a green laser (532 nm) in the visible spectrum with a power of 5 mW. 1.5 mg of sample is required per measurement. Spectroscopic measurements were performed at 1% power, with each measurement taken at 100-3200 cm⁻¹.-1 The procedure was performed within the spectral range, and each sample was accumulated over 10 cycles. Figure 3 shows the significant difference between the spectra of the treated and untreated samples. The characteristics of the two peaks of graphene (A and B, approximately 1350 cm⁻¹, respectively) were estimated from similar studies. -1 and 1600cm -1 The ) are shown on each curve. The A band of the spectrum is related to hybridization in the structure, and more precisely, to sp in the structure. 2 This relates to the level of interference typically caused by hybridization. The B band relates to the "interlayer" interactions typical of multilayer graphene structures. The lower frequency range (150-550 cm⁻¹) -1 The Raman band at 2500 cm⁻¹ provides further evidence of the difference between the treated sample and the GO control sample (GO curve in Figure 3). They show a lower carbon concentration in the treated sample, thus reinforcing the inference that a higher percentage of heterologous atoms were inserted. A weaker Raman 2A (2500 cm⁻¹) band provides further evidence of the difference between the treated sample and the GO control sample (GO curve in Figure 3). -1 The bandwidth also reflects the layered nature of graphene. The larger curve 2A, the closer the analyzed material is to the uncontaminated graphene structure. Therefore, again, the fact that it has a low signal demonstrates the superior quality of the doped graphene obtained. Finally, the ratio of the intensity of peak A to the intensity of peak B (I A / I B ) is a parameter commonly used to characterize the level of defects in the crystalline structure of carbonaceous materials. Allowing for the evaluation of defective morphology of the analyzed sheet, this parameter provides information about the effect of the tested (doping) method of the present invention on the structure of graphene. A / I B The higher the ratio, the better the doping performance. It has a variable value of 0.9-1.1 for GO (control sample) and 1.2-2.0 for the treated sample (depending on the microorganism used). I of graphene oxide doped with the microorganism Natrolimnovius sulfridukensis (AArc1) A / I B The ratio is approximately 1.48 (Figure 3).

[0051] (Example 2) Reduction of graphene oxide in N,S-DDG using cell medium containing Halodesulfural alkaem formicicum (HTSR1 strain isolated from Stromboli, Italy) 240g·L in a 1L Schott bottle -1 NaCl, 3g·L -1 K2HPO4, 0.5g·L -1 H4Cl, 1-5 mM MgCl2×6H2O, 1 ml·L -1 A 900 mL mineral medium was packed (per liter of culture medium) containing the following substances: HCl 0.01N (i.e., 10 mmol), 0.6 g CoCl2 × 6H2O, 30 mg CuCl2, 0.3 g FeCl2 × 4H2O, 1.14 g H3BO3, 4 g MnCl2 × 4H2O, 0.5 g Na2MoO4 × 2H2O, 0.3 g NiCl2 × 6H2O, and finally 0.42 g ZnCl2. After sterilization, 20-50 mg of yeast extract was added, 10 g / L -1 HEPES (final pH approximately 7.0), 30 mmol of thiosulfate, and 50 mmol of formate were added. 100 mL of cell suspension (10 7 cells mL -1 ) was added as the inoculum. The headspace of the bottle was washed 5 times with nitrogen and once with argon, and it was carefully sealed. 1.5 g of grapheoxide powder for doping was added to a 50 mL flask, and the flask was filled with 40 mL·h -1A culture bottle containing norprene tubing was connected to the culture bottle, with the interposition of an agitation pump operating at a certain speed. This pump allowed the growing medium to circulate from the bottle into the dopant flask, and then return to the culture bottle after passing through a Whatman Grade 1 paper filter with an 8 μm porosity. As previously done, the culture was carried out at 40°C for one month in static mode (without vigorous stirring). This time, the dopant flask was placed on a Stirrer Pro heating plate / magnetic stirrer adjusted to the parameters of 50°C and stirring at 250 rpm. As before, all products and reagents (N,S-DDG produced, obviously omitting GO) are water-soluble. Therefore, the separation of N,S-DDG from biomass was performed by centrifugation (4,000 × g, 5 min), followed by isotonic solution (240 g·L). -1 This could be easily carried out by two washes with 50 mL of water and filtration through Whatman Grade 1 paper with a porosity of 8 μm. The resulting material was then rinsed three times with Milli-Q® water through a BIO-PURE® Vacuum filters module. It was then dried in a vacuum oven, Type DZF-6010 Vacuum Drying Oven, at 60°C for 4 hours.

