Extracellular electron carriers

Abstract

To provide a new extracellular electron transfer material.SOLUTION: An extracellular electron transfer material is composed of a material containing a protein that contains sulfur and a β sheet structure.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an extracellular electron transfer material and the like. [Background technology]

[0002] Research and development of technologies for environmental purification and resource recovery using bioelectrochemical systems that directly supply electricity (derived from renewable energy) to microorganisms is becoming increasingly common. Geobacter and Schewanella bacteria are known to be able to transfer electricity between themselves and electrodes (electroactive microorganisms), but most bacteria are not electrically active and therefore require electricity to be supplied via extracellular electron carriers.

[0003] Extracellular electron transport has the effect of promoting a variety of anaerobic microbial reactions, including anaerobic dehalogenation, metal oxidation-reduction, metalloid oxidation-reduction, denitrification, nitrate reduction and ammonia production, carbon dioxide fixation and acetogenesis, methanogenesis, and nitrogen fixation.

[0004] Microbial fuel cells are systems that utilize a reaction that receives electricity from microorganisms. They generate electricity by receiving excess electrons from electrodes generated by the decomposition of organic matter dissolved in water. Research and development of this reaction is underway as a water treatment technology that does not require aeration. Here too, the use of extracellular electron carriers allows non-electroactive microorganisms to contribute to the microbial fuel cell reaction in the same way as electroactive microorganisms, increasing efficiency.

[0005] Research is also being conducted into the direct transfer of electricity from electron carriers to enzymes, but because enzymes are expensive and unstable, extracellular electron transfer techniques using microbial cells are considered more promising.

[0006] Known extracellular electron transfer substances include anthraquinone-2,6-disulfonic acid (AQDS), riboflavin, methyl viologen, benzyl viologen, hematin, neutral red, humic acid, and fulvic acid. However, these substances are difficult to use in actual bioreactors due to their water-solubility, which can lead to leaching during use in bioreactors. Increasing their concentration to improve efficiency (AQDS) can be harmful to the target microorganisms. Furthermore, the compounds themselves are toxic (or biologically active), which can lead to environmental pollution if released outdoors (viologens and neutral red). Therefore, attempts have been made to immobilize these water-soluble extracellular electron transfer substances on electrodes, but the long-term stability of the immobilization remains a challenge (the possibility of gradual leaching of harmful substances cannot be ignored), posing a significant obstacle to their practical application.

[0007] A desirable, non-toxic, solid extracellular electron carrier is iron-humic acid, which is made by insolubilizing alkali-soluble, acid-insoluble humic acid by binding it with iron ions (Patent Document 1). While these extracellular electron carriers have the advantage of being extractable from soil or sediment anywhere in the world, they have the problem that, because they are natural products, their activity is not consistent.

[0008] Furthermore, when constructing a bioelectrochemical system using a solid extracellular electron mediator, there are two methods: suspending it in a reactor or immobilizing it on an electrode. The latter method is preferable because it has higher electrical efficiency. Water-soluble extracellular electron mediators are immobilized using methods such as electropolymerization. Since immobilization changes electrical efficiency, it is necessary to establish an optimization method. Furthermore, in the case of silver epoxy, there are issues with the toxicity of silver and the limited range of application due to the redox potential of silver. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent No. 6410188 [Non-patent literature]

[0010] [Non-Patent Document 1] Lefevre, T., Rousseau, M.-E., Pezolet, M., 2007. Protein Secondary Structure and Orientation in Silk as Revealed by Raman Spectromicroscopy. Biophysical Journal 92, 2885-2895. Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a novel extracellular electron transfer material. [Means for solving the problem]

[0012] As a result of intensive research conducted by the present inventors in view of the above problems, That is, the present invention includes the following aspects.

[0013] Item 1. An extracellular electron transfer material comprising a material containing sulfur and a protein having a β-sheet structure.

[0014] Item 2. The extracellular electron transfer material according to Item 1, wherein the sulfur-binding structure in the protein is at least one selected from the group consisting of -Fe-S-, -SS-, -CS-, -SH, -SO, -SO2, and -SO3.

[0015] Item 3. The extracellular electron transfer material according to Item 1 or 2, wherein the content of the β-sheet structure in the protein is 2% by mass or more.

[0016] Item 4. The extracellular electron transfer material according to any one of Items 1 to 3, wherein the protein is at least one protein selected from the group consisting of fibroin protein and sericin protein.

[0017] Item 5. The extracellular electron transfer material according to any one of Items 1 to 4, wherein the material is a biologically derived material.

[0018] Item 6. The extracellular electron transfer material according to Item 5, wherein the biological material is at least one selected from the group consisting of silk thread, spider silk, oyster meat, and materials derived therefrom.

[0019] Item 7. The extracellular electron transfer material according to any one of Items 1 to 6, wherein the material is a water-insoluble material or a water-soluble material.

[0020] Item 8. A carrier comprising the extracellular electron transfer material according to any one of Items 1 to 7.

[0021] Item 9. The carrier according to Item 8, which is porous.

[0022] Item 10. The support according to Item 8 or 9, which is an electrode or an electrode material.

[0023] Item 11. The support according to Item 10, for use in a bioelectrochemical system or a microbial fuel cell.

[0024] Item 12. The carrier according to any one of Items 8 to 11, which is a carrier for cultivating microorganisms.

[0025] Item 13. A method for promoting a microbial reaction, comprising culturing a microorganism in a state in which the microorganism can be in contact with the extracellular electron transfer material according to any one of Items 1 to 7 or the carrier according to any one of Items 8 to 12.

[0026] Item 14. The method for promoting microbial reactions according to Item 13, wherein the microbial reactions are at least one selected from the group consisting of dehalogenation reactions, metal oxidation-reduction reactions, metalloid oxidation-reduction reactions, denitrification reactions, nitrate reduction reactions, carbon dioxide fixation reactions, methane production reactions, hydrogen production reactions, sulfate reduction reactions, nitrogen oxide fixation reactions, and nitrogen fixation reactions.

[0027] Item 15. A promoter for microbial reactions, comprising a material containing sulfur and a protein having a β-sheet structure.

[0028] Item 16. A method for promoting the growth of a microorganism, comprising culturing the microorganism in contact with the extracellular electron transfer material according to any one of Items 1 to 7 or the carrier according to any one of Items 8 to 12.

[0029] Item 17. A growth promoter for microorganisms, comprising a material containing sulfur and a protein having a β-sheet structure. [Effects of the Invention]

[0030] According to the present invention, a new extracellular electron transfer material can be provided. [Brief explanation of the drawings]

[0031] [Figure 1] The outline of the process for preparing sericin and fibroin from silkworm silk (cocoon balls) (Test Example 1-1) is shown below. [Figure 2] The mechanism of silk as an extracellular electron transfer (EET) material (hereinafter sometimes referred to as a "mediator") in Test Example 1-2 is shown below. Formate is the carbon source. [Figure 3]The dechlorination rate (μmol Cl-) L-1 d-1 of humin-dependent pentachlorophenol (PCP) dechlorination cultures is shown in Figure 1-1. From left to right, the following are: No mediator: culture without added mediator; humin: culture with 0.3 g of humin added per cocoon; Cocoon: culture with 30 mg of excised cocoons; Fibroin fiber: culture with 30 mg of fibroin fiber added; Cocoon-No C-source: culture with 30 mg of excised cocoons without added carbon source; and Fibroin fiber-No C-source: culture with 30 mg of fibroin fiber without added carbon source. The first four cultures were supplemented with a carbon source (10 mM formate). The mediator-free culture served as the negative control, and the humin-added culture served as the positive control. [Figure 4] 1 shows the dechlorination rate (μmol Cl−) L−1d−1 of the humin-dependent PCP-dechlorinating culture (Test Example 1-2). From left to right: No mediator: culture with no mediator added; Sericin: culture with 30 mg of lyophilized sericin added; Sericin-Centri-Insoluble: culture with the insoluble fraction (less than 30 mg) obtained by stirring 2 g of lyophilized sericin in 50 mL of purified water for 15 minutes and then centrifuging at 15,000 × g for 15 minutes; Sericin-Centri-Soluble: culture with the soluble fraction (less than 30 mg) of lyophilized sericin obtained by freeze-drying the soluble fraction obtained as the supernatant of the above centrifugation; Sericin-Filtrate: culture with the soluble fraction (less than 30 mg) of lyophilized sericin added, which was obtained by dissolving 120 mg of lyophilized sericin in 80 mL of mineral salts medium by stirring at room temperature for 15 minutes and then sterile-filtering using a 10 mL syringe and a membrane filter (pore size 0.22 μm, Millex-GV, Merck Millipore, Ireland). Cultures without added mediator served as negative controls. [Figure 5]The preparation process of different fractions of Bombyx mori (silkworm) silk (B. mori-silk) (A) and a photograph of the fractions in a dried state (B) are shown (Test Example 2-1). [Figure 6] B. The dissolution process of mori-silk fiber is shown (Test Example 2-1). [Figure 7] The dechlorination rate (µmol Cl-) L-1 d-1 of humin-dependent PCP-dechlorinating cultures is shown in Figure 2 (Test Example 2). The culture conditions were as follows: fibroin fiber (culture 1), fibroin-insoluble 1 (culture 2), fibroin-soluble 1 (culture 3), fibroin-insoluble 2 (culture 4), and fibroin-soluble 2 (culture 5). Except for culture 1 (air-dried fibroin fiber), cultures 2 to 5 used freeze-dried forms of different fibroin fractions. [Figure 8] An example of the procedure for recovering used insoluble fibroin 2 is shown below (Test Example 3-5). [Figure 9] The system used for the electrochemical oxidation-reduction of insoluble fibroin 2 is shown (Test Examples 3-5). [Figure 10] FT-IR spectra of soluble fibroin 2 (no EET function) and insoluble fibroin 2 (positive for EET function) are shown (Test Example 3). [Figure 11] FT-IR spectra of fibroin-insoluble 2 before and after application to a humin-dependent PCP-dechlorinating culture (Test Example 4). [Figure 12] The 13C CP / MAS NMR spectra of fibroin fiber, fibroin insoluble 1, fibroin insoluble 2, and fibroin soluble 2 are shown (Test Example 3). The symbol "o" indicates a material with EET function, and "x" indicates a material without EET function. β and r represent β-sheet and random coil, respectively. α, β, γ, δ, ε, and ξ indicate the atomic positions of C in the amino acid structure. The abbreviations for all amino acids shown in this figure are as follows: Ala = alanine, Gly = glycine, Ser = serine, Gln = glutamine, Tyr = tyrosine, and Phe = phenylalanine. [Figure 13] 13C CP / MAS NMR spectra of fibroin fiber and sericin (Test Example 3). The symbols in the figure are the same as those in FIG. 12. [Figure 14] The SK absorption near edge structure (XANES) spectrum of each reference compound is shown (Test Example 3). [Figure 15] The S K-edge XANES spectrum of the cocoon and fibroin fiber (A) and the spectrum comparison of cysteine ​​and cystine fibroin fiber are shown (Test Example 3). [Figure 16] SK absorption edge XANES spectra of different fractions of fibroin fiber (total fraction and soluble / insoluble 1 fraction) (A) and different fractions of soluble 1 fibroin (total fraction and soluble / insoluble 2 fraction) (B) are shown (Test Example 3). [Figure 17] SK absorption edge XANES spectra of cocoons (A) and fibroin fibers (B) before and after culture are shown (Test Example 3). [Figure 18] 1 shows the SK absorption edge XANES spectra of fibroin insoluble 1 (A) and fibroin insoluble 2 (B) before and after culture (Test Example 3). [Figure 19] 1A shows the C 1s XPS spectrum and B shows the S 2p XPS spectrum of electrochemically oxidized fibroin and reduced fibroin insoluble 2 (Test Example 3). [Figure 20] The dechlorination rates (μmol Cl-) L-1 d-1 of extracellular electron transfer material (EETM)-dependent PCP dechlorination cultures using different materials are shown in Test Example 4. The dechlorination rates (μmol Cl-) L-1 d-1 of NaOH / HF-pretreated fibroin fibers (Culture A), UV-pretreated cocoons (Culture B), UV-pretreated fibroin fibers (Culture C), and used fibroin insoluble 2 (Culture D) are also shown. [Figure 21] 1 shows 13C CP / MAS NMR spectra of fibroin fibers before and after NaOH / HF treatment (Test Example 4). [Figure 22] 1 shows the SK absorption edge XANES spectra of fibroin fiber before and after treatment with NaOH / HF (Test Example 4). [Figure 23] 1 shows the SK absorption edge XANES spectra of fibroin fiber and UV-treated fibroin fiber (Test Example 4). [Figure 24] Shown are cocoons and fibroin fibers (A, B) before and after ultraviolet treatment (Test Example 4). [Figure 25] The effects of cocoons and fibroin fibers on Fe(III)-reducing culture are shown (Test Example 5). [Figure 26] This shows acetic acid production in CO2-reducing culture (Test Example 5). Control 1 was a culture without the addition of a mediator, Control 2 was a culture with the addition of humin as a mediator, Control 3 was a culture using fibroin fiber without the addition of H2, Control 4 was a culture using fibroin fiber without the addition of microorganisms, and Control 5 was a culture without the addition of either H2 or microorganisms. [Figure 27] The results of comparing the acetylene reducing activity (ARA) of cultures using test conditions with cocoons (+) and without cocoons (-) (A) with the ARA of cultures using test conditions with fibroin fibers (+) and without fibroin fibers (-) (B) are shown (Test Example 5). [Figure 28] The function of silk as an EETM in each anaerobic microbial culture is shown. [Figure 29] The method for preparing a fibroin sponge and a fibroin-coated sponge is outlined below (Test Example 6). [Figure 30] The procedure for collecting a used fibroin-coated PU sponge is shown below (Test Example 6). [Figure 31]The dechlorination rates (µmol Cl-) L-1 d-1 of different cultures using each sponge are shown in Test Example 6. Sponge 1 was a fibroin sponge (100% fibroin, 18.4 ± 0.9 mg), Sponge 2 was a polyurethane (PU) sponge, Sponge 3 was a fibroin-coated PU sponge (fibroin content 7.0 ± 0.6 mg), Sponge 4 was a sea sponge (20 mg), Sponge 5 was a fibroin-coated sea sponge (fibroin content 4.2 ± 1.0 mg), Sponge 6 was raw cotton, and Sponge 7 was a fibroin-coated raw cotton (fibroin content 10.6 ± 1.0 mg). [Figure 32] 1 shows the dechlorination rate (new use / reuse) of the culture using the fibroin-coated PU sponge (Test Example 6). [Figure 33] The advantages of using a fibroin sponge and a fibroin-coated PU sponge are shown (Test Example 6). [Figure 34] The SK absorption edge XANES spectra (A) of fibroin soluble 1 and a sponge produced from fibroin soluble 1 (100% fibroin) and the SK absorption edge XANES spectra (B) of the fibroin sponge before and after culture are shown. [Figure 35] 1 shows the dechlorination rate of a heterologous culture using a heterologous material as an EET mediator (Test Example 7). [Figure 36] The concept of EETM materials is shown. The red circle indicates the core functional structure (a beta sheet with S-containing functional groups). Larger circles indicate a more complex material structure. Purple text indicates that these components are also present in natural materials in addition to the core functional structure. F atoms bonded to the exterior of the material indicate that it can be reinforced by fluorination. [Figure 37] As an example of bioelectrochemical culture using EETM materials, an H-type two-chamber bioelectrochemical culture system is shown. [Figure 38]Hydrogen production (A), methane production (B), and acetic acid production (C) after 48 hours of anaerobic cultivation in the cathode chamber of a bioelectrochemical cultivation system are shown. Condition 1: fibroin insoluble 2 and carbon dioxide-reducing culture medium were added and the culture was incubated at a potential of -600 mV (based on the standard hydrogen electrode). Condition 2: carbon dioxide-reducing culture medium alone was added and the culture was incubated at -600 mV (based on the standard hydrogen electrode). Condition 3: fibroin insoluble 2 alone was added and the culture was incubated at -600 mV (based on the standard hydrogen electrode). Condition 4: fibroin insoluble 2 and carbon dioxide-reducing culture medium were added and the culture was incubated without applying current. DETAILED DESCRIPTION OF THE INVENTION

