Construction and application of conductive biofilm

Abstract

The present invention provides a conductive biofilm comprising a microorganism having cilia or pili and a conductive polymer bound to the cilia or pili. The present invention also provides a composite comprising a photocatalyst layer and a biofilm comprising a microorganism having cilia or pili. The invention also provides an artificial Z-some system. The artificial Z-some system comprises a photocatalyst layer and the conductive biofilm provided by the invention.

Description

Technical Field

[0001] This invention relates to bioengineering and biomaterials. Specifically, it relates to conductive biomembranes, their application in transferring electrons in Z-scheme systems, and composite Z-scheme systems comprising said conductive biomembranes. Background Technology

[0002] Plants or algae convert light energy into chemical energy through photosynthesis, a reaction that utilizes a "Z-scheme" configuration for electron transfer. Photosystem II absorbs solar energy to generate electron / hole pairs; the holes oxidize water into oxygen, while photosystem I transfers electrons through redox protein clusters to the final electron acceptor—ferroredoxin. Inspired by nature, researchers have proposed using inorganic semiconductors to simulate nature and construct an artificial Z-scheme photosynthesis system, mimicking the natural use of light energy to drive the production of high-value-added chemicals. Artificially synthesized semiconductor materials possess excellent visible light absorption capabilities, potentially overcoming the efficiency limitations of natural photosynthesis. The artificial Z-scheme system requires two photocatalysts and an electron transfer material.

[0003] The electron transfer materials used in the Z-scheme water splitting catalyst in the prior art need to meet three key conditions: (1) achieving close contact between the hydrogen production catalyst (HEP) and the oxygen production catalyst (OEP); (2) excellent conductivity; and (3) strong stability. Electron mediators are the most commonly used electron transfer materials, but they have poor stability and their performance is strongly dependent on pH. Moreover, ionic electron mediators can lead to reverse reactions. Reduced graphene oxide (rGO), indium tin oxide nanoparticles (np-ITO), Ir nanoparticles, In@InOx particles and other solid conductive materials have also been used as electron mediator materials. However, the binding force between these conductive materials and the catalyst is not strong, which affects the stability and activity of the overall structure. Au layers can be prepared by vacuum evaporation and used as electron mediators. However, this method requires expensive equipment and is difficult to prepare on a large scale. Existing artificial systems do not have the living characteristics of natural materials such as self-growth and self-regeneration, which affects the sustainability and practical application of the system. Therefore, the preparation of sustainable, efficient and scalable conductive materials is still a major challenge in this field.

[0004] In artificial Z-scheme systems, existing electron transport materials face challenges such as cumbersome preparation, reverse reactions, use of precious metals, and poor sustainability.

[0005] Therefore, there is a need in the field to develop a sustainable electron transport material that is simple to prepare, has reduced reverse reaction, and does not use precious metals. Summary of the Invention

[0006] To meet the need for improvements to the Z-scheme system, the inventors developed a sustainable and scalable conformally attached conductive biofilm and its preparation method.

[0007] In a first aspect, the present invention provides a composite comprising a photocatalyst layer and a biofilm, the photocatalyst layer comprising a first photocatalyst and a second photocatalyst, wherein the first photocatalyst is capable of absorbing light and catalyzing an oxidation reaction (such as the oxidation of water), the second photocatalyst is capable of absorbing light and catalyzing a reduction reaction (such as the reduction of protons), and wherein the biofilm comprises microorganisms having cilia or pili, the cilia or pili comprising pili proteins having nucleation sites.

[0008] In some embodiments, the nucleation site comprises charged amino acids and / or hydrophobic domains. In some embodiments, the microorganism is modified to express modified cilia or fimbriae proteins, the modified cilia or fimbriae proteins comprising additional charged amino acids and / or hydrophobic domains. In some embodiments, the modified cilia or fimbriae proteins comprise a His tag. In some embodiments, in-situ polymerization of polymers is possible at the nucleation site.

[0009] In some embodiments, the microorganism is *Escherichia coli*. In some embodiments, the ciliary or fimbriae protein is CsgA protein.

[0010] In a second aspect, the present invention provides a method for preparing the complex of the present invention, comprising:

[0011] i) Provide a photocatalyst layer; and

[0012] ii) The microorganisms are grown on the photocatalyst layer to form a biofilm.

[0013] In a third aspect, the present invention provides a conductive biofilm comprising a microorganism having cilia or pili and a conductive polymer bound to the cilia or pili, wherein the cilia or pili comprise pili proteins having nucleation sites.

[0014] In some embodiments, the nucleation site comprises charged amino acids and / or hydrophobic domains. In some embodiments, the microorganism is modified to express modified cilia or fimbriae proteins, the modified cilia or fimbriae proteins comprising additional charged amino acids and / or hydrophobic domains. In some embodiments, the modified cilia or fimbriae proteins comprise a His tag. In some embodiments, in-situ polymerization of polymers is possible at the nucleation site.

[0015] In some embodiments, the microorganism is *Escherichia coli*. In some embodiments, the ciliary or fimbriae protein is CsgA protein.

[0016] In some embodiments, the conductive polymer is polypyrrole.

[0017] In a fourth aspect, the present invention provides a method for preparing the conductive biofilm of the present invention, comprising:

[0018] i) Cultivate the microorganisms to form a biofilm;

[0019] ii) Contact the biofilm with the monomers of the conductive polymer; and

[0020] iii) Inducing the polymerization of the monomer to form the conductive polymer in situ at the nucleation sites in the cilia or pili of the microorganism.

[0021] In a fifth aspect, the present invention provides an artificial Z-scheme system comprising a photocatalyst layer and a conductive biofilm, wherein the photocatalyst layer comprises a first photocatalyst and a second photocatalyst, wherein the first photocatalyst is capable of absorbing light and catalyzing oxidation reactions (such as the oxidation of water), and the second photocatalyst is capable of absorbing light and catalyzing reduction reactions (such as the reduction of protons), and wherein the biofilm comprises microorganisms having cilia or pili, wherein the cilia or pili comprise pili or pili proteins having nucleation sites.

[0022] In some embodiments, the nucleation site comprises charged amino acids and / or hydrophobic domains. In some embodiments, the microorganism is modified to express modified cilia or fimbriae proteins, the modified cilia or fimbriae proteins comprising additional charged amino acids and / or hydrophobic domains. In some embodiments, the modified cilia or fimbriae proteins comprise a His tag. In some embodiments, in-situ polymerization of polymers is possible at the nucleation site.

[0023] In some embodiments, the microorganism is *Escherichia coli*. In some embodiments, the ciliary or fimbriae protein is CsgA protein.