[0052] Here too, neither organic solvents nor acids are required for any of the N,S-DDG purification steps. The N,S-DDG obtained from the above procedure showed the following Raman spectrum (DDG-T curve): the area of ​​peak A was 2,692E+05 and the area of ​​peak B was 2,042E+05. In conclusion, in Example 2, the I of graphene oxide doped with the microorganism Halodesulfuracaeum formicicum (HTRS1) A / I B The ratio was approximately 1.32 (Figure 3).

[0053] (Example 3) Oxygen reduction reaction in an electrolytic cell using N,S-DDG prepared according to Examples 1 and 2 Here, the inventors measured the performance of graphene catalysts doped according to Examples 1 and 2 for reducing oxygen to hydrogen peroxide.

[0054] To obtain the percentage of H2O2 produced, the following electrochemical characterization measurements were performed in a three-electrode cell controlled by a bipotentiostat-galvanostat. For this purpose, a rotating ring-disk electrode (RRDE) consisting of a platinum ring and a glassy disk coated with a catalyst ink that was dried on the electrode was used as the working electrode. The ORR (oxygen reduction reaction) activity was tested at 1600 rpm in an O2 flow using polarization curves between 1.1 and 0.2 V relative to the RHE (reference hydrogen electrode). To detect H2O2 formation, measurements were performed using the ring electrode held at 1.2 V.

[0055] The electrochemical reduction of O2 with a good catalyst (such as platinum, known as a conductor) has no intermediate phase and directly reduces the oxygen molecule to water (H2O). Therefore, hydrogen peroxide is almost completely absent as a byproduct of the reaction. On the other hand, raw graphene oxide has the opposite properties; it is an excellent catalyst for the reduction of oxygen to hydrogen peroxide and an inhibitor of hydrogen peroxide reduction in water. Nevertheless, because this raw material does not conduct electricity well, the expected volume of H2O2 produced remains low, although it is a large proportion of water. The following experiments were carried out with electrodes consisting of 1 mg of graphene oxide (raw and doped with HTSR1 and AArc1 bioactive agents, respectively). (i) Using an HTSR1-doped graphene oxide sample, the inventors obtained yields of 92±3% H2O and 8±3% H2O2. (ii) Using AArc1-doped graphene oxide samples, the inventors obtained yields of 72±5% H2O and 28±5% H2O2. (iii) Using an HSR2-doped graphene oxide sample, the inventors obtained yields of 90±2% H2O and 10±2% H2O2. (iv) Finally, using the raw graphene oxide sample, the inventors obtained yields of 2.4 ± 0.3% H2O and 97.6 ± 3.7% H2O2.

[0056] Second, products of hydrogen peroxide (H2O2) produced by electrosynthesis were measured. (i) The HTSR1-doped graphene oxide sample product was evaluated as 4.37 mg·h per 1 mg of the doped material. -1 and evaluated. (ii) The Aarc1-doped graphene oxide product was evaluated as 32.1 mg·h per 1 mg of the doped material. -1 and evaluated. (iii) The HSR2-doped graphene oxide product was evaluated as 10.2 mg·h per 1 mg of the doped material. -1 and evaluated. (iv) The raw graphene oxide product was evaluated as 10.5 μg·h per 1 mg of the raw material. -1 and evaluated.