[0032] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0033] 1. Extracellular electron carriers In one aspect, the present invention relates to an extracellular electron transfer material (hereinafter sometimes referred to as "extracellular electron transfer material of the present invention") comprising a material (hereinafter sometimes referred to as "material of the present invention") containing a protein comprising sulfur and a β-sheet structure (hereinafter sometimes referred to as "protein of the present invention"). This will be described below.

[0034] Sulfur is not particularly limited as long as it is an atom constituting the protein of the present invention. In the protein of the present invention, sulfur is bonded to other atoms to form a sulfur-bonded structure. Examples of sulfur-bonded structures in the protein of the present invention include, but are not limited to, -Fe-S-, -SS-, -CS-, -SH, -SO, -SO2, and -SO3. The sulfur-bonded structure may be a single type or a combination of two or more types. The sulfur-bonded structure is identified by SK-edge X-ray absorption structure analysis. Specifically, it is identified according to the methods described in Test Examples 3-3 and 3-4.

[0035] The sulfur content in the protein of the present invention is not particularly limited and may be a trace amount. Examples of such a content include 0.001 to 1% by mass, 0.002 to 0.5% by mass, 0.004 to 0.2% by mass, 0.007 to 0.15% by mass, and 0.01 to 0.12% by mass. Ranges obtained by arbitrarily changing the upper and / or lower limits of these ranges are also exemplified herein. The sulfur content is measured by elemental composition analysis. Specifically, it is measured according to the method described in Test Example 2-1.

[0036] A β-sheet structure is a type of secondary structure of proteins. When peptide chains are connected in an almost fully extended state and several of these are stacked in parallel or antiparallel, a pleated sheet (pleated plate) structure is formed by hydrogen bonding between the O in the C=O group of one peptide chain and the H in the NH group of the adjacent strand. β-sheet structure can be structured in two ways: parallel sheets, in which the peptide chains are aligned in the same direction, and antiparallel sheets, in which the chains are aligned in opposite directions. In parallel β-sheets, the distance between hydrogen bonds is constant, while in antiparallel β-sheets, points with close and distant hydrogen bond distances alternate. The substituents of the amino acid side chains protrude alternately above and below this sheet. The presence or absence of a β-sheet structure in the protein of the present invention can be determined by Fourier transform infrared spectroscopy (FT-IR) or 13 It is identified by C CP / MAS NMR analysis, specifically, by the method described in Test Example 3-1 or Test Example 3-2.

[0037] The content of β-sheet structures in the protein of the present invention is not particularly limited, but is, for example, 2% by mass or more. From the viewpoint of extracellular electron transfer ability, the content is preferably 5% by mass or more, more preferably 10% by mass or more, even more preferably 20% by mass or more, even more preferably 30% by mass or more, particularly preferably 40% by mass or more, and especially preferably 45% by mass or more. The upper limit of the content is not particularly limited, and is, for example, 90% by mass, 80% by mass, 70% by mass, or 60% by mass. The content is measured by polarized microscopic Raman spectroscopy. Specifically, it is measured by the method described in Non-Patent Document 1.

[0038] The protein of the present invention may be a natural protein or an artificial protein. Specific examples of the protein of the present invention include fibroin protein and sericin protein.

[0039] A natural protein such as a fibroin protein may have a mutation introduced therein. Examples of mutations include substitution, deletion, insertion, and addition of an amino acid chain. Among these, substitution is preferred, and conservative substitution is more preferred.

[0040] As used herein, "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitution include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0041] The amino acid sequence after the mutation has, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity to the amino acid sequence before the mutation.

[0042] "Identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Thus, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul S F. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul S F. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on the BLAST algorithm. Specific techniques for these analysis methods are publicly known, and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0043] The protein of the present invention may be a molecule consisting of a polypeptide or a complex thereof, or may be a molecule consisting of a polypeptide or a complex thereof linked to another substance (e.g., a fluorescent substance, a radioactive substance, an inorganic particle, etc.).

[0044] The protein of the present invention may be chemically modified. The polypeptide constituting the protein of the present invention may have a carboxyl group (-COOH) or a carboxylate (-COO) at the C-terminus. -), amide (-CONH2), or ester (-COOR). Here, R in the ester may be, for example, a C alkyl group such as methyl, ethyl, n-propyl, isopropyl, or n-butyl. 1-6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3-8 Cycloalkyl groups such as phenyl and α-naphthyl 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups; α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 An aralkyl group; a pivaloyloxymethyl group, etc. are used. In the polypeptide constituting the protein of the present invention, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, for example, the above-mentioned C-terminal esters are used as the ester. Furthermore, in the polypeptide constituting the protein of the present invention, the amino group of the N-terminal amino acid residue may be protected by a protecting group (for example, a C-protecting group such as a formyl group or an acetyl group). 1-6 C such as alkanoyl 1-6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1-6 C such as alkanoyl group 1-6 Also included are those protected with an alkyl group (such as an acyl group).

[0045] The proteins of the present invention may be those to which known proteins or peptides such as protein tags or signal sequences have been added. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, and fluorescent protein tag.

[0046] The protein of the present invention may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is pharmaceutically acceptable, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0047] The protein of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0048] In one embodiment of the present invention, the protein of the present invention can be produced by cell culture using genetic engineering techniques. In this embodiment, the protein of the present invention can be produced, for example, by culturing cells containing a polynucleotide comprising a coding sequence for the protein of the present invention (the polynucleotide of the present invention).

[0049] The polynucleotide of the present invention may contain other sequences in addition to the coding sequence of the protein of the present invention. The polynucleotide of the present invention contains the coding sequence of the protein of the present invention in a state that allows the protein of the present invention to be expressed. Examples of other sequences include a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, an origin of replication, a coding sequence for a reporter protein (e.g., a fluorescent protein), a coding sequence for a drug resistance gene, etc.

[0050] The polynucleotide of the present invention preferably comprises an expression cassette for the protein of the present invention. The expression cassette comprises a promoter and a coding sequence for the protein of the present invention under the control of the promoter. The coding sequence is typically positioned downstream of the promoter so that it is under the control of the promoter. Examples of promoters that can be used in expression cassettes include CMV-IE (cytomegalovirus early gene-derived promoter), SV40 ori, retroviral LTP, SRα, EF1α, and β-actin promoters. The promoter is operably linked to the coding sequence. Here, "operably linked to the promoter" is synonymous with "the coding sequence is positioned under the control of the promoter," and typically means that the coding sequence is linked to the 3' end of the promoter directly or via another sequence. A poly(A) addition signal sequence is positioned downstream of the coding sequence. Transcription is terminated using the poly(A) addition signal sequence. Examples of poly(A) addition signal sequences that can be used include the SV40 poly(A) addition sequence and the poly(A) addition sequence of the bovine growth hormone gene.

[0051] The polynucleotide of the present invention may be a linear polynucleotide or a circular polynucleotide (e.g., a vector). The vector may be a plasmid vector or a viral vector. The vector may also be, for example, a cloning vector or an expression vector. Examples of expression vectors include vectors for prokaryotic cells such as Escherichia coli or actinomycetes, and vectors for eukaryotic cells such as yeast cells, insect cells, or mammalian cells. More specifically, viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, and Sendai viral vectors. Examples of non-viral vectors include various plasmid vectors, liposomal vectors, positively charged liposomal vectors, YAC vectors, and BAC vectors.

[0052] In one embodiment of the present invention, from the viewpoint of production costs, etc., a protein in a biologically derived material can be used as the protein of the present invention. Therefore, the material of the present invention containing the protein of the present invention is preferably a biologically derived material.

[0053] Biologically derived materials are materials produced by organisms, materials constituting organisms, or materials derived therefrom (i.e., materials obtained by treating or processing them as starting materials), and are not particularly limited insofar as they are concerned.

[0054] Examples of biologically derived materials include, preferably, silk thread, spider silk, oyster meat, oyster secretions (preferably secretory adhesive substances), sea anemones, mussel byssus, carp egg mantles, and materials derived therefrom. Among these, silk thread, spider silk, oyster meat, and materials derived therefrom are more preferred, and silk thread and materials derived therefrom are even more preferred. These are described below.

[0055] Cocoon filaments are threads spun by arthropods, such as moths, spiders, mayflies, bees, ants, caddisflies, etc. Among these, moths are preferred, and silkworm moths are particularly preferred.

[0056] The cocoon filaments can be used as they are, or can be treated or processed before use. The method of treatment or processing is not particularly limited as long as the protein of the present invention is not completely removed, and examples thereof include cutting, degumming (sericin removal), dialysis, solid-liquid separation, drying, etc.

[0057] The degree of cutting is not particularly limited as long as it can be made into a form suitable for use as an extracellular electron transfer material or a form suitable for other treatments.

[0058] Degumming can be carried out, for example, by treating the cocoon filaments with a high-temperature alkaline aqueous solution. Specifically, the cocoon filaments can be treated in a sodium carbonate aqueous solution (e.g., 0.02 to 0.1 M) at 60 to 100°C for about 1 to 8 hours. After degumming, the filaments can be washed with water, dried, etc. as needed. Fibroin fibers and water-soluble sericin can be obtained by degumming.

[0059] Dialysis is typically performed on fibroin fibers after degumming. For example, fibroin fibers are dissolved in a hot aqueous ethanol solution and then dialyzed with water. The ethanol concentration in the aqueous ethanol solution is, for example, 7 to 40%, preferably about 15 to 30%. The temperature of the aqueous ethanol solution is, for example, 50 to 90°C, preferably 65 to 85°C. The amount of the aqueous ethanol solution is, for example, 3 to 20 parts by mass, preferably 5 to 10 parts by mass, per part by mass of fibroin fibers. The molecular weight cutoff of the dialysis membrane is, for example, 5 to 30 kDa, preferably 8 to 20 kDa. The dialysis time is, for example, 1 to 10 days. After dialysis, solid-liquid separation (preferably centrifugation) can be performed to separate the fibroin fibers into an insoluble fraction and a soluble fraction. Both fractions can be used as the material of the present invention. After dialysis or solid-liquid separation, the fibers can be washed with water, dried (air-dried, freeze-dried, etc.), or the like, as needed.