[0024] In some embodiments, the conductive polymer is polypyrrole.

[0025] In a sixth aspect, the present invention provides a method for preparing the artificial Z-scheme system of the present invention, comprising:

[0026] i) Provide a photocatalyst layer;

[0027] ii) Allow the microorganisms to grow on the photocatalyst layer to form a biofilm;

[0028] iii) Contacting the biofilm with the monomers of the conductive polymer; and

[0029] iv) Inducing the polymerization of the monomer to form the conductive polymer in situ at the nucleation sites in the cilia or pili of the microorganism.

[0030] In the artificial Z-scheme system prepared by the method of the present invention, the formed biofilm is conformally attached to the photocatalyst layer. Attached Figure Description

[0031] Figure 1 This demonstrates the preparation principle and morphological characterization of the conductive biofilm of Escherichia coli.

[0032] Figure 2 The test results show the conformal adhesion coating based on conductive biofilm and its conductivity properties.

[0033] Figure 3 This diagram illustrates the process of building a manual Z-scheme system.

[0034] Figure 4 This demonstrates the morphological characterization of the artificial Z-scheme and the performance testing of photocatalytic water splitting.

[0035] Figure 5 Stability tests of an artificial Z-scheme system containing conductive biofilms of varying sizes are shown. Invention Details

[0036] I. Definition

[0037] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, and microbiology-related terms and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. For example, the standard recombinant DNA and molecular cloning techniques used in this invention are well known to those skilled in the art and are described more fully in the following literature: Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0038] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0039] As used herein, a "photocatalyst" is a catalyst capable of absorbing light and catalyzing oxidation or reduction reactions. Exemplary photocatalysts for catalyzing oxidation reactions include, but are not limited to, BiVO4, LaTiO2N, Bi4NbO8Cl, TiO2 (such as rutile TiO2), and Mo:BiVO4. Exemplary photocatalysts for catalyzing reduction reactions include, but are not limited to, SrTiO3:La,Rh, and LaMg. 1 / 3Ta 2 / 3 O2N, ZrO2 / LaMg 1 / 3 Ta 2 / 3 O2N, GaLa5Ti2Cu 0.9 Ag 0.1 S5O7, P:Zn 0.5 Cd 0.5 S 1–x TiO2 / CdSZnSe:CGSe, Cu2O, and Rh:SrTiO3. The photocatalyst can also be a photocatalyst for carbon fixation or for nitrogen fixation. Examples of photocatalysts for carbon or nitrogen fixation are known in the art (see, for example, Y. Xu et al., Carbon-based nanostructures for emerging photocatalysis: CO2 reduction, N2 fixation, and organic conversion, Trends in Chemistry, 2022).

[0040] As used herein, the term "biofilm" refers to an organized community of microorganisms (such as bacteria) that is attached to the surface of living or inanimate objects and encapsulated in extracellular macromolecules (such as polypeptides and polysaccharides). Bacteria in biofilms are more resistant to environmental stresses, antibiotics, and host immune defense mechanisms than free-floating bacteria.

[0041] As an example of biofilm-forming bacteria, Escherichia coli is a Gram-negative bacterium that can express cilia or fimbriae proteins (such as CsgA protein) to form cilia or fimbriae outside the bacterial cell.

[0042] As used herein, "conductive biofilm" refers to a biofilm with conductive properties, which can be prepared, for example, by forming a composite of the biofilm and a conductive material (such as a conductive polymer). The conductive polymer can be bound to the biofilm by in-situ polymerization at nucleation sites in the biofilm (such as cilia or fimbriae therein).

[0043] In the context of biofilms, peptides or polypeptides (such as cilia or fimbriae) in this paper, "nucleation sites" refer to charged amino acid residues and / or hydrophobic domains.

[0044] As used herein, the term "peptide" means a chain containing at least two amino acids linked by peptide bonds. The term "polypeptide" is interchangeable with "protein" and means a chain containing ten or more amino acid residues. All peptides and polypeptides are written in left-to-right order, showing the direction from the amino terminus to the carboxyl terminus.

[0045] The term "amino acid" includes both naturally occurring and non-natural amino acids found in proteins. For the conventional nomenclature (single-letter and three-letter) for naturally occurring amino acids in proteins, see Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0046] As used herein, the term "fusion polypeptide" is a recombinant product comprising two or more peptide fragments not present in a single natural polypeptide. These fragments may be fused directly or via a linker, such as a flexible linker (e.g., a GS linker). Typically, fusion polypeptides can be generated by expressing a polynucleotide containing nucleotide sequences encoding two or more peptide fragments and a linker (if present) in a desired order.

[0047] As used herein, the term "polynucleotide" generally refers to a nucleic acid molecule (e.g., 100 nucleotides or more than 30 knucleotides in length) and a sequence that is complementary (antisense) or identical (sense) to a fragment or molecule of messenger RNA (mRNA) or miRNA. The term can also refer to transcribed or non-transcribed DNA or RNA molecules.

[0048] As used herein, the term "polynucleotide construct" refers to a single-stranded or double-stranded polynucleotide isolated from a naturally occurring gene or modified to contain a non-naturally occurring nucleic acid fragment. When a polynucleotide construct contains a control sequence required to express the coding sequence of the present invention, the polynucleotide construct contains an "expression cassette".

[0049] As used herein, the term "exogenous polynucleotide" refers to a nucleotide sequence that is not derived from its host. It may be identical to or heterologous to the host's DNA. One example is the sequence of interest inserted into a vector. Such exogenous DNA sequences can originate from a variety of sources, including DNA, cDNA, synthetic DNA, and RNA. Exogenous polynucleotides also encompass DNA sequences encoding antisense oligonucleotides.

[0050] As used herein, the term "expression cassette" refers to a multinucleotide fragment containing a coding polypeptide operatively linked to another nucleotide sequence provided for the expression of the multinucleotide, such as a control sequence.

[0051] As used herein, the term "coding" means that the polynucleotide directly specifies the amino acid sequence of its protein product. The boundaries of a coding sequence are generally defined by an open reading frame (ORF), which typically begins with an ATG start codon or another start codon (e.g., GTG, TTG) and ends with a stop codon (e.g., TAA, TAG, TGA). The coding sequence can be DNA, cDNA, or a recombinant nucleotide sequence.

[0052] As used herein, the term “expression” includes any steps involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0053] "Control sequences" include all elements necessary or beneficial to the expression of the polynucleotides encoding the polypeptides of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or may be native or foreign to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, enhancers, signal peptide sequences, and transcription terminators. Control sequences include at least a promoter and a signal for terminating transcription and translation.