[0057] The higher production rate of H2O2 by graphene modified with AArc1 may be due to the fact that this material is a slightly less effective catalyst for the reduction of O2 to H2O, and this material is conductive in any case. Based on this, graphene modified with AArc1 seems to be a more efficient material for generating H2O2, while graphene modified with HSTR1 is considered to be more efficient for generating H2O from O2. In either case, the productivity of graphene modified with AArc1 and graphene modified with HSTR1 is substantially higher than that of the control graphene oxide (3150 times and 970 times higher, respectively, that is, 3 times higher).

[0058] (Example 4) Cyclic voltammetry analysis The electrodes listed above were modified with different materials: two types of electrodes were modified with reduced GO; one was modified with the microorganisms listed above, identified by the HSR2 code, grown on acetate (curve 2) and pirubate (curve 3); one was modified with HSR6 accelerated with formate (curve 4); and one was modified with AArc-S grown on sucrose (curve 5). For all microorganisms, elemental sulfur was used as the electron acceptor (Figure 4A). In Figure 4, instead, electrodes modified with GO reduced with AArc-1 grown on polysulfide and electrodes modified with GO reduced with HTSR1 grown on thiosulfate were used.

[0059] All electrodes modified according to the present invention exhibit yields associated with the enlarged area caused by graphene doped in the method of the present invention. This result clearly demonstrates, on the one hand, that the doping procedure was correct, and on the other hand, that it was effective compared to the control represented by undoped GO (curve 1) in both graphs. In graph A, the amplitude of the curve area is larger than the amplitude of the curve in graph B, but it should be noted that in the case of graph A, as previously described, elemental sulfur was used as the electron acceptor, which involves washing the doped graphene with contaminants. In contrast, in the case of graph B, the acceptor used does not involve the use of contaminants.

[0060] (Example 5) Photoelectric X-ray spectroscopy This technique enables the characterization of solid surfaces by studying the energy of electrons emitted by the solid when irradiated with X-ray photons. In this way, information about the state of chemical bonding and the concentration of atoms on the surface can be obtained. The equipment used included a hemispherical electron analyzer, a 5x electron intensifier detector (Channeltron®), and Mg(K αThe spectrophotometer VG ESCALAB 200R (VG-Scientific) consists of an anode X-ray emission source (=1253.6 eV), has an operating chamber pressure of less than 9 Torre, and operates at 12 kV and 10 mA. The elements carbon, oxygen, nitrogen, and sulfur on the catalyst surface, as well as their oxidation states, were analyzed using XPS technique.

[0061] Table 3 below details the values ​​obtained from the atomic surface ratios O / C, N / C, and S / C of all catalysts. It may be noted that the treatment of graphene oxide with the halobacteria of the present invention results in the reduction of graphene oxide, thereby reducing surface oxygen and increasing the content of sulfur and nitrogen heteroatoms in the sample treated with the bacterial HTSR of the present invention. In particular, samples treated with HTRS1 had the highest sulfur content, while samples treated with HSR2 had the highest nitrogen content. An intermediate value for sulfur, nitrogen, and oxygen was obtained in samples treated with HSR6.

[0062] [Table 3] Furthermore, it should be noted that the O / C ratio of the three samples treated with the microorganisms of the present invention is less than half that of graphene oxide. This means that the conductivity is twice that of these samples.

[0063] Figure 5 shows the XPS spectra of untreated graphene oxide samples obtained from three samples treated with HSR2, HSR6, and HTRS1, respectively, for a mixture of three different elements: A) carbon 1s, B) nitrogen 1s, and C) sulfur 2p. Spectra A) shows the C1s zone for all samples, outlining their distribution in four subgroups corresponding to the oxidation states of graphite, hydroxyl, epoxy, and carboxylic acid, respectively. Subgroups with binding energies less than approximately 284.53 eV correspond to graphite carbon (CC), while the latter subgroups correspond to higher oxidation states with higher binding energies. The greatest difference between graphene oxide and samples treated with HSR microorganisms is indicated by the amplitude of the curve for the oxidized carbon subgroups (C=O at 287.66 eV, CO, COC, and CO at 286.55 eV). This is significantly lower in the treated samples than in graphene oxide. This indicates significant reduction in graphene oxide in the presence of microorganisms.