[0060] When the soluble fraction obtained above is left in solution at a low temperature (e.g., 0 to 10°C) for a certain period of time (e.g., 1 week to 3 months), an insoluble matter is generated. The insoluble matter is subjected to solid-liquid separation (preferably centrifugation) to obtain an insoluble fraction, which can also be used as a material of the present invention. After solid-liquid separation, the fraction can be washed with water, dried (air-dried, freeze-dried, etc.), etc., as needed.

[0061] The material of the present invention can be a water-soluble material, but is preferably a water-insoluble material from the viewpoint of forming a carrier, as described below. Here, a "water-insoluble material" refers to a material in which the majority (e.g., 50% by mass or more, preferably 60% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, still more preferably 90% by mass or more, and particularly preferably 95% by mass or more) remains solid without dissolving even when left in a sufficient amount of water at 30°C for 20 days.

[0062] The material of the present invention can be fluorinated, for example, by treatment with hydrogen fluoride.

[0063] The material of the present invention may be one type alone or a combination of two or more types.

[0064] The content of the protein of the present invention in the material of the present invention is not particularly limited, but is, for example, 2% by mass or more, preferably 10% by mass or more, more preferably 20% by mass or more, even more preferably 50% by mass or more, still more preferably 70% by mass or more, particularly preferably 80% by mass or more, especially more preferably 90% by mass or more, and particularly preferably 95% by mass or more.

[0065] The state of the material of the present invention may be a solid, a solution, a semi-solid (such as a gel), or the like.

[0066] An extracellular electron transfer material is a material that can mediate electrons and is used to receive (or facilitate) the receipt of electrons produced by cells, or to transfer (or facilitate) the delivery of electrons to cells. As will be explained in "3. Method for Promoting Microbial Reactions" below, extracellular electron transfer materials can promote various microbial reactions (e.g., dehalogenation reactions, metal oxidation-reduction reactions, metalloid oxidation-reduction reactions, denitrification reactions, nitrate reduction reactions, carbon dioxide fixation reactions, methane production reactions, hydrogen production reactions, sulfate reduction reactions, nitrogen oxide fixation reactions, nitrogen fixation reactions, etc.).

[0067] 2. Carrier In one aspect, the present invention relates to a carrier (sometimes referred to herein as "the carrier of the present invention") comprising the extracellular electron transfer material of the present invention. This will be described below.

[0068] The carrier is not particularly limited as long as it can support a microorganism. The carrier of the present invention includes one composed mainly (for example, 35% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 95% by mass or more) of the extracellular electron transfer material of the present invention (carrier 1 of the present invention), and one composed of a carrier and the extracellular electron transfer material of the present invention present on the carrier (carrier 2 of the present invention).

[0069] The shape of the carrier is not particularly limited and may be spherical, pellet-like, rod-like, rectangular, etc. The carrier may be porous in order to increase the surface area for supporting the microorganisms.

[0070] The carrier 1 of the present invention can be obtained by using the extracellular electron transfer material of the present invention as is or by drying it. When the extracellular electron transfer material of the present invention is in a solid form, it can be used as is as the carrier 1 of the present invention. When the extracellular electron transfer material of the present invention is in a solution form, it can be dried to a solid form and used as the carrier 1 of the present invention. There are no particular limitations on the method for producing the porous carrier 1 of the present invention. For example, the solution form of the extracellular electron transfer material of the present invention can be mixed with water-soluble particles, followed by drying to solidify the extracellular electron transfer material of the present invention, and the water-soluble particles can then be removed by a water washing treatment or the like to obtain a porous carrier of the present invention in which the areas where the water-soluble particles were present have become pores.

[0071] The support (constituent support) constituting the support 2 of the present invention is not particularly limited, and examples thereof include resins, carbon materials, fiber constituent materials, metals, electrode materials, and the like. More specifically, examples of the resin include polyurethane (PU) resin, polyvinyl alcohol (PVA) resin, polyethylene terephthalate (PET) resin, polybutylene terephthalate (PBT) resin, polyethylene naphthalate resin, modified polyester resin, polyethylene (PE) resin, polypropylene (PP) resin, polystyrene resin, cyclic olefin resin, polyvinyl chloride resin, polyvinylidene chloride resin, polyvinyl butyral (PVB) resin, polyether ether ketone (PEEK) resin, polysulfone (PSF) resin, polyphenylene sulfide (PPS) resin, polyethersulfone (PES) resin, polycarbonate (PC) resin, polyamide resin, aromatic polyamide (PPA) resin, polyimide resin, polyamideimide (PAI) resin, polyetherimide (PEI) resin, polymethylpentene (PMP) resin, acrylic resin, triacetyl cellulose (TAC) resin, polyarylate (PAR), and liquid crystal polymer (LCP). Examples of carbon materials include carbon fibers (e.g., PAN-based carbon fibers, pitch-based carbon fibers, carbon nanotubes, etc.), carbon black, activated carbon, hard carbon, soft carbon, mesoporous carbon, graphene, carbon nanotubes, and fullerenes. Examples of fiber materials include rayon, polynosic, cupra, lyocell, acetate, cotton, hemp, flax, sheep wool, mohair, cashmere, camel, llama, alpaca, vicuna, angora, glass fibers (e.g., glass wool, glass fiber, etc.), and mineral fibers (e.g., chrysotile asbestos, white asbestos, blue asbestos, brown asbestos, direct asbestos, tremolite asbestos, and volcanic asbestos). Examples of metal and electrode materials include carbon materials such as carbon black, activated carbon, graphite, and conductive carbon; and metal materials such as platinum, gold, silver, copper, palladium, chromium, aluminum, and nickel, as well as alloys and metal halides. The carrier 2 of the present invention can be obtained by attaching (for example, coating) the extracellular electron transfer material of the present invention to a constituent carrier.

[0072] As will be explained in "3. Method for Promoting Microbial Reactions" below, the carrier of the present invention can promote various microbial reactions (e.g., dehalogenation reactions, metal oxidation-reduction reactions, metalloid oxidation-reduction reactions, denitrification reactions, nitrate reduction reactions, carbon dioxide fixation reactions, nitrogen fixation reactions, etc.). From this perspective, the carrier of the present invention is preferably a carrier for microbial cultivation. Furthermore, microbial reactions may be carried out under the supply of electrical energy, and from the viewpoint of being suitable for use in such cases, the carrier of the present invention is preferably an electrode or electrode material. These can be suitably used in bioelectrochemical systems or microbial fuel cells.

[0073] 3. Methods for promoting microbial reactions In one aspect, the present invention relates to a method for promoting a microbial reaction (sometimes referred to herein as the "method of the present invention"), which comprises culturing a microorganism in a state in which the microorganism can be in contact with the extracellular electron transfer material of the present invention or the carrier of the present invention. This method will be described below.

[0074] The microorganisms (target microorganisms) used in the methods of the present invention are not particularly limited, as long as they are capable of microbial reactions (reactions occurring within the microorganism) that are promoted by receiving or donating electrons. Examples of such microbial reactions include dehalogenation, metal oxidation-reduction, metalloid oxidation-reduction, denitrification, nitrate reduction, carbon dioxide fixation, methanogenesis, hydrogen production, sulfate reduction, nitrogen oxide immobilization, and nitrogen fixation. Examples of the microorganisms include carbon dioxide-fixing bacteria (e.g., acetogens, methanogens, formate-producing bacteria, etc.), dechlorinating bacteria, metal-reducing bacteria, metalloid-oxidizing bacteria, metalloid-reducing bacteria, denitrifying bacteria, nitrate-reducing bacteria, hydrogen-producing bacteria, sulfate-reducing bacteria, and nitrogen-fixing bacteria.

[0075] The bacteria used in the method of the present invention may be wild-type bacteria or mutant bacteria, including bacteria into which mutations have been artificially introduced.

[0076] The bacteria used in the method of the present invention may be isolated bacteria (e.g., purchased from a microorganism collection institution or collected from the environment and obtained by isolation procedures), or bacterial groups collected from the environment. Acetogens, methanogens, and the like are widely distributed in the environment and can be easily obtained, for example, from soil (preferably flooded soil).

[0077] The medium used in the method of the present invention contains the substances used in the above-mentioned microbial reaction. As an example, the case where an inorganic carbon source used in a carbon dioxide fixation reaction is used will be described.

[0078] The inorganic carbon source is not particularly limited as long as it is carbon dioxide or a compound or ion capable of generating carbon dioxide. Examples of the inorganic carbon source include carbon dioxide, carbonate ions, bicarbonate ions, carbon monoxide, etc. The inorganic carbon source can be used alone or in combination of two or more.

[0079] A medium containing an inorganic carbon source can be obtained by a conventional method. For example, carbon dioxide can be dissolved in the medium by exposing the medium to carbon dioxide. Alternatively, a medium containing an inorganic carbon source can be obtained by dissolving a compound that generates carbonate ions or bicarbonate ions in water in the medium. Such compounds are not particularly limited, but include bicarbonates and carbonates such as sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, magnesium carbonate, and calcium carbonate. These compounds can be used alone or in combination of two or more.

[0080] The carbon dioxide concentration in the medium is, for example, 1 to 500 mmol / L, preferably 5 to 200 mmol / L, more preferably 10 to 150 mmol / L, and even more preferably 20 to 100 mmol / L.

[0081] When using other substances used in microbial reactions, the substances to be used, their concentrations, etc. can be determined in the same manner as above or according to known microbial reaction conditions.

[0082] In addition to the substances used in the microbial reaction, the medium may contain other components necessary for the medium. Examples of such components include buffers, carbon sources, nitrogen sources, inorganic components, vitamins, etc. Various buffers that can be used in culture media can be used as buffers. Nitrogen sources are not particularly limited, but include inorganic nitrogen sources such as inorganic ammonium salts and nitrates. Inorganic components are sources of elements necessary for growth (e.g., P, S, K, Na, Mg, Ca, Fe, Cu, Zn, Mn, W, Se, etc.). Inorganic components can be added as chlorides, sulfates, phosphates, nitrates, and other salts containing these elements.

[0083] The atmospheric conditions for culture are not particularly limited. Preferably, culture is carried out under anaerobic conditions. Specifically, for example, the oxygen concentration in the culture atmosphere is preferably 5% or less, more preferably 1% or less, even more preferably 0.1% or less, and even more preferably 0.01% or less. In one embodiment of the present invention (for example, when a carbon dioxide fixation reaction is carried out), it is preferable that the culture atmosphere contains carbon dioxide. This allows carbon dioxide, which is a carbon source, to be continuously supplied to the medium.

[0084] The culture temperature is not particularly limited and can be set appropriately depending on the optimum temperature of the microorganism used. The culture temperature can be set, for example, within ±10°C, preferably ±5°C, more preferably ±2°C, and even more preferably ±1°C around the optimum temperature. The upper limit of the culture temperature can be set appropriately depending on the temperature at which the microorganism can grow. Examples of such upper limits are 90°C, 70°C, 60°C, 50°C, 40°C, and 38°C. The lower limit of the culture temperature can also be set appropriately depending on the temperature at which the microorganism can grow. Examples of such lower limits are 5°C, 10°C, 15°C, and 20°C.

[0085] The culture method is not particularly limited. For example, static culture can be employed.

[0086] The method of the present invention can be used both in the absence of an electrical energy supply (non-electrical culture) and in the presence of an electrical energy supply (electrical culture). In the former case, the decomposition reaction of an organic carbon source or hydrogen (H) by another microorganism can be used via an extracellular electron mediator as an electron source for promoting the microbial reaction. Furthermore, the target microorganism can simultaneously utilize the electrons from the extracellular electron mediator along with the organic carbon source and hydrogen.

[0087] In one aspect, the present invention relates to a promoter for a microbial reaction, which comprises a material containing a protein having sulfur and a β-sheet structure.

[0088] 4. Methods for promoting microbial growth In one aspect, the present invention relates to a method for promoting the growth of a microorganism, which comprises culturing the microorganism in a state in which the microorganism can be in contact with the extracellular electron transfer material of the present invention or the carrier of the present invention. The microorganism, culture, etc. are the same as those described in "3. Method for promoting a microbial reaction."

[0089] In one aspect, the present invention relates to a microbial growth promoter comprising a material containing a protein comprising sulfur and a β-sheet structure. [Example]

[0090] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0091] Hereinafter, extracellular electron transfer substances are sometimes referred to as "EETM" (Extracellular electron transfer material). Extracellular electron transfer is also sometimes referred to as "EET" (Extracellular electron transfer).

[0092] Test Example 1. Identification of extracellular electron carriers <Test Example 1-1. Silkworm cocoons and fibroin fibers> Bombyx mori silkworm cocoons were purchased. Sericin was solubilized by degumming, then dialyzed and freeze-dried. Specifically, the cocoons were boiled in Na2CO3 solution (0.05 M) at ~80°C for 4 h. The Na2CO3 solution was replaced with a new one after the third hour of boiling. The Na2CO3 solution was collected and dialyzed for 4 days using a cellulose membrane tube (tube diameter 23.8 mm, molecular weight cutoff 12–14 kDa). The collected Na2CO3 solution was then freeze-dried for 5 days to obtain water-soluble sericin. The degummed fibroin fibers were washed with tap water for 30 min and then five times with distilled water. The fibroin fibers were air-dried for 4 days and finally stored in plastic bags for further use (Figure 1).