[0054] For example, the control sequence can be a suitable promoter sequence, a nucleotide sequence of a polynucleotide that is recognized by the host cell to express the polypeptide encoding the present invention. The promoter sequence contains a transcriptional control sequence that mediates polypeptide expression. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, such as the lac operon of *E. coli*. Promoters also include mutant, truncated, and heterozygous promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to those of the host cell.

[0055] As used herein, the term “operably linked” refers to a configuration in which a control sequence is positioned appropriately relative to the coding sequence of a polynucleotide sequence, thereby directing the expression of the polypeptide coding sequence.

[0056] Various manipulations can be performed on polynucleotides encoding polypeptides of interest to improve polypeptide expression. Manipulation of polynucleotides (e.g., codon optimization) is desired or necessary before insertion into a vector, depending on the expression vector or host. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0057] As used herein, the term “recombinant” refers to a nucleic acid, vector, polypeptide, or protein that is produced using DNA recombination (cloning) methods and is different from the natural or wild-type nucleic acid, vector, polypeptide, or protein.

[0058] As used herein, the term "hybridization" refers to nucleotide sequences that have at least about 90%, preferably at least about 95%, more preferably at least about 96%, and even more preferably at least 98% homology with each other, and typically remain hybridized with each other under given stringent hybridization and washing conditions.

[0059] In this invention, to determine the percentage similarity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., a gap may be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). Then, the amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage similarity between two sequences is a function of the number of shared identical positions (i.e., percentage similarity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length.

[0060] Those skilled in the art will know that various computer programs can be used to determine the similarity between two sequences.

[0061] "Percentage of identity" or "percentage of sequence identity" refers to a comparison between the amino acids of two polypeptides or between the nucleotides of two polynucleotides, where, when optimally aligned, the two polypeptides or polynucleotides have approximately the specified percentage of identical amino acids. For example, "95% identity" means a comparison between the amino acids of two polypeptides or between the nucleotides of two polynucleotides, where, when optimally aligned, 95% of the amino acids in the two polypeptides or 95% of the nucleotides in the two polynucleotides are identical.

[0062] Those skilled in the art are familiar with various hybridization conditions, such as strict hybridization conditions and highly strict hybridization conditions. See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, NY; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, NY.

[0063] Of course, the polynucleotides of the present invention do not include polynucleotides that hybridize only with poly A sequences (such as the 3' end poly(A) of mRNA) or poly T (or U) residues.

[0064] As used herein, the term "host cell" refers to, for example, microbial, yeast, insect, and mammalian cells, which can or have served as a recipient for a vector. The term includes the progeny of the original cell that has been transduced. Therefore, as used herein, "host cell" generally refers to a cell that has been transduced with a foreign DNA sequence. It is understood that, due to natural, accidental, or intentional mutations, the progeny of a single parent cell may not necessarily be morphologically identical to the original parent or identical in genomic or total DNA complementarity.

[0065] II. Biofilms conformally attached to photocatalysts

[0066] The inventors discovered that the biofilm can conformally adhere to the photocatalyst, thereby improving the contact between the photocatalyst layer and the conductive layer.

[0067] Therefore, the present invention provides a composite comprising a photocatalyst layer and a biofilm, wherein the photocatalyst layer comprises a first photocatalyst and a second photocatalyst, wherein the first photocatalyst is capable of absorbing light and catalyzing oxidation reactions (such as the oxidation of water), the second photocatalyst is capable of absorbing light and catalyzing reduction reactions (such as the reduction of protons), and wherein the biofilm comprises microorganisms having cilia or pili, wherein the cilia or pili comprise pili proteins having nucleation sites.

[0068] In some embodiments, the nucleation site comprises charged amino acids and / or hydrophobic domains. For example, the charged amino acids include positively charged amino acids (such as lysine, arginine, and histidine) and negatively charged amino acids (such as glutamic acid and aspartic acid).

[0069] Furthermore, the cilia or fimbriae can be modified to increase the number of nucleation sites therein. In some embodiments, the microorganism is modified to express modified cilia or fimbriae (recombinant cilia or fimbriae) containing additional charged amino acids and / or hydrophobic domains. For example, the microorganism contains a polynucleotide encoding the modified cilia or fimbriae. In some embodiments, the microorganism does not express wild-type cilia or fimbriae. In some embodiments, the modified cilia or fimbriae contains a tag of charged amino acids and / or hydrophobic domains, such as a His tag (e.g., a 6×His or 7×His tag). In some embodiments, the modified cilia or fimbriae includes the tag fused to the N-terminus of the wild-type cilia or fimbriae. In some embodiments, the modified cilia or fimbriae includes the tag fused to the C-terminus of the wild-type cilia or fimbriae. In some embodiments, the modified cilia or fimbriae contain a tag fused to the N-terminus of the wild-type cilia or fimbriae and a tag fused to the C-terminus of the wild-type cilia or fimbriae.

[0070] In some implementations, in-situ polymerization of the polymer can occur at the nucleation site.

[0071] Examples of microorganisms that can be used to form the biofilm include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris, Ganoderma lucidum mycelium, Caulobacter crescentus, Shewanella oneidensis, Geobacter, and Aspergillus niger.

[0072] In some embodiments, the microorganism is *Escherichia coli*. In some embodiments, the ciliary protein is CsgA protein. For example, the microorganism is *E. coli* expressing a fusion protein of a His tag and CsgA protein (CsgA-His), thereby the *E. coli* having cilia comprising CsgA-His. In some embodiments, the *E. coli* is modified not to express wild-type CsgA protein. Thus, the *E. coli* has cilia composed of CsgA-His.

[0073] In some embodiments, the CsgA comprises the amino acid sequence of amino acids 50-158 of SEQ ID NO:1, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with amino acids 50-158 of SEQ ID NO:1.

[0074] In some embodiments, the CsgA-His comprises the amino acid sequence of amino acids 43-165 of SEQ ID NO:1, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with amino acids 43-165 of SEQ ID NO:1.

[0075] In some embodiments, the microorganism comprises an expression cassette for expressing cilia or fimbriae proteins, the expression cassette containing a promoter operatively linked to a nucleotide sequence encoding the cilia or fimbriae proteins. A signal peptide (e.g., amino acids 1-42 of SEQ ID NO:1) may also be added to guide the secretion of the expressed cilia or fimbriae proteins. In some embodiments, the nucleotide sequence encodes the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:1.