[0064] The spectra at energy level N1s (Figure 5B) were equally divided into three peaks. The lowest binding energy peak (approximately 399 eV) corresponds to pyrimidine nitrogen, followed by pyrolysis nitrogen at approximately 400 eV, and graphite nitrogen at approximately 402 eV. The peak with the highest intensity is pyrimidine nitrogen in all three processed samples. The observed components are similar in all three samples, with the exception of HSR2, where the proportion of N-pyrrole nitrogen is slightly higher.

[0065] The S2p energy level spectra for the HTSR samples in Figure 5C) were separated into four components, with the exception of the graphene oxide sample, which was kept under a single group. For nitrogen, the S2p signal is very low in graphene oxide. The peaks obtained in the HSR samples at approximately 163–164 eV and approximately 166 eV are attributed to sulfur in the atomic chains of graphene, thiophene, and oxidized thiophene (C-SO, C-SO2), respectively. Alternatively, disulfuric acid (SO4) 2- ), sulfite (SO3 2- ) or thiosulfite (S2O3 2-The oxidation of sulfur in its higher state (as in the form of ) is thought to correspond to a bond energy of over 167 eV.

[0066] Therefore, the graph clearly demonstrates the superior performance in reducing graphene, particularly when treated with the bacteria of the present invention having identified forms such as pyrrole nitrogen and thiophene sulfur, and the increased content of heteroatoms N and S on the surface of graphene, even compared to similar processes based on the use of other microorganisms.

Claims

1. A method for producing graphene doped with nitrogen and sulfur atoms (N,S-DDG), comprising the steps of: - providing a microorganism of the class Halobacteria that is strictly anaerobic and sulfite reducing and capable of surviving at salinity conditions of more than 200 g L-1 and at a pH of 7.0 to 10.0 at 20°C to 50°C; - hydrogen (H) as electron donor in amounts up to 100 mmol 2 ), acetate (C 2 H 4 O 2 ), formate (CH 2 O 2 ), glycerol (C 3 H 8 O 3 ), glucose (C 6 H 12 O 6 ), sucrose (C 12 H 22 O 11 ) and other similar sugars, lactate (C 3 H 6 O 3 ), short chain fatty acids (C 4 -C 9 ) and / or pyruvate (C 3 H 4 O 3 ) and, as an electron acceptor, elemental sulfur (S 8 °), polysulfide (-S-S 6 -S-), thiosulfate (S 2 O 3 2- ), dimethyl sulfoxide (CH 3 ) 2 SO and tetrathionate (S 4 O 6 2- S selected from the group consisting of 2- Cultivating the microorganism in a medium containing one or more of the more oxidized sulfur forms, wherein the medium further comprises a nitrogen source; - contacting a solution of graphene oxide (GO) with said medium containing said microorganisms for a time sufficient to obtain a doping with nitrogen and sulfur; - washing the graphene to remove both the organic phase and the nitrogen- and sulfur-containing molecules that have not reacted with the oxidized graphene; A method comprising:

2. 2. The method of claim 1, wherein the microorganism is selected from the genera Halalkaliarcaeum, Halanaeroarchaeum, Halodesulfurarchaeum, Halarchaeoglobus, Natranaeroarchaeum and Natronolimnobius.

3. The medium is 240 g L -1 of NaCl, 3g・L -1 K 2 H.P.O. 4 , 0.5g / L -1 NH 4 Cl, 1-5 mM MgCl 2 x6H 2 It contains O, is sterile, and has a concentration of 20-50 mg / L. -1 of yeast extract, 1 ml / L -1 of acid trace metal solution, 1 mL L -1 The method according to claim 1 or 2, wherein the Se / W alkaline solution and vitamin mix are added.