[0093] <Test Example 1-2. EETM-dependent pentachlorophenol (PCP) dechlorination culture> A humin-dependent PCP dechlorination culture was developed and maintained for several years. In this culture, dechlorination occurred only when humin (as a solid-phase EET material extracted from soil) was added. The cocoons and fibroin fibers were confirmed to have EET function when they were able to replace humin and successfully maintain the activity of this culture. Figure 2 shows the mechanism of extracellular electron transfer in the cocoons and fibroin fibers in a humin-dependent PCP dechlorination culture, which functions as an EET-dependent PCP dechlorination culture. Each 60 mL serum bottle contained 20 mL of mineral medium (containing 0.02 mL of trace element solution), 10 mM formate, 0.2 mL of vitamin solution, 20 μM PCP, and 0.2 mM of the reducing agent titanium nitrilotriacetic acid (Ti-NTA).

[0094] Composition of mineral medium (per L): 1 g NH4Cl, 0.05 g CaCl2.2H2O, 0.1 g MgCl2.6H2O, 0.4 g HPO4, 1 mL trace element SL-10 solution, 1 mL Se / W solution, 1 mL resathrin 0.1% and 15 mM MOPS buffer (pH 7.2).

[0095] Composition of vitamin solution (per liter): biotin 2 mg, folic acid 2 mg; pyridoxine hydrochloride 10 mg, thiamine hydrochloride·2H2O, riboflavin 5 mg, nicotinic acid 5 mg, D-Ca-pantothenic acid 5 mg, cyanocobalamin 5 mg, p-aminobenzoic acid 5 mg, lipoic acid 10 mg, nicotinamide 5 mg, hemin 5 mg, 1,2-naphthoquinone 5 mg.

[0096] Composition of trace element SL-10 solution (per L): 10 mL HCl (25%), 1.5 g FeCl3·6H2O, 70 mg ZnCl2, 0.1 g MnCl2·4H2O, 6 mg H3BO3, 0.19 g CoCl2·6H2O, 2 mg CuCl2·2H2O, 24 mg NiCl2·6H2O, 36 mg Na2MoO4·2H2O.

[0097] The medium was inoculated by transferring 5% (v / v) of humin. 30 mg of cocoons, 30 mg of fibroin fibers, or 30 mg of sericin were added to the medium for testing. The cultures were grown at 30°C under dark conditions for 20 days. PCP and its metabolite concentrations were measured using a GCMS-QP2010 (Shimadzu Corporation, Kyoto, Japan). As shown in Figure 2, humin-dependent pentachlorophenol dechlorination cultures were replaced by humin, and EETMs were identified as those that could maintain PCP dechlorination activity in the culture medium.

[0098] Based on Figure 2, one may question whether a carbon source is truly necessary (since the cocoons and fibroin fibers themselves are entirely organic). To examine whether the cocoons and fibroin fibers function as a carbon source, we performed tests without adding a carbon source in EETM-dependent PCP dechlorination cultures.

[0099] <Test Example 1-3. Results regarding cocoons and fibroin> Dechlorination rate of PCP dechlorination culture ((μmol Cl - )L -1 d -1) was calculated from the metabolite concentrations during the 20-day culture period. Cultures that did not produce any PCP metabolites were determined to have a dechlorination rate of 0. The EET functions of the cocoons and fibroin fibers are shown in Figure 3.

[0100] No mediator: The absence of dechlorination activity in the mediator-free humin-dependent PCP dechlorinating culture confirmed that this culture functioned as an EETM-dependent PCP dechlorinating culture. The actual PCP concentration in this culture was 23.4 ± 1.2 μM (the original planned concentration was 20 μM as described in Materials and Methods). Based on the actual PCP concentration in the "no mediator" culture, the highest dechlorination rate achieved when all PCP was dechlorinated to produce phenol was 5.78 ± 0.3 μmol Cl - ) L -1 d -1 )

[0101] Humin: Anaerobic PCP dechlorination was observed under positive control conditions. In the EETM-dependent PCP dechlorination culture using humin (0.3 g / 20 mL), the dechlorination rate was 4.2 ± 0.3 (μmol Cl - ) L -1 d -1 ) was.

[0102] Cocoon: Culture using a cocoon (a piece of cocoon, 30 mg) yielded 4.6 ± 0.4 (μmol Cl - ) L -1 d -1 ) PCP was dechlorinated.

[0103] Fibroin fiber: Cultures using fibroin fiber (30 mg) had a mean concentration of 5.1 ± 1.7 μmol Cl - )L -1 d -1 ) PCP was dechlorinated.

[0104] These four points confirm that both the cocoon and the fibroin fiber have EET function. In other words, both the cocoon and the fibroin fiber are EETM.

[0105] Cocoon-No C-source: Cultures using cocoons (a piece of cocoon, 30 mg) without the addition of a carbon source had a yield of 3.8 ± 1.2 (μmol Cl - ) L -1 d -1 ) PCP was dechlorinated.

[0106] Fibroin fiber - No C-source: In cultures with fibroin fiber (30 mg) added without the addition of a carbon source, no PCP dechlorination activity was observed.

[0107] These two points suggest that sericin bound to the exterior of fibroin fibers can be used as a carbon source for microorganisms in the absence of other external carbon sources. Furthermore, the inactivity of the cultures with and without fibroin fibers suggests that fibroin fibers function as an EETM but not as a carbon source, i.e., the degradation of fibroin fibers by microorganisms is slow (or not decomposed within 20 days of culture).

[0108] <Test Example 1-4. Results for water-soluble sericin> The EET function of freeze-dried sericin is shown in Figure 4.

[0109] No mediator: The humin-dependent PCP-dechlorinating culture without a mediator had no dechlorinating activity, confirming that this culture functioned as an EETM-dependent PCP-dechlorinating culture.

[0110] Sericin: The culture containing 30 mg of freeze-dried sericin showed a mean concentration of 4.5 ± 4.6 μmol Cl - ) L -1 d -1 ) PCP was dechlorinated.

[0111] Sericin-Centri-insoluble: The culture to which the lyophilized sericin water-insoluble fraction (less than 30 mg) was added had a concentration of 5.5 ± 2.0 (μmol Cl - ) L -1 d -1 ) PCP was dechlorinated.

[0112] Sericin-Centri-Soluble: Cultures containing less than 30 mg of lyophilized sericin water-soluble fraction showed a mean concentration of 3.3 ± 3.6 (μmol Cl - ) L -1 d -1 ) PCP was dechlorinated.

[0113] Sericin-Filtrate: The culture containing less than 30 mg of lyophilized sericin filtrate (water-soluble) had a mean concentration of 4.3 ± 3.6 (μmol Cl - ) L -1 d -1 ) PCP was dechlorinated.

[0114] Combined with the results of Test Examples 1-3 (FIG. 3), it was suggested that sericin functions as both a carbon source and an EETM in the EETM-dependent PCP dechlorination culture.

[0115] Test Example 2. Extracellular electron transport function of different fractions of fibroin fibers <Test Example 2-1. Materials and Methods> The steps for preparing the fibroin solution and different silk fractions are described with reference to Figures 5 and 6. Finely chopped fibroin fibers were dissolved in a CaCl2:ethanol:water mixture with a molar ratio of 1:2:8 at 70-80°C. The volume of the solution was 100 mL per 15 g of fibroin fibers. The fibroin solution was then dialyzed in a cellulose membrane tube (tube diameter 23.8 mm, molecular weight cutoff 12-14 kDa) for 4 days. The distilled water was changed twice daily. Finally, the mixture was centrifuged at 12,000 × g (15 min) to obtain fibroin insoluble fraction 1 and fibroin soluble fraction 1. Fibroin insoluble fraction 1 was washed twice with distilled water, centrifuged at 12,000 × g (15 min), and then freeze-dried for 2 days.

[0116] Soluble fibroin 1 was processed in two ways. In the first processing method, fibroin soluble 1 was freeze-dried for 7 days to obtain a dried form. This solid form of soluble fibroin 1 was used in the EETM-dependent PCP dechlorination culture in this experiment. In the second processing method, soluble fibroin 1 was poured into a polyethylene bottle and stored in a refrigerator for 1 month to allow coagulation and precipitation. Centrifugation (15,000 × g, 15 min) separated the soluble fibroin 1 into two fractions: insoluble fibroin 2 and soluble fibroin 2. Insoluble fibroin 2 was then treated in the same manner as insoluble fibroin 1. soluble fibroin 2 was freeze-dried for 7 days to obtain a dried product.

[0117] These fractions (in dried form) were then tested in an EETM-dependent PCP dechlorination culture under the same culture conditions as those for the cocoon / fibroin fiber / sericin test described in Test Example 1. Each material was added at a rate of 30 mg per 20 mL of EETM-dependent PCP dechlorination culture medium and tested in the EETM-dependent PCP dechlorination culture medium.

[0118] The elemental composition (CHNS) of cocoons, fibroin fibers, and other forms of silk was determined using a PerkinElmer 2400 Series II CHNS / O analyzer (PerkinElmer Japan). Larger pieces (i.e., cocoons and fibroin fibers) were randomly cut into the smallest possible particles with scissors. All samples were pre-dried in an oven at 105 °C for one day and then placed in a desiccator overnight.

[0119] <Test Example 2-2. Results> The results are shown in FIG.

[0120] Culture using fibroin fiber described in Test Example 1 (culture 1): The dechlorination rate of this culture was 5.1 ± 1.7 (μmol Cl - ) L -1 d -1 ) was.

[0121] Culture 2 with fibroin-insoluble 1: The dechlorination rate of this culture was 4.8 ± 2.4 (μmol Cl - ) L -1 d -1 ) was.

[0122] Culture 3 with soluble fibroin: The dechlorination rate of this culture was 5.2 ± 0.7 μmol Cl - ) L -1 d -1 As mentioned in the Materials and Methods section, fibroin soluble 1 here refers to a freeze-dried solution of fibroin soluble 1. After being placed in a culture bottle, it remained solid even during the 20-day culture period. Because fibroin soluble 1 contains fibroin insoluble 2 and fibroin soluble 2 (completely soluble fraction) due to its structure, it is possible that a portion of the 30 mg of freeze-dried fibroin soluble 1 dissolved in the culture medium.

[0123] Culture 4 using fibroin-insoluble 2: The dechlorination rate of this culture was 5.0 ± 1.1 (μmol Cl - ) L -1 d -1 ) was.

[0124] Culture with soluble fibroin 2 (culture 5): No dechlorination was performed. Lyophilized soluble fibroin 2 dissolved completely after being added to the medium.

[0125] [Table 1]

[0126] Table 1 shows the elemental composition (CHNS) of cocoons, fibroin fibers, and different fractions of fibroin fibers. Fibroin fibers have a slightly higher total element content than cocoons (i.e., 44.48% C for fibroin fibers and 3.39% C for cocoons). All fractions of fibroin fibers (soluble / insoluble 1 and 2) have similar elemental compositions to fibroin fibers, except for fibroin insoluble 1. The measurement error was ±0.3%, making it difficult to compare the S content. However, XANES analysis confirmed that both materials contained sulfur in their structures (Test Example 3).

[0127] Test Example 3: Analysis of why silk has extracellular electron transfer function <Test Example 3-1. FT-IR analysis> To obtain Fourier transform infrared spectroscopy (FT-IR) spectra, a JASCO FT-IR-6100 spectrometer was used. The KBr pellet method was used. Specifically, the freeze-dried sample was ground in a mortar for 10 minutes, mixed with KBr, and then pressed with a stainless steel die set until the sample mixture became transparent. The measurement conditions were wavenumbers from 4500 to 500 cm. -1 , resolution 4cm -1 The cumulative number of measurements was 8. In this analysis, only freeze-dried fibroin insoluble 2 and freeze-dried fibroin soluble 2 were measured.

[0128] <Test Example 3-2. 13 C CP / MAS NMR analysis> Fibroin fiber, fibroin insoluble 1, fibroin soluble 1, fibroin soluble 2, fibroin insoluble 2 13C CP / MAS NMR spectra were obtained using an ECA-700 spectrometer (JEOL Ltd., Tokyo, Japan). Hexamethylbenzene was used as a standard compound to confirm the instrument's good condition. Samples were packed into zirconia sample tubes (JEOL Ltd., No. 780239954) and measured under the following conditions: relaxation time of 1 s, contact time of 2 ms, sweep speed of 70.4 kHz, resonance frequency of 176 MHz, and 80,000 scans per sample. Two sample packing methods were used: (1) air-dried fibroin fiber, freeze-dried soluble fibroin 1, freeze-dried insoluble fibroin 1, and freeze-dried soluble fibroin 2. These samples were filled into zirconia sample tubes using a pair of tweezers (approximately 2 mm of the sample tube was emptied in order to close the cap); (2) Since the freeze-dried fibroin insoluble 2 itself was in powder form after freeze-drying, the freeze-dried fibroin insoluble 2 powder was poured into the sample tube using a small stainless steel spatula.