[0076] The nucleotide sequence encoding the cilia or pili proteins can be modified to improve expression in host cells. In some embodiments, the expression cassette comprises the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:2.

[0077] In some embodiments, the promoter is an inducible promoter. For example, the promoter is selected from promoters derived from the lactose operon, promoters derived from the tetracycline operon, metal ion-induced promoters, light-induced promoters, and heme-induced promoters.

[0078] In some embodiments, the promoter is a tetracycline-induced promoter, for example, the promoter comprises the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:3.

[0079] In some embodiments, the expression cassette comprises the nucleotide sequences of SEQ ID NO:3 and SEQ ID NO:2.

[0080] In some embodiments, the first photocatalyst can absorb visible light. In some embodiments, the second photocatalyst can absorb visible light.

[0081] In some embodiments, the first photocatalyst is selected from BiVO4, LaTiO2N, Bi4NbO8Cl, TiO2 (such as rutile TiO2) and Mo:BiVO4.

[0082] In some embodiments, the second photocatalyst is selected from SrTiO3:La,Rh, and LaMg. 1 / 3 Ta 2 / 3 O2N, ZrO2 / LaMg 1 / 3 Ta 2 / 3 O2N, GaLa5Ti2Cu 0.9 Ag 0.1 S5O7, P:Zn 0.5 Cd 0.5 S 1–x TiO2 / CdSZnSe:CGSe, Cu2O and Rh:SrTiO3.

[0083] In some embodiments, the first photocatalyst comprises bismuth vanadate (BiVO4), such as Mo:BiVO4. In some embodiments, the second photocatalyst comprises strontium titanate (SrTiO3), such as Rh:SrTiO3.

[0084] In some embodiments, the photocatalyst is a photocatalyst for carbon fixation.

[0085] In some embodiments, the photocatalyst is a photocatalyst for nitrogen fixation.

[0086] The present invention also provides a method for preparing the complex of the present invention, comprising:

[0087] i) Provide a photocatalyst layer; and

[0088] ii) The microorganisms are grown on the photocatalyst layer to form a biofilm.

[0089] In some embodiments, step i) includes forming the photocatalyst layer by coating the first and second photocatalysts onto the surface of a substrate. For example, coating can be performed by placing one or more of the following solutions or suspensions onto the substrate surface:

[0090] a) A solution or suspension containing the first and second photocatalysts, or

[0091] b) A solution or suspension containing the first photocatalyst and a solution or suspension containing the second photocatalyst.

[0092] In some embodiments, step ii) includes culturing the substrate coated with the photocatalyst layer in a culture medium containing the microorganisms.

[0093] Suitable conditions for culturing biofilms are known to those skilled in the art. For example, the microorganism is *Escherichia coli*, and the culture medium is M63 medium. In some embodiments, *E. coli* is grown at 30°C for 48 hours.

[0094] III. Conductive biofilm

[0095] The inventors discovered that conductive polymers can be polymerized in situ at nucleation sites in biofilms, thereby imparting conductivity to the biofilms.

[0096] Therefore, the present invention provides a conductive biofilm comprising a microorganism having cilia or pili and a conductive polymer bound to the cilia or pili, wherein the cilia or pili comprise pili proteins having nucleation sites.

[0097] In some embodiments, the nucleation site comprises charged amino acids and / or hydrophobic domains. For example, the charged amino acids include positively charged amino acids (such as lysine, arginine, and histidine) and negatively charged amino acids (such as glutamic acid and aspartic acid).

[0098] Furthermore, the cilia or fimbriae can be modified to increase the number of nucleation sites therein. In some embodiments, the microorganism is modified to express modified cilia or fimbriae containing additional charged amino acids and / or hydrophobic domains. For example, the microorganism contains a polynucleotide encoding the modified cilia or fimbriae. In some embodiments, the microorganism does not express wild-type cilia or fimbriae. In some embodiments, the modified cilia or fimbriae contains a tag of charged amino acids and / or hydrophobic domains, such as a His tag (e.g., a 6×His or 7×His tag). In some embodiments, the modified cilia or fimbriae includes the tag fused to the N-terminus of the wild-type cilia or fimbriae. In some embodiments, the modified cilia or fimbriae includes the tag fused to the C-terminus of the wild-type cilia or fimbriae. In some embodiments, the modified cilia or fimbriae contain a tag fused to the N-terminus of the wild-type cilia or fimbriae and a tag fused to the C-terminus of the wild-type cilia or fimbriae.

[0099] In some implementations, in-situ polymerization of the polymer can occur at the nucleation site.

[0100] Examples of microorganisms that can be used to form the biofilm include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris, Ganoderma lucidum mycelium, Sterculia crescentis, Shewanella, Geobacterium and Aspergillus niger.

[0101] In some embodiments, the microorganism is *Escherichia coli*. In some embodiments, the ciliary protein is CsgA protein. For example, the microorganism is *E. coli* expressing a fusion protein of a His tag and CsgA protein (CsgA-His), thereby the *E. coli* having cilia comprising CsgA-His. In some embodiments, the *E. coli* is modified not to express wild-type CsgA protein. Thus, the *E. coli* has cilia composed of CsgA-His.

[0102] In some embodiments, the CsgA comprises the amino acid sequence of amino acids 50-158 of SEQ ID NO:1, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with amino acids 50-158 of SEQ ID NO:1.

[0103] In some embodiments, the CsgA-His comprises the amino acid sequence of amino acids 43-165 of SEQ ID NO:1, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with amino acids 43-165 of SEQ ID NO:1.

[0104] In some embodiments, the microorganism comprises an expression cassette for expressing cilia or fimbriae proteins, the expression cassette containing a promoter operatively linked to a nucleotide sequence encoding the cilia or fimbriae proteins. A signal peptide (e.g., amino acids 1-42 of SEQ ID NO:1) may also be added to guide the secretion of the expressed cilia or fimbriae proteins. In some embodiments, the nucleotide sequence encodes the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:1.

[0105] The nucleotide sequence encoding the cilia or pili proteins can be modified to improve expression in host cells. In some embodiments, the expression cassette comprises the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:2.

[0106] In some embodiments, the promoter is an inducible promoter. For example, the promoter is selected from promoters derived from the lactose operon, promoters derived from the tetracycline operon, metal ion-induced promoters, light-induced promoters, and heme-induced promoters.

[0107] In some embodiments, the promoter is a tetracycline-induced promoter, for example, the promoter comprises the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:3.

[0108] In some embodiments, the expression cassette comprises the nucleotide sequences of SEQ ID NO:3 and SEQ ID NO:2.