4. The acid trace metal solution contained the following substances (per liter of medium): HCl 0.01N, 0.6 g CoCl 2 x6H 2 O, 30 mg CuCl 2 , 0.3 g FeCl 2 ×4H 2 O, 1.14 g H 3 B.O. 3 , 4 g MnCl 2 ×4H 2 O, 0.5 g Na 2 MoO 4 ×2H 2 0, 0.3 g NiCl 2 x6H 2 O and 0.42 g ZnCl 2 The vitamin mix contains 1 mg of B per liter of deionized water. 12 vitamins, 20 mg biotin, 20 mg folic acid, 50 mg nicotinic acid, 50 mg p-aminobenzoic acid, 50 mg calcium pantothenate, 100 mg pyridoxine x HCl, 50 mg riboflavin, 50 mg thiamine and 50 mg thioctic acid, said Se / W alkaline solution containing the following substances (per liter of 0.01N NaOH): 2 mg Na 2 SeO 3 and 4 mg Na 2 WO 4 x 1.5H 2 The method of claim 3 , wherein the compound comprises O.

5. The medium is 10 g L -1 The method of claim 3 or 4, further comprising HEPES.

6. The medium is a mixture of two media, the first medium being 240 g L -1 of NaCl, 5 g L -1 of KCl, 2 g L -1 K 2 H.P.O. 4 , 0.5 g L -1 NH 4 The second medium contains 190 g L -1 Na 2 CO 3 , 30g / L -1 NaHCO 3 , 16g·L -1 of NaCl, 5.0 g L -1 8 mM KCl, 8 mM NH 4 Cl, 1.0 g / L -1 K 2 H.P.O. 4 and both media contained 1 mM MgCl 2 x6H 2 O, 1mL / L -1 Acid trace metal solution, vitamin mix, 1mL / L -1 Alkaline solution of Se / W, 20 mg L -1 3. The method according to claim 1 or 2, wherein a yeast extract of

7. The step of contacting graphene oxide (GO) with a cell culture medium comprises contacting the GO with a cell culture medium at a temperature between 20° C. and 50° C. for a time period between 10 and 30 days with or without stirring at a concentration of 2 mg / mL -1 The method according to any one of claims 1 to 6, wherein the method is carried out by contacting graphene oxide in the form of a powder at a concentration of up to 1000000000.

8. The method of any one of claims 1 to 7, wherein the step of washing the graphene comprises separation of the organic material from the doped graphene by centrifugation and / or filtration.

9. A plant for carrying out the method according to any one of claims 1 to 8, comprising a first tank (1) for storing and maintaining / growing a microorganism according to claim 1 or 2 which reduces graphene oxide, at least a second tank (2) hydraulically connected to the first tank for mixing and doping said oxide with said microorganism, temperature control and adjustment means (3) for the first tank (1), pH control and adjustment means (4) for the first tank, temperature control and adjustment means (5) for the second tank, means for separating an organic phase from the doped graphene, means for washing the doped graphene (7) and means for drying the doped and washed graphene (8).

10. Graphene doped with nitrogen and sulfur by the method of any one of claims 1 to 8, comprising, as a percentage of the total percentage of atoms, 1% to 9% nitrogen atoms and 0.3% to 15% sulfur atoms, with more than 90% of the nitrogen atoms being present as pyrrolic nitrogen.

11. 11. The graphene of claim 10, wherein greater than 40% of the sulfur atoms are present as thiophenol sulfur.

12. 12. Use of the doped graphene according to claim 10 or 11 for producing nanomaterials used as electronic and electrochemical components, electrodes of electrochemical cells, analytical systems, purification systems, nanocomponents for the medical field, telecommunications field, aviation field, aerospace field, robotics field, eco-sustainable macro materials as machine parts, automotive parts, aeronautical parts, aerospace parts and robotics parts.

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