[0129] <Test Example 3-3. SK-edge X-ray absorption structure analysis> All SK near-edge structure (XANES) spectra of silk cocoons, fibroin fibers, and other fractions were acquired at BL6N1 (0.85–6 keV) at the Aichi Synchrotron Radiation Center. Prior to pellet sample preparation for XANES analysis, the cocoons, air-dried fibroin fibers, and freeze-dried fibroin fractions (except freeze-dried fibroin insoluble 2 and freeze-dried fibroin soluble 2) were finely chopped (as small as possible) with scissors. A 5 mm diameter stainless steel die set was used to prepare the pellet samples. Briefly, the material (100%, without any other chemicals, i.e., Cu powder, boron nitride, etc.) was placed in the die set and pressed at a pressure of 500 kg per piece using a hydraulic press (PIKE Pixie 181-1400, PIKE Technologies, Inc., Wisconsin, USA). The pellet samples were then attached to a sample plate with double-sided carbon tape. The measurement conditions were as follows: He gas at 1 atmosphere, X-ray incident angle 45° relative to the surface normal. oThe measurements were performed in partial fluorescence yield (PFY) mode with an irradiation area of ​​1 × 3 mm. Energy calibration was performed using the white line position (~2841.7 eV) of the SK absorption edge spectrum of SO4. Data processing was performed using Athena software (version 0.9.26).

[0130] <Test Example 3-4. K-edge X-ray absorption structure analysis of C 1S and S 2p> C 1s and S 2p X-ray photoelectron spectroscopy (XPS) spectra of electrochemically reduced or oxidized fibroin insoluble 2 were obtained using measurements at BL 7U (0.3-0.85 keV) at the Aichi Synchrotron Radiation Center. For the measurements, samples were mixed with copper powder at a sample:Cu ratio of 1:1 (v / v) and formed into 5 mm diameter pellets. Briefly, the samples were mixed with Cu powder (75 μm, purity 99.95%) at a 1:1 (v / v) ratio by grinding in a ceramic mortar for 20 min. The mixture was then placed in a mold set and pressed with a hydraulic press (PIKE Pixie 181-1400, PIKE Technologies, Inc., Wisconsin, USA) at a force of 500 kg per mold. The pellet samples were then attached to a sample plate using double-sided carbon tape and a screw nut with a washer attached to the bottom. The XPS measurement conditions were as follows: 1.08 × 10 -8 The vacuum pressure was 0.02 Pa, the step size energy was 0.02 eV, and the X-ray incidence angle relative to the surface normal was 0. The Au 4f XAFS spectrum was used to calibrate the applied photon energy. Data processing was performed using CasaXPS software (version 2.3.15, Casa software Ltd., Teignmouth, UK).

[0131] <Test Example 3-5. Analysis of changes in the redox state of S in silk> To investigate the changes in the redox state of S in silk, two methods were performed.

[0132] Method 1: Effect of incubation in culture. Materials were compared before and after 20 days of incubation in an EETM-dependent PCP dechlorination culture. The mediator used was collected for material characterization. Ethanol (10 mL, 99.5%) was injected into the culture bottle to kill the microorganisms. Spent cocoons and fibroin fibers were agitated twice (5 min) in acetone, air-dried, and then washed several times with distilled water. Finally, air-dried. Figure 8 shows the preparation procedure for spent cocoons (fibroin-insoluble 2 is shown as an example).

[0133] Method 2: Effect of electrochemical oxidation / reduction of fibroin insoluble 2 (lyophilized sample). Fibroin insoluble 2 was electrochemically oxidized (+400 mV vs. Ag / AgCl) or reduced (-600 mV vs. Ag / AgCl) for 15 hours using an H-shaped two-chamber vessel (separated by a Nafion membrane (N117, DuPont Fuel Cells, Wilmington, DE, USA)). One chamber consisted of a Pt working electrode and an Ag / AgCl reference electrode, filled with phosphate buffer (pH 7, 0.1 M) containing 2 g / L fibroin insoluble 2. The other chamber consisted of a Pt counter electrode and a 3.3 M KCl solution. The entire two-chamber system was made anaerobic by N2 sparging for 2 hours, after which the redox treatment was carried out for 15 hours using a potentiostat (HSV-110, Hokuto Denko Corporation, Osaka, Japan). After the treatment, the oxidized / reduced fibroin insoluble 2 was collected, washed with ultrapure water (oxygen-free), and finally air-dried. This electrochemical treatment was carried out in an anaerobic glove box. Figure 9 shows an overview of the H-shaped two-chamber vessel system used for the electrochemical redox treatment of the sample.

[0134] To understand the redox state of S in the cocoons, fibroin fibers, and fibroin fractions, various reference compounds were measured. The reference compounds were lipoic acid, sulfur, cysteine, oxidized glutathione, reduced glutathione, methionine sulfoxide, sulfamic acid, sulfanilic acid, ferrozine disodium salt, 2-morpholinoethanesulfonic acid, and SO4. Because the sulfur content in the reference compounds was too high to obtain clear S K-edge XANESE spectra, all reference compounds were ground in a ceramic mortar for 20 min and diluted with boron nitride powder at a ratio of reference compound:boron nitride = 1:20 (v / v). The pellet formation procedure was the same as described above.

[0135] <Test Example 3-6. Results> The FT-IR spectra of water-soluble fibroin 2 and insoluble fibroin 2 are shown in Figure 10, and the correspondence between each absorption and the functional group is shown in Table 2.

[0136] [Table 2]

[0137] 3280, 3078, 2960, 2880, 1445, 1335, 1310 cm observed in the spectrum -1 There are differences in the FT-IR spectra of fibroin insoluble 2 (with EET function) and fibroin soluble 2 (without EET function) (Figure 10), especially in the 900–1700 cm -1 The difference in absorption indicating the β-sheet structure in this region was observed (enlarged view in Figure 10): 1620–1631 cm , which indicates Amide I (β-sheet). -1 absorption, 1220~1230cm indicating Amide III (β-sheet) -1 Absorption of -CH2(AG) n (β-sheet) 975cm -1 (A represents alanine, and G represents glycine.) These results indicate that fibroin contains a β-sheet structure when it exhibits EET function in EETM-dependent PCP-dechlorinating cultures.

[0138] Figure 11 shows the FT-IR spectra of fibroin insoluble 2 and spent fibroin insoluble 2. The two are very similar (almost identical), and no changes were observed. Spent fibroin insoluble 2 was collected after 20 days of culture in the EETM-dependent PCP dechlorination culture, washed with acetone and ethanol, and air-dried. This strongly supports the idea that fibroin insoluble 2, as described below, exhibits EETM function in repeated cultures.

[0139] fibroin 13 The results of the C CP / MAS NMR analysis are shown in Figure 12. The various amino acids contained in fibroin fiber, fibroin insoluble 1, fibroin insoluble 2, and fibroin soluble 2 were observed by C CP / MAS NMR analysis. These were alanine, glycine, serine, glutamine, tyrosine, and phenylalanine (details are shown in Figure 12). The peak at a chemical shift of 33 ppm (shown as (1) in Figure 12) indicated the presence of wax in fibroin insoluble 1. This fraction (fibroin insoluble 1) was considered insoluble due to the presence of wax. It is not possible to define the individual single peaks in the region from 105 ppm to 120 ppm. According to the literature, this chemical shift region corresponds to Tyr Cε. The shoulder peaks at 110.5 ppm (No. 2) and 112.5 ppm (No. 3) may suggest other redox states of Tyr Cε (Ar-OH and Ar=O with Ar as the aromatic ring) influenced by the surrounding local structure. 13Comparing the C CP / MAS NMR spectra, as shown in Figure 12, suggested that the material with EET function contained β-sheet structures that were not present in fibroin soluble 2 (which lacked EET function). During the incubation period of fibroin soluble 1, almost all of the β-sheets in fibroin soluble 1 aggregated to form larger particles, i.e., fibroin insoluble 2. Clearly, peaks at 20 ppm (derived from AlaC β-β sheets) and 64 ppm (derived from SerC β-β sheets) appear in the spectrum of fibroin insoluble 2 but are absent in the spectrum of fibroin soluble 2. Meanwhile, random coil-derived AlaC β (16 ppm) and SerC β (61 ppm) were detected in the spectrum of fibroin soluble 2. Furthermore, C α (55 ppm) from Ser, Gln, Tyr, and Phe were all detected only in fibroin insoluble 2 but not in fibroin soluble 2. In the chemical shift region from 105 ppm to 120 ppm, soluble fibroin 2 had only one peak at 115.5 ppm. In addition, the spectrum of insoluble fibroin 2 had two peaks, Ala Cβ (172.4 ppm) and GlyCβ (169.6 ppm), while the spectrum of soluble fibroin 2 showed GlyCβ (171.8 ppm) derived from random coil.

[0140] Sericin 13 The C CP / MAS NMR spectrum is shown in Figure 13, comparing it with that of fibroin fibers. Sericin also contains the amino acids alanine, glycine, serine, glutamine, tyrosine, and phenylalanine (also contained in fibroin fibers). A peak of AlaCβ (derived from β-sheet) at a chemical shift of 20 ppm was observed in both sericin and fibroin fibers. This strongly supports the idea that sericin also functions as an EETM. Furthermore, SerCβ (derived from β-sheet) at 64 ppm was not observed in sericin, while SerCβ derived from random coils was detected.

[0141] The results of the SK absorption edge X-ray absorption structure analysis are shown in FIGS.

[0142] The S K-edge XANES spectra of various reference compounds are shown in Figure 14. Three oxidation / reduction state regions of S were observed: reduced-S (<2475 eV, i.e., -SS-, -CS-, -SH), intermediate-S (2475 to 2480 eV, i.e., -S=O, -SO2), and oxidized-S (>2480 eV, i.e., -SO3, -SO4).

[0143] Figure 15A shows that both the cocoon and fibroin fiber contain reduced forms of sulfur (compared to the spectra of standard compounds shown in Figure 14): the cocoon shows a white line at 2472.2 eV, and the fibroin fiber shows a white line at 2472.4 eV. Comparing the S K-edge XANES spectrum of the fibroin fiber with the spectrum of cysteine / cystine (Figure 15B), the sulfur in the fibroin fiber is likely in the chemical form of -SH or -SS- bonds. Although fibroin fiber and sericin contained only trace amounts of sulfur-containing amino acids (cysteine / cystine), these amino acids may contribute to the EET function of silk. This study is the first to perform S K-edge XANES analysis of sulfur in silk (cocoon, fibroin fiber, and other soluble / insoluble fractions).

[0144] The S K-edge XANES spectra of the soluble and insoluble fractions of fibroin fibers are shown in Figure 16. Sulfur was present in a reduced state in fibroin fibers (white line at 2472.4 eV), fibroin insoluble 1 (white line at 2472.4 eV), fibroin soluble 1 (white line at 2472.8 eV), and fibroin insoluble 2 (white line at 2472.8 eV). Potential sulfur bond structures representing the white lines in this range are -SS-, -CS-, and -SH, as shown in the spectra of standard compounds. Three energy levels representing white lines were observed in the spectrum of fibroin soluble 2 (2472.8, 2475.5, and 2480.2 eV). The sulfur bond structures of fibroin soluble 2 were -SS-, -CS-, -SH (peak at 2472.8 eV), -S=O (peak at 2475.5 eV), and -SO3 (peak at 2480.2 eV). The SK-edge absorption spectrum of fibroin insoluble 1 (the most crystalline part of the fibroin fiber) was similar to that of fibroin fiber. This is thought to be due to incomplete dissolution of the fibroin fiber. Both fibroin soluble 1 and fibroin insoluble 1 supported the activity of the EETM-dependent PCP dechlorination culture. Fibroin insoluble 2 showed a spectrum similar to that of fibroin soluble 1. This is thought to be the reason why both fibroin soluble 1 and fibroin insoluble 2 possessed EETM function. The oxygenated sulfur contained in fibroin soluble 2 partially explains why this fraction did not possess EET function. This sulfur may have been oxygenated during the 1-month storage of fibroin soluble 1 (-S=O at 2475.5 eV and -SO3 at 2480.2 eV) or may have originated from some unknown S-containing structure present in fibroin soluble 1.

[0145] The SK-edge XANES spectrum of the spent cocoons was nearly identical to that of the fresh cocoons (Figure 17A). A white line was observed at 2472.2 eV. The SK-edge spectra of the fresh and spent cocoons are thought to reflect the sulfur contained in sericin. To maintain the shape of the cocoons during culture, they were not autoclaved before culture. Even after 20 days of culture, the fibroin fibers remained covered with sericin (observed). Sericin is partially consumed by the bacteria as a carbon source (as described in Test Example 1, sericin can function as a carbon source during culture). Thus, the two spectra in Figure 17A are nearly identical. This high similarity suggests that the detected sulfur is not derived from the culture medium or from the bacterial cells during sample preparation. In Figure 17B, the SK-edge spectrum of the spent fibroin fibers showed a change in the sulfur redox state compared to the fresh fibroin fibers before use. Oxygenated sulfur appeared in the spent fibroin fibers (probably -S=O at 2475.5 eV and -SO3 at 2480.2 eV).