[0109] Examples of the conductive polymer include, but are not limited to, polypyrrole, polyaniline, polythiophene, polyacetylene, and polyphenylacetylene. In some embodiments, the conductive polymer comprises polypyrrole.

[0110] The present invention also provides a method for preparing the conductive biofilm of the present invention, comprising:

[0111] i) Cultivate the microorganisms to form a biofilm;

[0112] ii) Contact the biofilm with the monomers of the conductive polymer; and

[0113] iii) Inducing the polymerization of the monomer to form the conductive polymer in situ at the nucleation sites in the cilia or pili of the microorganism.

[0114] In some embodiments, step i) includes culturing the microorganism in a culture medium. Suitable conditions for culturing biofilms are known to those skilled in the art. For example, the microorganism is *Escherichia coli*, and the culture medium is M63 medium. In some embodiments, *Escherichia coli* is grown at 30°C for 48 hours.

[0115] In some embodiments, step ii) includes mixing the solution containing the monomer with the biofilm.

[0116] In some embodiments, the conductive polymer is polypyrrole, and the monomer is pyrrole. In some embodiments, step iii) includes providing Fe. 3+ This induces pyrrole to polymerize in situ at nucleation sites in the cilia or fimbriae of the microorganism to form polypyrrole.

[0117] Those skilled in the art will understand that steps ii) and iii) can be performed simultaneously, or step iii) can be performed after step ii).

[0118] IV. Manual Z-scheme System

[0119] The inventors discovered that the conductive biofilm of the present invention can conformally adhere to the photocatalyst layer to achieve good electron transfer, thereby generating a highly efficient artificial Z-scheme system.

[0120] Therefore, the present invention provides an artificial Z-scheme system comprising a photocatalyst layer and a conductive biofilm, wherein the photocatalyst layer comprises a first photocatalyst and a second photocatalyst, wherein the first photocatalyst is capable of absorbing light and catalyzing oxidation reactions (such as the oxidation of water), and the second photocatalyst is capable of absorbing light and catalyzing reduction reactions (such as the reduction of protons), and wherein the biofilm comprises microorganisms with cilia or pili, wherein the cilia or pili comprise pili proteins with nucleation sites.

[0121] In some embodiments, the nucleation site comprises charged amino acids and / or hydrophobic domains. For example, the charged amino acids include positively charged amino acids (such as lysine, arginine, and histidine) and negatively charged amino acids (such as glutamic acid and aspartic acid).

[0122] Furthermore, the cilia or fimbriae can be modified to increase the number of nucleation sites therein. In some embodiments, the microorganism is modified to express modified cilia or fimbriae containing additional charged amino acids and / or hydrophobic domains. For example, the microorganism contains a polynucleotide encoding the modified cilia or fimbriae. In some embodiments, the microorganism does not express wild-type cilia or fimbriae. In some embodiments, the modified cilia or fimbriae contains a tag of charged amino acids and / or hydrophobic domains, such as a His tag (e.g., a 6×His or 7×His tag). In some embodiments, the modified cilia or fimbriae includes the tag fused to the N-terminus of the wild-type cilia or fimbriae. In some embodiments, the modified cilia or fimbriae includes the tag fused to the C-terminus of the wild-type cilia or fimbriae. In some embodiments, the modified cilia or fimbriae contain a tag fused to the N-terminus of the wild-type cilia or fimbriae and a tag fused to the C-terminus of the wild-type cilia or fimbriae.

[0123] In some implementations, in-situ polymerization of the polymer can occur at the nucleation site.

[0124] Examples of microorganisms that can be used to form the biofilm include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris, Ganoderma lucidum mycelium, Sterculia crescentis, Shewanella, Geobacterium and Aspergillus niger.

[0125] In some embodiments, the microorganism is *Escherichia coli*. In some embodiments, the ciliary protein is CsgA protein. For example, the microorganism is *E. coli* expressing a fusion protein of a His tag and CsgA protein (CsgA-His), thereby the *E. coli* having cilia comprising CsgA-His. In some embodiments, the *E. coli* is modified not to express wild-type CsgA protein. Thus, the *E. coli* has cilia composed of CsgA-His.

[0126] In some embodiments, the CsgA comprises the amino acid sequence of amino acids 50-158 of SEQ ID NO:1, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with amino acids 50-158 of SEQ ID NO:1.

[0127] In some embodiments, the CsgA-His comprises the amino acid sequence of amino acids 43-165 of SEQ ID NO:1, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with amino acids 43-165 of SEQ ID NO:1.

[0128] In some embodiments, the microorganism comprises an expression cassette for expressing cilia or fimbriae proteins, the expression cassette containing a promoter operatively linked to a nucleotide sequence encoding the cilia or fimbriae proteins. A signal peptide (e.g., amino acids 1-42 of SEQ ID NO:1) may also be added to guide the secretion of the expressed cilia or fimbriae proteins. In some embodiments, the nucleotide sequence encodes the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:1.

[0129] The nucleotide sequence encoding the cilia or pili proteins can be modified to improve expression in host cells. In some embodiments, the expression cassette comprises the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:2.

[0130] In some embodiments, the promoter is an inducible promoter. For example, the promoter is selected from promoters derived from the lactose operon, promoters derived from the tetracycline operon, metal ion-induced promoters, light-induced promoters, and heme-induced promoters.

[0131] In some embodiments, the promoter is a tetracycline-induced promoter, for example, the promoter comprises the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:3.

[0132] In some embodiments, the expression cassette comprises the nucleotide sequences of SEQ ID NO:3 and SEQ ID NO:2.

[0133] In some embodiments, the first photocatalyst can absorb visible light. In some embodiments, the second photocatalyst can absorb visible light.

[0134] In some embodiments, the first photocatalyst is selected from BiVO4, LaTiO2N, Bi4NbO8Cl, TiO2 (such as rutile TiO2) and Mo:BiVO4.

[0135] In some embodiments, the second photocatalyst is selected from SrTiO3:La,Rh, and LaMg. 1 / 3 Ta 2 / 3 O2N, ZrO2 / LaMg 1 / 3 Ta 2 / 3 O2N, GaLa5Ti2Cu 0.9 Ag 0.1 S5O7, P:Zn 0.5 Cd 0.5 S 1–x TiO2 / CdSZnSe:CGSe, Cu2O and Rh:SrTiO3.

[0136] In some embodiments, the first photocatalyst comprises bismuth vanadate (BiVO4), such as Mo:BiVO4. In some embodiments, the second photocatalyst comprises strontium titanate (SrTiO3), such as Rh:SrTiO3.

[0137] In some embodiments, the photocatalyst is a photocatalyst for carbon fixation.