[0146] In Figure 18A, the sulfur in fibroin insoluble 1 was present in a reduced state (white line at 2472.4 eV). Possible sulfur bond structures representing this white line were -SS-, -CS-, and -SH. The sulfur in the material after one culture use was oxygenated, as evidenced by the three white lines observed in the spectrum of spent fibroin insoluble 1 (2472.8, 2475.5, and 2480.2 eV). The sulfur bond structures in spent fibroin insoluble 1 were -SS-, -CS-, -SH (for the peak at 2472.8 eV), -S=O (for the peak at 2475.5 eV), and -SO3 (for the peak at 2480.2 eV). In Figure 18B, the sulfur in fibroin insoluble 2 was also present in a reduced state (white line energy at 2472.4 eV). The SK-edge XANES spectrum of spent fibroin insoluble 2, which had been used once in culture, showed clear differences compared to fresh fibroin insoluble 2. In addition to a white line energy peak at 2472.4 eV, there was also a white line energy peak at 2480.2 eV (-SO3). This change in the sulfur redox state suggests that S-containing functional groups contribute to the EET function of fibroin insoluble 2. It should be noted that the SK-edge spectra of spent fibroin fiber (Figure 17B) and spent fibroin insoluble 2 (Figure 18B) indicate that the white line energy peak at 2480.2 eV observed in spent fibroin insoluble 2 is due to a change in the sulfur redox state in this material, not contamination. Despite this significant change in sulfur redox state, spent fibroin insoluble 2 was still able to function as an EETM in an EETM-dependent PCP dechlorination culture (Experiment 4). This suggests that the S-containing functional groups in fibroin-insoluble 2 remain functional even after oxygenation during 20 days of culture.

[0147] Because fibroin insoluble 2 was electrochemically reduced / oxidized in phosphate buffer (HPO4 / KH2PO4), potassium contamination in the sample could not be avoided (Figure 19A). However, an important result was that identical K 2p XPS spectra were obtained for both oxidized and reduced fibroin insoluble 2. This suggests that there is no (or the same level of) spectral shift due to charge accumulation during sample measurement. Therefore, the difference in binding energy between the oxidized and reduced fibroin insoluble 2 C 1s XPS spectra in Figure 19A is likely due to the difference in the redox state of carbon. That is, the carbon in the oxidized fibroin insoluble 2 is more oxidized than that in the reduced fibroin insoluble 2 of the same material. Figure 19B shows the S 2p XPS spectrum. Due to the low S content and lower analytical sensitivity compared to XANES measurements, the S 2p XPS spectrum obtained had a higher noise level than the S K-edge XANES spectrum. However, there was a clear difference in the S 2p XPS spectra between the oxidized and reduced fibroin insoluble 2 (Figure 19B). The sulfur in the oxidized fibroin insoluble 2 was more oxidized.

[0148] From these findings, we conclude that the core functional structure in silk is a sulfur-containing β-sheet protein. Natural products that contain β-sheets and sulfur-containing functional groups in their protein structure are all expected to have extracellular electron transfer functions.

[0149] Test Example 4. Stability of extracellular electron transfer function of silk <Test Example 4-1. Material Processing> To confirm the stability of the EET function of silk materials, three methods were carried out.

[0150] Method 1: Strong UV irradiation. The cocoons and fibroin fibers were placed in a transparent tube (SuperClear Centrifuge Tube, 100% virgin polypropylene, Labcon, California, USA) and stored in a clean bench for one week. The UV lamp used in the clean bench generated UV light at a wavelength of 253.7 nm (model NEC GL-30, Hotalux, Tokyo, Japan). The distance from the UV lamp to the material was approximately 55 cm.

[0151] Method 2: Chemical Treatment (NaOH / HF). Fibroin fibers were treated using a procedure similar to that used in the humin extraction process. Briefly, they were washed six times with 0.1 N NaOH and 2% HF. The washing procedure consisted of two washes with NaOH, four with HF, two with NaOH, two with HF, and finally two with NaOH. Each wash was performed for 24 hours on a mechanical shaker, followed by centrifugation at 8000 × g for 15 minutes, after which the wash solution was replaced. This was followed by 10 washes with distilled water. The fifth wash was neutralized, and the fibers were air-dried for four days.

[0152] Method 3: Reuse of spent mediator Spent fibroin-insoluble 2 (cultured in pentachlorophenol (PCP) dechlorination culture) was recovered using a method similar to that described in Figure 8. This fraction was reused in PCP dechlorination culture to confirm whether EET function remained.

[0153] Using the materials treated by any of methods 1 to 3, the dechlorination rate was measured in the same manner as in Test Example 1. The results are shown in FIG.

[0154] <Test Example 4-2. Effect of NaOH / HF Cleaning> The dechlorination rate of the EETM-dependent PCP-dechlorinating culture using fibroin fibers was 5.1±1.7 μmol Cl - ) L -1 d -1 ) (Figure 3), and the dechlorination rate of the NaOH / HF-treated fibroin fibers was 3.9 ± 1.3 ((μmol Cl - ) L-1 d -1 ) (Figure 20).

[0155] 13 C CP / MAS NMR analysis confirmed that the carbon chemical structure of the fibroin fibers remained almost unchanged before and after NaOH / HF treatment (Figure 21), which explains why the fibroin fibers retained their EET ability even after NaOH / HF treatment.

[0156] Figure 22 shows the redox state of sulfur in fibroin fibers (white line at 2472.4 eV). The sulfur bond structures indicated by this white line are -SS-, -CS-, and -SH. The reduced state of sulfur, indicated by the white line at 2472.8 eV, was also observed after treatment with NaOH / HF. The white line at 2475.6 eV (-S=O) was more pronounced in the fibroin fibers treated with NaOH / HF. This change may explain the lower dechlorination rate of the culture treated with NaOH / HF compared to that of fresh fibroin fibers. In general, fluorinated materials are more persistent in the environment than the original materials. HF washing is known to cause fluorination of organic compounds. Visual observation revealed that the fibroin fibers treated with NaOH / HF were whiter than the intact fibroin fibers.

[0157] <Test Example 4-3. Effect of UV treatment> Cultures using UV-treated cocoons and UV-treated fibroin fibers showed weaker activity. The dechlorination rates of these cultures were 0.76±1.2 and 0.31±0.4 (μmol Cl - ) L -1 d -1) (Culture B and C in Figure 20). Strong UV rays could destroy the functional structure of both the cocoons and the fibroin fibers. With the same UV exposure period, the functional structure of the fibroin fibers (not coated with sericin on the outside) was more destroyed than that of the cocoons. It can be understood that the binding of sericin to the outside of the fibroin fibers reduces the effect of UV rays on the fibroin fibers.

[0158] The SK-edge XANES spectra in Figure 23 show the change in the sulfur redox state between fresh and UV-treated fibroin fibers. As shown in Figure 24, a color change in the cocoons and fibroin fibers due to UV treatment was observed.

[0159] Prolonged (7 days) irradiation with strong UV light changed the structure of the fibroin fibers (Figure 23). The increase in the relative intensity of the white lines at 2475.6 eV and 2480.2 eV in the SK absorption edge spectrum of UV-treated fibroin fibers suggests the oxygenation of sulfur in the structure (-S=O and -SO3, respectively).

[0160] <Test Example 4-4. Recycled fibroin insoluble 2> After one incubation in the PCP dechlorination culture, fibroin-insoluble 2 functioned well even when reused in a new culture. The dechlorination rate of the EETM-dependent PCP dechlorination culture using fresh fibroin-insoluble 2 was 5.0 ± 1.1 (µmol Cl). - ) L -1 d -1 ) (Figure 7), and the used fibroin insoluble 2 was 5.2 ± 0.6 ((μmol Cl - ) L -1 d -1) (Figure 20). This suggests the possibility of using silk waste for this purpose (as EETM). FT-IR spectra (Figure 11) showed that there was almost no change in functional groups between fresh and used fibroin insoluble 2 (Test Example 3). Similarly, the difference in the redox state of sulfur was examined with reference to Figure 18B (Test Example 3).

[0161] Test Example 5. Silk as an EETM in other microbial cultures <Test Example 5-1. Fe (III) reducing culture> Humin-dependent PCP dechlorination cultures have been shown to have the ability to reduce Fe(III). Therefore, we used this culture as an inoculum to examine the EET function for Fe reduction in cocoons and fibroin fibers. The medium composition was identical to that of EETM-dependent PCP dechlorination cultures, except that 20 μM PCP was replaced with 4 mM amorphous Fe(III) oxide (FeOOH). Cultures using these materials confirmed that cocoons and fibers possessed EET function when they produced higher Fe(II) levels than mediator-free cultures up to the third generation. Because this culture was not mediator-dependent, higher Fe(II) production indicated EETM function. Fe(II) concentrations were measured by the phenanthroline method at 510 nm using a spectrophotometer (U-1900, Hitachi, Tokyo, Japan). For colorimetric measurement, 1 mL of sample (filtered through a membrane filter with a pore size of 0.2 μm) was mixed with 1 mL of 10% hydroxylammonium chloride, 1 mL of 0.2% o-phenanthroline, and 2.5 mL of acetate buffer.

[0162] <Test Example 5-2. CO2 reduction culture> CO2-reducing cultures using humin as a mediator were maintained by subculturing at 14-day intervals. Each 60 mL serum bottle contained 20 mL of mineral salts medium (containing 0.02 mL of trace element solution), 0.2 mL of vitamin solution, 0.2 mM Ti-NTA, and test material (30 mg of cocoons or 30 mg of fibroin fibers). The pH was adjusted to 7.0 ± 0.2 by bubbling the medium in the sealed culture bottle with a 4:1 N2:CO2 mixture for 1 hour. The headspace was then replaced with a 4:1 H2:CO2 mixture for 15 minutes.

[0163] Composition of mineral salts medium (per L): NH4Cl 1g, CaCl2·2H2O 0.05g, MgCl2·6H2O 0.1g, K2HPO4 4g, NaHCO3 4g, trace element SL-10 solution 1mL, Se / W solution 1mL, and Resuslin 0.1% 1mL.

[0164] Composition of trace element SL-10 solution (per L): 10 mL HCl (25%), 1.5 g FeCl3·6H2O, 70 mg ZnCl2, 0.1 g MnCl2·4H2O, 6 mg H3BO3, 0.19 g CoCl2·6H2O, 2 mg CuCl2·2H2O, 24 mg NiCl2·6H2O, 36 mg Na2MoO4·2H2O.

[0165] Composition of vitamin solution (per liter): Biotin 2 mg, Folic acid 2 mg; Pyridoxine hydrochloride 10 mg, Thiamine hydrochloride 2H2O, Riboflavin 5 mg, Nicotinic acid 5 mg, D-Ca-Pantothenic acid 5 mg, Cyanocobalamin 5 mg, P-Aminobenzoic acid 5 mg, Lipoic acid 10 mg, Nicotinamide 5 mg, Hemin 5 mg, 1,2-Naphthoquinone 5 mg.

[0166] <Test Example 5-3. N fixed culture> <Test Example 5-3-1. Accumulation of N-fixed consortium> The function of EETs for promoting N fixation in cocoons and fibroin fibers was confirmed using previously enriched N-fixing anaerobic cultures in modified nitrogen-deficient Ashby (MNDA) medium. The composition of MNDA medium was mannitol (20 g L), KHPO (0.2 g L), and HCl (0.1 g L). -1 ), MgSO4·7H2O (0.2g L -1 ), NaCl (0.2g L -1 ), K2SO4 (0.1g L -1 ), CaCO3 (5g L -1 ) 20 mL of MNDA medium was placed in a 50 mL culture vial and sealed with a butyl rubber stopper and aluminum cap. The medium in the culture vial was bubbled with N2 gas, and the headspace was flushed with N2 gas to create an anaerobic condition. 2 mL of the enriched consortium was inoculated into the prepared anaerobic medium. Three strains were subcultured to enrich for nitrogen-fixing microbial communities: a fibroin fiber culture using MNDA medium supplemented with 1.5 g / L fibroin fiber, a cocoon culture using MNDA medium supplemented with 1.5 g / L cocoons, and a mediator-free culture using MNDA medium alone. Each enrichment culture was tested in triplicate. After inoculation, all cultures were statically cultured at 30°C for 2 weeks. After incubation, N fixation activity was measured using the acetylene reduction assay (ARA).

[0167] <Test Example 5-3-2. Acetylene reduction assay> A commonly used method for confirming the N fixation activity of N-fixing cultures is the ARA. This was performed using 10 ml glass vials. MNDA medium bubbled with helium gas was used for the ARA. Two sets of 10 ml glass vials containing 2 ml of MNDA medium bubbled with helium gas were prepared anaerobically (one set supplemented with 10 mg of cocoon / fibroin fiber per vial, the other set without any silk material). Two ml of each consortium was then inoculated into two sets of vials, and 200 μL of acetylene gas was injected as a substrate. The cultures were incubated at 30°C for 3 days under static conditions. After 3 days of incubation, 100 μL of gaseous sample was collected from the headspace using a gas-tight syringe (Pressure-Lok®, VICI Precision Analytical Syringe, Baton Rouge, LA, USA). The concentrations of acetylene and ethylene in the samples were measured using a GC-14B (Shimadzu Corporation, Kyoto, Japan) equipped with a molecular sieve 5A column (60 / 80 mesh, 3 m long) and a Porapak N column (50 / 80 mesh, 2 m long) connected in series and a flame ionization detector.

[0168] <Test Example 5-4. Results> The addition of cocoons and fibroin fibers increased Fe(II) concentrations compared to mediator-free cultures (Figure 25), suggesting the EET function of cocoons and fibroin fibers. Note that, unlike mediator-dependent PCP dechlorination cultures, the Fe(III) reduction process occurred even in the absence of mediator.

[0169] The effects of cocoons and fibroin fibers in CO2-reduced culture are shown in Figure 26. Both cocoons and fibroin fibers produced more acetic acid in CO2-reduced culture than cultures without mediator (Control 1). This indicates that they exerted EET function in the reaction of CO2 reduction to acetic acid production. Furthermore, when cultures were performed using the same amounts of cocoons and fibroin fibers (30 mg / piece), the culture using fibroin fibers produced more acetic acid, suggesting that the EET function for this reaction is primarily present in the fibroin fibers, not sericin.

[0170] Figure 27 shows that the addition of cocoons and fibroin fibers to the test medium significantly (p<0.05) promoted ARA. However, the effect of subculture using cocoons and fibroin on microbial enrichment was smaller than the effect of adding cocoons and fibroin fibers on ARA. This trend was consistent across all three generations of cultures tested. The promotion of ARA by the addition of cocoons and fibroin fibers suggests that both cocoons and fibroin fibers acted as electron mediators to promote ARA. When tested without the addition of fibroin fibers, no significant difference in ARA results was observed between cultures enriched without mediator addition and those enriched with fibroin fibers (Figure 27B). This is likely due to the lack of differences in microbial community structure between the two cultures over three generations of enrichment. However, the microbial cultures enriched using cocoons (cocoon cultures) showed a slightly higher ARA compared to cultures without a mediator (Figure 27A). In conclusion, both the cocoons and fibroin fibers functioned as electron mediators, promoting the ARA of the cultures.

[0171] The functions of silk (cocoons and fibroin fibers) demonstrated above are shown in Figure 28. Dehalogenation and iron reduction are useful for environmental bioremediation, etc. Reduction of CO2 to acetic acid is useful for curbing global warming and producing chemicals from CO2, etc. N2 fixation is useful in the fertilizer manufacturing industry, etc.

[0172] Test Example 6: Fibroin sponge / fibroin-coated sponge <Test Example 6-1. Materials and Methods> The fibroin soluble 1 solution used in this study is shown in Figure 6. Immediately after dialyzing and centrifuging this fibroin soluble 1 solution at 12,000 × g (15 min), fibroin sponges and fibroin-coated sponges were prepared from this fibroin soluble 1 solution. Initially, 15 g of fibroin fiber was dissolved in 100 mL of solution (CaCl2:ethanol:water = 1:2:8 molar ratio). Fibroin insoluble 1 (the insoluble portion of the fibroin fiber) accounted for approximately 5% of the total weight of the fibroin fiber. Therefore, the concentration of fibroin soluble 1 was estimated to be 14.25% (wt / vol). At this state, the fibroin soluble 1 solution was at room temperature and considered a fresh solution.

[0173] Four types of sponges were prepared using the following procedure (Figure 29).

[0174] First sponge (100% fibroin sponge). Step 1: The soluble fibroin 1 solution was poured into a mold PTFE container (1 x 1 x 5 cm, a container wrapped in a PTFE sheet) until it was about half full. Step 2: NaCl particles were then added until the mold volume was full. The mold was left to stand in a desiccator for 4 days. Step 3: The sponge-like contents were removed from the mold with a small stainless steel spatula and submerged in a beaker containing 1 L of distilled water to dissolve and remove the NaCl, yielding a 100% fibroin sponge (the distilled water was replaced with fresh water six times over two days). Step 4: The sponge was allowed to air dry for two days.

[0175] The second sponge (fibroin-coated polyurethane sponge) and the third sponge (fibroin-coated sea sponge) were prepared. Polyurethane (PU) sponges or sea sponges were cut into cubes (0.5 × 0.5 × 0.5 cm). The fibroin soluble solution 1 used to prepare the coated sponges was the same as that used to prepare the fibroin sponges described above. The coating process was as follows: Step 1: A 200 mL beaker containing 20 mL of fibroin soluble solution 1 and the sponge to be coated were prepared. Step 2: The PU sponge or sea sponge was immersed in the fibroin soluble solution 1 solution by hand (wearing laboratory gloves) for 5 minutes. The PU sponge or sea sponge was then removed, pressed firmly with the hand to remove excess fibroin solution, and air-dried for 12 hours. Step 3: Step 2 was repeated once more, followed by air-drying. Step 4: The fibroin-coated PU sponge or fibroin-coated sea sponge was stirred in distilled water for 15 minutes to remove any potential soluble fibroin. Step 5: Air-dried for 2 days. The fibroin-coated sponges were named fibroin-coated PU sponge and fibroin-coated sea sponge, respectively.

[0176] The fourth sponge (fibroin-coated raw cotton sponge) was prepared by purchasing raw cotton and manually removing unnecessary parts such as seeds and skin. The raw cotton was used as is without cutting. The fibroin coating process was carried out using the same procedure as for the second and third sponges. The resulting sponge was named the fibroin-coated raw cotton sponge. When used in testing, it was cut into pieces according to the required amount.

[0177] These sponges were tested in EETM-dependent PCP dechlorination cultures. The culture conditions were the same as those described in Test Example 1. For fibroin sponges (100% fibroin): one sponge was used per culture bottle. For fibroin-coated PU sponges and fibroin-coated sea sponges: two sponges were used per culture bottle. 20 mg of fibroin-coated raw cotton sponges were used per culture bottle.

[0178] To examine the stability of the EET-functionalized sponges, used sponges were collected from old cultures (FIG. 30), washed, and reused in new cultures.

[0179] In Experiment 3, we confirmed that sulfur-containing functional groups contribute to the EET function of cocoons, fibroin fibers, and the soluble fraction of fibroin. Furthermore, to understand the changes in the sulfur redox state in these materials, we performed S K-edge XANES analysis of fibroin sponges and used fibroin sponges. The cleaning procedure for used fibroin sponges was the same as that described in Figure 30.

[0180] <Test Example 6-2. Results> The amount of fibroin in each sponge was as follows: 100% fibroin sponge (0.5 x 0.5 x 0.5 cm): Contains 18.4 ± 0.9 mg of fibroin. Fibroin-coated PU sponge (0.5 x 0.5 x 0.5 cm): Contains 3.5 ± 0.3 mg of fibroin. Fibroin-coated sea sponge (0.5 x 0.5 x 0.5 cm): Contains 2.1 ± 0.5 mg of fibroin. Fibroin-coated raw cotton sponge (20 mg): Contains 10.6 ± 1.0 mg of fibroin.

[0181] The EET function of each prepared sponge in the EETM-dependent PCP dechlorination culture is shown in FIG.

[0182] The results for the 100% fibroin sponge were as follows: The dechlorination rate for the culture using the 100% fibroin sponge (18.4±0.9 mg fibroin) was 4.9±2.0 ((μmol Cl - ) L -1 d -1 ) (Figure 31). The dechlorination rate of the culture using 30 mg of fibroin fiber was 5.1 ± 1.7 ((μmol Cl - ) L -1 d -1) (Figure 3). Comparing the weight of EETM in the culture (18.4 mg for fibroin sponge and 30 mg for fibroin fiber) and the similarity in the dechlorination rates between the culture-treated fibroin sponge and the culture-treated fibroin fiber suggested two possible reasons: (1) approximately 40% of the fibroin fiber weight contains S-containing β-sheets for EET function (Example 7), or (2) the porous structure of the fibroin sponge helps promote the adherent growth of bacteria.

[0183] The results for the fibroin-coated PU sponges are as follows: The culture using a PU sponge (without fibroin coating) was inactive (Sponge 2, Figure 31). For this culture, two PU sponges (each sponge: 0.5 x 0.5 x 0.5 cm) were used in one culture bottle. In the culture using two fibroin-coated PU sponges of the same size (Sponge 3, Figure 31), the dechlorination rate was 3.2 ± 0.4 (µmol Cl - ) L -1 d -1 The total amount of fibroin added to the cultures was 7.0 ± 0.6 mg for both sponges. The dechlorination rate was lower than that of the 100% fibroin sponge, but the dechlorination rate per unit mass of fibroin was higher for the fibroin-coated PU sponge.

[0184] The results for the fibroin-coated sponges are as follows: The culture using a sponge (without fibroin coating) was inactive (Sponge 4, Figure 31). For this culture, two sponges (cut into 0.5 x 0.5 x 0.5 cm pieces) were used in one culture bottle. When two fibroin-coated sponges of the same size (Sponge 5, Figure 31) were cultured, the dechlorination rate was 4.8 ± 0.7 (µmol Cl - ) L -1 d -1 The total amount of fibroin added to the culture was 4.2±1.0 mg for two pieces. The dechlorination rate per unit mass of fibroin was higher for the fibroin-coated sea sponge than for the fibroin-coated PU sponge.

[0185] The results for the fibroin-coated raw cotton sponge are as follows: There was no activity in the culture with 20 mg of raw cotton (without fibroin coating) added (Sponge 6, Figure 31). On the other hand, when 20 mg of fibroin-coated raw cotton sponge (Sponge 7, Figure 31) was used for the culture, the dechlorination rate was 3.9 ± 2.4 (µmol Cl - ) L -1 d -1 ) One 20 mg piece of fibroin-coated raw cotton sponge was added to the culture, containing 10.6 ± 1.0 mg of fibroin. The dechlorination rate per unit mass of fibroin was lower than that of the fibroin-coated PU sponge and the fibroin-coated sea sponge, but higher than that of the 100% fibroin sponge. This is thought to be because the use of the coated sponge increases the surface area per unit mass of fibroin, thereby increasing the surface area for microbial attachment.

[0186] The stability of the EET function of the fibroin-coated PU sponge was evaluated by a reuse test, and the results are as follows: The dechlorination rate of the culture using the fibroin-coated PU sponge was 3.2 ± 0.4 (μmol Cl - ) L -1 d -1 After culturing this culture for one cycle (20 days), the fibroin-coated PU sponge was collected / washed (Figure 30) and reused in the EETM-dependent PCP dechlorination culture, resulting in a dechlorination rate of 3.2 ± 0.3 ((μmol Cl - ) L -1 d -1 ). The results in Figure 32 suggest that the EET function of the fibroin-coated PU sponge is maintained even after one culture. These results also suggest that the fibroin-coated PU sponge degrades slowly (it does not degrade within 40 days (the total of the first and second culture periods) or degrades sufficiently slowly). Based on the above, the advantages of using a fibroin sponge and a fibroin-coated PU sponge are summarized in Figure 33.

[0187] The redox state of S in the fibroin sponge before and after use was as follows. The redox state of S in the fibroin sponge (100% fibroin) was reduced (white line energy 2472.8 eV, -SS-, -CS-, -SH) (Figure 34A). Once incubated in the EETM-dependent PCP dechlorination culture, a portion of S transitioned to an intermediate redox state (2475.6 eV, -S=O) (Figure 34B). The K-edge absorption spectrum of S was almost identical to that of soluble fibroin 1 (Figure 34A). This reflects the fact that the fibroin sponge was prepared from soluble fibroin 1. This suggests the following two points: (1) fibroin sponges, like soluble fibroin 1, should (or must) possess EET functions; and (2) the sulfur in soluble fibroin 1 is not easily oxygenated by air. The change in the redox state of the fibroin sponge before and after incubation (Figure 34B) suggests that sulfur-containing functional groups are involved in EET function, further supporting the first point.

[0188] Test Example 7. Comparative material as an extracellular electron transfer material <Test Example 7-1. Materials and Methods> Here, we hypothesized that materials with functional structures containing β-sheets and S possess EET functions, and conducted experiments using several materials. The test conditions using an EETM-dependent PCP dechlorination culture were the same as in Test Example 1.

[0189] The first was amino acids. Based on the amino acid composition in fibroin fibers, commercially available amino acids were purchased from Sigma-Aldrich and individually tested. These were cystine, cysteine, serine, threonine, alanine, tyrosine, and glycine. 3 mg of each amino acid was used in a culture bottle. Two culture bottles (duplicate) were cultured.

[0190] The second is peptides. Three short peptides (purchased from Sigma-Aldrich) were tested: glycine-tyrosine, tyrosine-tyrosine, and phenylalanine-tyrosine. These were tested separately. 3 mg of each peptide was used per culture bottle. Duplicate culture bottles were prepared.

[0191] The third sample was oysters. Oysters were purchased from Amazon. They were stored overnight at room temperature to thaw. The shells and meat were then separated and washed five times with distilled water. These were placed in separate mortars and dried in an oven at 105°C for two days. The dried oyster shells were then crushed into small pieces with a hammer. The dried oyster meat was then attached to the mortar after drying. A stainless steel spatula was used to scrape the oyster meat from the mortar. This resulted in a dried oyster meat powder (powder with a particle size distribution). Both materials were tested separately. 30 mg of each material was used per culture bottle. Three culture bottles were cultured for each material (triplicate cultures).