[0138] In some embodiments, the photocatalyst is a photocatalyst for nitrogen fixation.

[0139] Examples of the conductive polymer include, but are not limited to, polypyrrole, polyaniline, polythiophene, polyacetylene, and polyphenylacetylene. In some embodiments, the conductive polymer comprises polypyrrole.

[0140] The present invention also provides a method for preparing the artificial Z-scheme system of the present invention, comprising:

[0141] i) Provide a photocatalyst layer;

[0142] ii) Allow the microorganisms to grow on the photocatalyst layer to form a biofilm;

[0143] iii) Contacting the biofilm with the monomers of the conductive polymer; and

[0144] iv) Inducing the polymerization of the monomer to form the conductive polymer in situ at the nucleation sites in the cilia or pili of the microorganism.

[0145] In some embodiments, step i) includes forming the photocatalyst layer by coating the first and second photocatalysts onto the surface of a substrate. For example, coating can be performed by placing one or more of the following solutions or suspensions onto the substrate surface:

[0146] a) A solution or suspension containing the first and second photocatalysts, or

[0147] b) A solution or suspension containing the first photocatalyst and a solution or suspension containing the second photocatalyst.

[0148] In some embodiments, step ii) includes culturing the substrate coated with the photocatalyst layer in a culture medium containing the microorganisms.

[0149] Suitable conditions for culturing biofilms are known to those skilled in the art. For example, the microorganism is *Escherichia coli*, and the culture medium is M63 medium. In some embodiments, *E. coli* is grown at 30°C for 48 hours.

[0150] In some embodiments, step iii) includes contacting the solution containing the monomer with the biofilm, for example, placing the photocatalyst-biofilm composite in the solution containing the monomer.

[0151] In some embodiments, the conductive polymer is polypyrrole, and the monomer is pyrrole. In some embodiments, step iv) includes providing Fe. 3+ This induces pyrrole to polymerize in situ at nucleation sites in the cilia or fimbriae of the microorganism to form polypyrrole.

[0152] Those skilled in the art will understand that steps iii) and iv) can be performed simultaneously, or step iv can be performed after step iii).

[0153] The artificial Z-scheme system of the present invention can be used for total water splitting, artificial photosynthesis, or artificial nitrogen fixation.

[0154] This invention provides a method for splitting water, comprising contacting water with an artificial Z-scheme system under light irradiation, for example, wherein the first photocatalyst is selected from BiVO4, BiVO4:Mo, LaTiO2N, Bi4NbO8Cl, TiO2 (such as rutile TiO2), and Mo:BiVO4, and the second photocatalyst is selected from SrTiO3:La,Rh, and LaMg. 1 / 3 Ta 2 / 3 O2N, ZrO2 / LaMg 1 / 3 Ta 2 / 3 O2N, GaLa5Ti2Cu 0.9 Ag 0.1 S5O7, P:Zn 0.5 Cd 0.5 S 1–x TiO2 / CdSZnSe:CGSe, Cu2O and Rh:SrTiO3.

[0155] The present invention also provides a method for fixing carbon dioxide, comprising contacting carbon dioxide with the artificial Z-scheme system under illumination, wherein the photocatalyst is a photocatalyst for carbon fixation.

[0156] The present invention also provides a method for nitrogen fixation, comprising contacting nitrogen gas with the artificial Z-scheme system under illumination, wherein the photocatalyst is a photocatalyst for nitrogen fixation.

[0157] Beneficial effects

[0158] This invention utilizes in-situ synthesis of conductive polymers via biofilms, achieving the preparation of conductive biofilms and avoiding the use of precious metals. Furthermore, the layer-by-layer assembly method avoids energy-intensive processes such as sintering. Using the conformally attached conductive biofilm as an electron transfer material, effective connections between photocatalysts in the z-scheme system can be achieved. Since the biofilm is regenerable, the sustainability of material preparation is greatly improved. In summary, this invention successfully develops conformally attached conductive biofilms, which have excellent application prospects in the fields of catalysis and energy. Example

[0159] The technical solution of the present invention will be described in detail through the following embodiments. It should be understood that the purpose of the following embodiments is only to illustrate the principles and effects of the present invention, and not to limit the present invention in any way.

[0160] Unless otherwise specified, all materials and reagents used in the embodiments of this application are commercially available. Experimental methods not specifying particular conditions in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0161] Preparation of M63 culture medium: Dissolve 15.6g of M63 powder in 1L of deionized water, adjust the pH to 7.0, and autoclave at 120℃ for 20 minutes. Before use, add the following components (final concentration): 1mM MgSO4, 0.2% w / v glucose, 34μg / mL chloramphenicol, and 250ng / mL tetracycline.

[0162] Preparation of polypyrrole (PPy) reaction solution: Add 8 mL of 50 mM FeCl3 solution and 32 μl of pyrrole to 42 mL of deionized water.

[0163] Example 1: Preparation of conductive biofilm

[0164] Escherichia coli MG 1655PROΔcsgA ompR234 (also known as Tc) was cultured in LB medium containing 34 μg / mL chloramphenicol. Receiver / CsgA His The expression cassette, containing CsgA-His encoding SEQ ID NO:1, and the expression cassette containing SEQ ID NO:3 operatively linked to SEQ ID NO:2 (see X. Wang, et al., Programming Cells for Dynamic Assembly of Inorganic Nano-Objects with Spatiotemporal Control. Adv Mater 30, e1705968 (2018)), was cultured at 37°C and 220 rpm for 12 h. Bacterial cells were collected by centrifugation (5,000 g, 10 min) and resuspended in an equal volume of M63 medium as a seed culture, which was then added to the M63 medium in the culture dish at a volume ratio of 1:100.

[0165] The culture dish was placed in a 30°C incubator for 48 hours, allowing bacteria to secrete amyloid fibrils under tetracycline induction, forming a biofilm tightly adhering to the bottom of the dish. The culture medium was discarded, the biofilm was washed twice with deionized water, and then 25 mL of PPy reaction solution was added. The reaction was carried out at room temperature for 8 hours. Figure 1 As shown in the top image, polypyrrole can be synthesized in situ on the surface of a biofilm through ferric oxidation polymerization, thereby forming a conductive biofilm.

[0166] Discard the PPy reaction solution and wash twice with deionized water. Centrifuge (4,000g, 5 min) to collect the conductive biofilm and resuspend it in PBS solution. Take 10 μL and drop it onto a TEM grid for morphological characterization by TEM. Specifically, absorb excess solution with filter paper and wash the TEM grid twice by adding 10 μL of ddH2O to the grid and quickly blotting it dry on the filter paper. Then, place the TEM grid with 5 μL of 2wt% solution for negative staining of the sample for 1 min. Then, remove excess uranyl acetate and dry the grid under an infrared lamp for 30 min. Obtain TEM images using a JEM-1400plus operating at an accelerating voltage of 120 kV or a JEM-2100plus operating at an accelerating voltage of 200 kV.

[0167] like Figure 1 As shown in the lower image, PPy nanoclusters are interconnected to form a conductive PPy network structure.

[0168] Example 2: Testing the conductivity of conductive biofilms

[0169] The purpose of this embodiment is to test the electrical conductivity of polypyrrole bonded to a biofilm.

[0170] A polyethylene terephthalate (PET) plate (1×5 cm) was placed at the bottom of a petri dish, and a conductive biofilm, i.e., a conformally attached conductive coating, was prepared on the PET plate according to the method described in Example 1. The PET plate was then dried overnight in a drying oven at 37°C.

[0171] The dried PET plates were observed by SEM. Images were acquired in secondary electron imaging (SEI) mode using a Zeiss SUPRA 55 SAPPHIRE scanning electron microscope operating at an accelerating voltage of 5 kV.

[0172] like Figure 2 As shown in the left image, the conductive biofilm exhibits a porous structure and is interconnected through a network structure.

[0173] Furthermore, the conductivity of the conductive biofilm in the dry state was determined by current-voltage testing. Specifically, using an electrochemical workstation (CHI660E, Shanghai Chenhua), cyclic voltammetry was used to record the IV curves of the biofilm-coated PET plates, with a voltage range of -5V to 5V. PET plates coated with biofilms without bound polypyrrole were used as controls.

[0174] like Figure 2 As shown in the right image, the conformally attached conductive biofilm exhibited superior conductivity compared to the control.

[0175] Example 3: Application of conductive biofilms

[0176] The purpose of this embodiment is to verify the application prospects of conformally attached conductive biofilm coatings. To this end, the inventors conducted research on photocatalytic overall water splitting.

[0177] Mo:BiVO4 was prepared as follows: 6 mmol Bi(NO3)5·5H2O (Adamas, 99%) and 6 mmol NH4VO3 (Macklin, 99.95%) were dissolved in 20 mL of 2M HNO3 and 20 mL of 2M NaOH solution, respectively. For Mo doping, stoichiometric amounts of NaMoO4 (Collins, 99.95%) were added to the NH4VO3 solution (Mo:Mo+V = 0.04 mol%). The two solutions were then mixed under vigorous stirring. 4 mL of ethylene glycol (Aladdin, 99%) was added to the resulting orange precipitate and stirred for 30 min. The pH was then adjusted to 2, and the mixture was stirred for another 30 min. Finally, the suspension was kept at 473 K for 24 h. After several rounds of centrifugation (5,000 g, 10 min) and washing with distilled water, the sample was dried overnight in an oven at 333 K.

[0178] Rh:SrTiO3 was synthesized via a one-step solid-state reaction. Specifically, SrCO3 (Macklin, 99.95%), rutile TiO2 (Mackline, 99.99%), and Rh2O3 (Cerametek Materials, 99.8%) powders were mixed in a mortar at a Sr:Ti:Rh molar ratio of 1.05:0.94:0.06 and then milled with ethanol for at least 1 hour. The mixture was spread onto an alumina plate and calcined in a muffle furnace at 1273 K in air for 6 hours.

[0179] like Figure 3 As shown, a photocatalyst mixed suspension containing Mo:BiVO4 and Rh:SrTiO3 was drop-coated onto a glass slide to form a thin layer. After annealing at 300°C for 1 hour, the slide was placed at the bottom of a petri dish. A conformally attached conductive biofilm was then formed on the surface of the photocatalyst layer according to the method described in Example 1, forming an artificial Z-scheme system. After drying at room temperature, the artificial Z-scheme system was transferred to another glass plate using double-sided tape.

[0180] As described in Example 2, cross-sections of the artificial Z-scheme system are imaged using SEM. Figure 4As shown in the left image, the artificial Z-scheme system has a layered structure, including a photocatalyst layer with a thickness of approximately 10 μm and a conductive biofilm layer with a thickness of approximately 30 μm. The conformal attachment of the conductive biofilm to the photocatalyst facilitates electron transfer between the catalysts, thereby enabling photocatalytic total water splitting.

[0181] A visible light-driven total water splitting experiment was conducted in ultrapure water using a 300W xenon lamp (λ>420nm) for top irradiation.

[0182] like Figure 4 As shown in the right image, the ratio of hydrogen to oxygen produced by the reaction is 2:1, confirming the photocatalytic water splitting capability of the artificial Z-scheme system.

[0183] Furthermore, the inventors discovered that if the substrate is changed to ITO conductive glass (conductive biofilm without conformal attachment), the electron transfer efficiency is low due to the inability of the conductive layer and catalyst layer to achieve conformal attachment, which greatly reduces the overall water splitting performance, demonstrating the excellent effect of conformally attached conductive biofilm.

[0184] Example 4: Conductive biofilm prepared on a large scale

[0185] The cultivation of biofilms requires only simple equipment, thus facilitating large-scale preparation. The purpose of this embodiment is to verify the application prospects of large-scale prepared conductive biofilms.

[0186] like Figure 5 As shown in the left image, different sizes (2.25, 4, 9, and 12.25 cm, respectively) were prepared according to the method in Example 3. 2 An artificial Z-scheme system was developed, and its performance in photocatalytic water splitting was tested.

[0187] like Figure 5 As shown in the left image, the catalytic efficiency of the artificial Z-scheme system of different sizes is comparable, with a standard deviation of only 2.5%, indicating that the performance of the artificial Z-scheme system remains stable after scaling up. This proves that the conductive biofilm of the present invention can be prepared on a large scale.

[0188] In addition, the inventors conducted photocatalytic performance tests under different background pressures, such as... Figure 5 As shown in the right image, the catalytic activity remained above 91% when the background pressure increased from 4.5 kPa to 100 kPa.

[0189] The inventors prepared a reference artificial Z-scheme system using metal (titanium) as the electron transfer material. When the background pressure increased from 4.5 kPa to 100 kPa, the reference artificial Z-scheme system could only maintain a catalytic efficiency of 39%.

[0190] It is evident that, under atmospheric pressure, the photocatalytic performance of the conductive biofilm-based artificial Z-scheme system of this invention surpasses that of the Ti-based photocatalytic system. This result indicates that the conductive biofilm coating does not undergo reverse reactions and possesses industrial production potential.