[0192] The fourth is human hair. The question here is whether a material containing high S-containing functional groups but lacking β-sheets in its structure still possesses EET function. Human hair was chosen to test this question. It contains 4.3 to 6.7% sulfur in its structure, and its protein structure is primarily α-keratin. Undyed hair was cut into small particles with scissors and directly used for culture. 30 mg of hair was used per culture bottle. Two culture bottles (duplicates) were cultured.

[0193] The fifth group is humin and humic acid, which are known to possess EET functions. Previously, it was suggested that the binding of proteins with lignin and peptidoglycan is the pathway for the formation of humic substances (humic substances are divided into acid-alkali insoluble humin, alkali-soluble humic acid, and acid-alkali soluble fulvic acid based on their solubility in acidic and alkalinity). Furthermore, recent studies have suggested that in nature, humic substances encapsulate proteins to preserve their function. Humin (as a solid-phase mediator) has been reported to possess EET functions in PCP dechlorination cultures. Humic acid (as a soluble mediator) did not support the activity of PCP-dechlorination cultures, but insoluble Fe-humic acid complexes did. In this study, the initial pH conditions were adjusted to 4, using the previously reported conditions for preparing Fe-humic acid complexes. Fe 2+ The mixture containing Sigma-Aldrich humic acid was incubated at 30°C in the dark for 7 days without pH adjustment. The resulting precipitate was collected by centrifugation (15,000 × g, 15 minutes), washed with distilled water, neutralized, and freeze-dried. Humin and iron-humic acid were tested separately. 0.3 g of humin or 0.1 g of Fe-humate was used per culture bottle. Three culture bottles (three replicates) were cultured for each material.

[0194] <Test Example 7-2. Results> Based on previous reports, information on the β-sheet content of the samples is as follows: Fibroin fiber: 50% (Non-Patent Document 1: Measurements by polarized Raman microscopy). This information is consistent with the discussion of the fibroin sponge in Test Example 6 (comparison of the weight of the EETM during culture (18.4 mg of fibroin sponge and 30 mg of fibroin fiber) and the similarity in the dechlorination rate of the 100% fibroin sponge and fibroin fiber used for culture may indicate that approximately 40% of the weight of the fibroin fiber contains S-containing β-sheets for EET function). Oyster body: adductor muscle protein beta-sheet content 11.2%, visceral protein beta-sheet content 18.4%. · Oyster shell: No beta sheet content. Human hair: no β-sheet content. Figure 35 shows the PCP dechlorination rate of EETM-dependent PCP dechlorination cultures supplemented with each material. Cultures supplemented with amino acids or peptides had no activity in any of the cultures. Oyster shells did not support the activity of the cultures, but oyster bodies (after drying in an oven at 105°C for 2 days) strongly supported the microbial dechlorination process (dechlorination rate 5.6 ± 1.0 (µmol Cl - ) L -1 d -1 Human hair (mainly α-keratin, high S content) did not support the activity of this culture. This means that to function as an EETM, it is necessary to incorporate both β-sheet and S-containing functional groups. Humin: Dechlorination rate 4.2±0.3 ((μmol Cl - ) L -1 d -1 Iron-humate 1 (no cysteine ​​added during the complexation process): Dechlorination rate 1.9±0.5 (μmol Cl - ) L -1 d -1 ).

[0195] Test Example 8. Coating on electrodes The fibroin soluble 1 solution used in this study is shown in Figure 6. After dialysis and centrifugation at 12,000 × g (15 min), fibroin-coated electrodes were fabricated from this fibroin soluble 1 solution. First, 15 g of fibroin fibers were dissolved in 100 mL of chemical solution (CaCl2:ethanol:water = 1:2:8 molar ratio). Fibroin insoluble 1 (the insoluble portion of the fibroin fibers) accounted for approximately 5% of the total weight of the fibroin fibers. Therefore, the concentration of the fibroin insoluble 1 solution was estimated to be 14.25% (wt / vol). In this state, the fibroin soluble 1 solution was at room temperature and considered a fresh solution. A carbon plate (Niraco, Tokyo, Japan) measuring 5 × 2.5 × 0.1 cm was prepared. This carbon plate was immersed in 1 N hydrochloric acid for 4 h, then washed with tap water for 30 min and then washed 10 times with ultrapure water before use.

[0196] The coating procedure was as follows: A 100 mL beaker containing 50 mL of fibroin soluble solution 1 was prepared. It was then immersed in 1N hydrochloric acid for 4 hours at room temperature under normal aerobic conditions, and then washed with tap water for 30 minutes. A carbon plate was immersed in fibroin soluble solution 1 for 5 minutes. The carbon plate was then rotated upside down for another 5 minutes. The fibroin-coated carbon plate was then retrieved with tweezers and held vertically over the beaker for 2 minutes to remove excess fibroin. Finally, the fibroin-coated carbon plate was air-dried for 2 days.

[0197] Test Example 9: Carbon dioxide fixation using a bioelectrochemical system using fibroin <Test Example 9-1 Materials and Methods> An H-shaped two-compartment culture vessel separated by a Nafion membrane (N117) was used (Figure 37). The cathode chamber contained a reference electrode (Ag / AgCl), a working electrode (platinum, 0.8 mm diameter x 1 m length), 200 mL of culture medium, a stir bar, and 0.3 g of fibroin-insoluble 2. The reference electrode was capped with a tube filled with 3.3 M KCl solution. The anode chamber contained a counter electrode (platinum, 0.8 mm diameter x 1 m length) and a stir bar in 220 mL of culture medium. The composition of the culture medium (per L) was 0.35 g K2HPO4, 0.23 g KH2PO4, 1 g NH4Cl, 0.05 g CaCl2.2H2O, 0.1 g MgCl2.6H2O, 4 g NaHCO3, 1 mL of trace element SL-10 solution, and 1 mL of Se / W solution.

[0198] The Nafion membrane, platinum electrode, H-shaped two-chamber fermenter, and other components used here were pretreated as follows. The Nafion membrane was cut into 4.5 cm x 4.5 cm pieces and treated as follows: First, it was treated with 3% hydrogen peroxide at approximately 80°C for 1 hour, then gently boiled in 0.5 M sulfuric acid for 1 hour, rinsed with ultrapure water, boiled in ultrapure water at 80°C for 0.5 hours, rinsed with ultrapure water, and stored in ultrapure water. The platinum electrode was treated as follows: It was soaked in 80% sulfuric acid for 12 hours, rinsed with tap water for 30 minutes, and ultrasonically cleaned in ultrapure water for 10 minutes. Next, it was soaked in 2N NaOH solution for 4 hours, rinsed with tap water for 30 minutes, ultrasonically cleaned in ultrapure water for 30 minutes, and finally rinsed with ultrapure water. The H-type two-tank culture tank and the stirrers placed therein were washed with ultrapure water and then sterilized in an autoclave at 121°C for 15 minutes.

[0199] After assembling the bioelectrochemical system, a mixture of nitrogen and carbon dioxide (N2:CO2 = 50:50) was bubbled in for 2 hours to create anaerobic conditions. Next, 20 mL of carbon dioxide-reducing culture was added to the cathode chamber (approximately 10% inoculation). This culture was maintained in the laboratory as a carbon dioxide-reducing culture using humin obtained from Kamashima soil as EETM. After inoculation, the cathode chamber turned slightly pink. This is likely due to the resazurin contained in the inoculated culture being added together with the residual oxygen in the bioelectrochemical system without the addition of a reducing agent. The pink color disappeared approximately half a day after the bioelectrochemical system was started. The culture conditions were as shown in Table 3 below. The culture was performed at 30°C, with the stirrer rotating only in the cathode chamber (fibroin insoluble 2 was in suspension). Under closed-circuit conditions, the redox potential of the cathode chamber was set to -600 mV (relative to the hydrogen standard electrode) using a potentiostat (HSV-110, Hokuto Denko Inc., Osaka, Japan). After 24 and 48 hours of culture, 250 μL of gas was collected from the headspace and measured for hydrogen and methane using a gas chromatograph equipped with a differential calorimeter and a flame ionization detector. Culture medium was collected at the start of culture and after 48 hours of culture and subjected to acetic acid analysis. 0.3 mL of the collected culture medium was filtered through a 0.22 μm pore size membrane filter and diluted with 0.9 mL of ultrapure water (1 / 4 concentration) and analyzed by high-performance liquid chromatography (Shimadzu, LC-10AT, Kyoto, Japan). A 6 mM HClO4 solution was used as the mobile phase. Organic acids were detected at UV 440 nm using a post-column method with 0.1 mM bromothymol blue / 30 mM Na2HPO4 solution.

[0200] [Table 3]

[0201] <Test Example 9-2. Results> Figure 38 shows the time course of hydrogen and methane and the amount of acetic acid produced after 48 hours.

[0202] Condition 1 (with fibroin insoluble 2, with microbial inoculation, closed-circuit culture at an oxidation-reduction potential of -600 mV (based on the standard hydrogen electrode)): Hydrogen generation, thought to be from water electrolysis, was observed in the system. After 24 hours, the amount of hydrogen in the cathode chamber reached 17.16 μmol. This is lower than the amount of hydrogen generated in condition 3, likely because it was used for methane and acetic acid production. After 24 hours, 1.7 μmol of methane was produced. After 48 hours, this amount decreased to 1.14 μmol. 10.8 μmol of methane and 120.1 μmol of acetic acid were produced. Most of the hydrogen is thought to have been consumed by the microorganisms. (4 moles of hydrogen are required to produce 1 mole of methane by microorganisms, and 4 moles of hydrogen are required to produce 1 mole of acetic acid.)

[0203] Condition 2 (no fibroin insoluble 2, with microbial inoculation, oxidation-reduction potential -600mV (closed circuit culture based on standard hydrogen electrode): After 24 hours, 11.77 μmol of hydrogen was produced (similar to condition 1). 0.8 μmol of methane was detected, suggesting hydrogen utilization by the inoculated microorganisms. After 48 hours, the amount of hydrogen produced increased to 22.82 μmol. 1.02 μmol of methane was detected, but no acetic acid production was observed. This indicates that, unlike condition 1, the amount of hydrogen utilized by microorganisms was small.

[0204] Condition 3 (with fibroin insoluble 2, no microbial inoculation, oxidation-reduction potential -600mV (closed circuit culture based on standard hydrogen electrode): The amount of hydrogen produced was 56.64 μmol in 24 hours and 74.26 μmol in 48 hours. No production of methane or acetic acid was observed.

[0205] Condition 4 (with fibroin-insoluble 2, with microbial inoculation, open-circuit culture): After 48 hours, no hydrogen or acetic acid production was observed. Very little methane production was detected (0.001 μmol).

[0206] Comparing the results of the bioelectrochemical systems operated under the above four conditions, it was shown that insoluble fibroin 2 functioned as an EETM for the microbial reaction of carbon dioxide reduction in the bioelectrochemical system. Based on the amounts of methane and acetic acid produced, it is interpreted that a greater amount of hydrogen (or the equivalent electrons) than the detected hydrogen was supplied to the microbial carbon dioxide reduction reaction via insoluble fibroin 2. The hydrogen generation under condition 3 is thought to be due to the electrolysis of water. The decrease in the generation rate after 48 hours compared to 24 hours is thought to be due to an increase in pressure in the cathode chamber or inhibition by oxygen generation in the anode chamber.

Claims

1. An extracellular electron transfer material comprising a material containing a protein having sulfur and a β-sheet structure, The protein is at least one selected from the group consisting of fibroin protein and sericin protein, and / or The material is a biological material, and the biological material is at least one selected from the group consisting of silk thread, spider silk, oyster meat, and materials derived therefrom; Extracellular electron transporter.

2. The sulfur bond structure in the protein is -Fe-S-, -S-S-, -C-S-, -S-H, -SO-, -SO 2 - and - SO 3 The extracellular electron transfer material according to claim 1, which is at least one selected from the group consisting of:

3. 3. The extracellular electron transfer material according to claim 1, wherein the content of the β-sheet structure in the protein is 2% by mass or more.

4. The extracellular electron transfer material according to any one of claims 1 to 3, wherein the material is a water-insoluble material or a water-soluble material.

5. A carrier comprising the extracellular electron transfer material according to any one of claims 1 to 4.

6. The support according to claim 5, which is porous.

7. The support according to claim 5 or 6, which is an electrode or an electrode material.

8. 8. The support of claim 7 for use in a bioelectrochemical system or a microbial fuel cell.

9. The carrier according to any one of claims 5 to 8, which is a carrier for cultivating microorganisms.

10. A method for promoting a microbial reaction, comprising culturing a microorganism in a state in which the microorganism can be in contact with the extracellular electron transfer material according to any one of claims 1 to 4 or the carrier according to any one of claims 5 to 9.

11. 11. The method according to claim 10, wherein the microbial reaction is at least one selected from the group consisting of a dehalogenation reaction, a metal oxidation-reduction reaction, a metalloid oxidation-reduction reaction, a denitrification reaction, a nitrate reduction reaction, a carbon dioxide fixation reaction, a methane production reaction, a hydrogen production reaction, a sulfate reduction reaction, a nitrogen oxide fixation reaction, and a nitrogen fixation reaction.

12. A method for promoting the growth of microorganisms, comprising culturing microorganisms in a state in which they can come into contact with the extracellular electron transfer material according to any one of claims 1 to 4 or the carrier according to any one of claims 5 to 9.

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