[0191] These results confirm that the system prepared in this invention has good catalytic stability and potential for large-scale production.

[0192] sequence

[0193] SEQ ID NO:1CsgA-His amino acid sequence:

[0194] MKLLKVAAIAAIVFSGSALAGVVPQYGGGGNHGGGGNNSGPNHHHHHHHSELN

[0195] IYQYGGGNSALALQTDARNSDLTITQHGGGNGADVGQGSDDSSIDLTQRGFGN

[0196] SATLDQWNGKNSEMTVKQFGGGNGAAVDQTASNSSVNVTQVGFGNNATAHQYH

[0197] HHHHHH

[0198] SEQ ID NO:2CsgA-His nucleotide sequence:

[0199] ATGAAACTTTTAAAAGTAGCAGCAATTGCAGCAATCGTATTCTCCGGTAGCGC

[0200] TCTGGCAGGTGTTGTTCCTCAGTACGGCGGCGGCGGTAACCACGGTGGTGGCG

[0201] GTAATAATAGCGGCCCAAATCACCATCACCATCACCACCACCATTCTGAGCTGAAC

[0202] ATTTACCAGTACGGTGGCGGTAACTCTGCACTTGCTCTGCAAACTGATGCCCG

[0203] TAACTCTGACTTGACTATTACCCAGCATGGCGGCGGTAATGGTGCAGATGTTG

[0204] GTCAGGGCTCAGATGACAGCTCAATCGATCTGACCCAACGTGGCTTCGGTAAC

[0205] AGCGCTACTCTTGATCAGTGGAACGGCAAAAATTCTGAAATGACGGTTAAACA

[0206] GTTCGGTGGTGGCAACGGTGCTGCAGTTGACCAGACTGCATCTAACTCCTCCG

[0207] TCAACGTGACTCAGGTTGGCTTTGGTAACAACGCGACCGCTCATCAGTACCAC

[0208] CATCACCATCACCACCATTAA

[0209] SEQ ID NO:3 Tetracycline - inducible promoter

[0210] TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCA

[0211] CATCAGCAGGACGCACTGACC

Claims

1. A composite comprising a photocatalyst layer and a biofilm, the photocatalyst layer comprising a first photocatalyst and a second photocatalyst, wherein the first photocatalyst is capable of absorbing light and catalyzing an oxidation reaction, the second photocatalyst is capable of absorbing light and catalyzing a reduction reaction, and wherein the biofilm comprises microorganisms having cilia or pili, the cilia or pili comprising pili proteins having nucleation sites.

2. The complex of claim 1, wherein the nucleation site comprises charged amino acids and / or hydrophobic domains.

3. The complex of claim 1 or 2, wherein the microorganism is modified to express a modified cilia or fimbriae protein, the modified cilia or fimbriae protein comprising additional charged amino acids and / or hydrophobic domains.

4. The complex of claim 3, wherein the modified cilia or fimbriae protein comprises a His tag.

5. The composite of any one of claims 1-4, wherein in situ polymerization of the polymer is possible at the nucleation site.

6. The complex of any one of claims 1-5, wherein the microorganism is Escherichia coli.

7. The complex of claim 6, wherein the cilia or fimbriae protein is CsgA protein.

8. A method for preparing the complex of any one of claims 1-7, comprising: i) Provide a photocatalyst layer; and ii) The microorganisms are grown on the photocatalyst layer to form a biofilm.

9. A conductive biofilm comprising a microorganism having cilia or pili and a conductive polymer bound to the cilia or pili, wherein the cilia or pili comprise pili proteins having nucleation sites.

10. The conductive biofilm of claim 9, wherein the nucleation sites comprise charged amino acids and / or hydrophobic domains.

11. The conductive biofilm of claim 9 or 10, wherein the microorganism is modified to express modified cilia or fimbriae proteins, the modified cilia or fimbriae proteins comprising additional charged amino acids and / or hydrophobic domains.

12. The conductive biofilm of claim 11, wherein the modified cilia or fimbriae contain a His tag.

13. The conductive biofilm of any one of claims 9-12, wherein in-situ polymerization of polymers can be carried out at the nucleation site.

14. The conductive biofilm of any one of claims 9-13, wherein the microorganism is Escherichia coli.

15. The conductive biofilm of claim 14, wherein the cilia or fimbriae protein is CsgA protein.

16. The conductive biofilm of any one of claims 9-15, wherein the conductive polymer is polypyrrole.

17. A method for preparing a conductive biofilm according to any one of claims 9-16, comprising: i) Cultivate the microorganisms to form a biofilm; ii) Contact the biofilm with the monomers of the conductive polymer; and iii) Inducing the polymerization of the monomer to form the conductive polymer in situ at the nucleation sites in the cilia or pili of the microorganism.

18. An artificial Z-scheme system comprising a photocatalyst layer and a conductive biofilm, the photocatalyst layer comprising a first photocatalyst and a second photocatalyst, wherein the first photocatalyst is capable of absorbing light and catalyzing an oxidation reaction, the second photocatalyst is capable of absorbing light and catalyzing a reduction reaction, and wherein the biofilm comprises microorganisms having cilia or pili, the cilia or pili comprising pili proteins having nucleation sites.

19. The artificial Z-scheme system of claim 18, wherein the nucleation site comprises charged amino acids and / or hydrophobic domains.

20. The artificial Z-scheme system of claim 18 or 19, wherein the microorganism is modified to express modified cilia or fimbriae proteins, the modified cilia or fimbriae proteins comprising additional charged amino acids and / or hydrophobic domains.

21. The artificial Z-scheme system of claim 20, wherein the modified cilia or fimbriae protein comprises a His tag.

22. The artificial Z-scheme system of any one of claims 18-21, wherein in-situ polymerization of polymers is possible at the nucleation site.

23. The artificial Z-scheme system of any one of claims 18-22, wherein the microorganism is Escherichia coli.

24. The artificial Z-scheme system of claim 23, wherein the cilia or fimbriae protein is CsgA protein.

25. The artificial Z-scheme system of any one of claims 18-24, wherein the conductive polymer is polypyrrole.

26. A method for preparing an artificial Z-scheme system according to any one of claims 18-25, comprising: i) Provide a photocatalyst layer; ii) Allow the microorganisms to grow on the photocatalyst layer to form a biofilm; iii) Contacting the biofilm with the monomers of the conductive polymer; and iv) Inducing the polymerization of the monomer to form the conductive polymer in situ at the nucleation sites in the cilia or pili of the microorganism.

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