Three-dimensional microbial flora biological hybrid system for efficiently generating electricity by utilizing lignocellulose hydrolysate

Abstract

The invention provides a three-dimensional microbial flora biological hybrid system capable of efficiently generating electricity by utilizing lignocellulose hydrolysate, and belongs to the technical field of biological batteries. According to the invention, an artificial microbial flora SLB composed of S.oneidensis, Lactococcus lactis and Bacillus subtilis is preliminarily constructed, the bacteria in the artificial microbial flora SLB are respectively subjected to genetic engineering modification, then a carbon cloth anode is replaced by a carbon felt anode, a dispersion liquid GO is added, the electricity generation and conductivity of an anode electroactive biological membrane are further optimized, and the electrochemical performance of the anode electroactive biological membrane is further improved. And finally, a three-dimensional microbial flora biological hybrid system S7L1B1 (at) CFamp capable of directly utilizing the lignocellulose hydrolysate to generate power is constructed. And GO. According to the present invention, the MFC technology is introduced, such that the MFC technology has good lignocellulose hydrolysate degradation and biological power generation capacity, such that the new approach is developed for the high-value utilization of the lignocellulose resource, the solid theoretical basis and the practical guidance are provided, and the application development of the MFC technology in the lignocellulose resource conversion field is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biobatteries, and in particular to a three-dimensional microbial flora biohybrid system for efficiently generating electricity using lignocellulose hydrolysate. Background Art

[0002] With the continued decline in the Earth's reserves of non-renewable resources, such as petroleum, and the increasingly severe global climate and environmental challenges caused by their utilization, the urgent task is to fully and efficiently develop and utilize the vast reserves of renewable resources. As the primary biomass resource, lignocellulosic biomass offers multiple advantages, including widespread geographical distribution, diverse and abundant sources, relatively low cost, significant renewable properties, environmental friendliness, low pollution levels, and net carbon dioxide emissions. In recent years, the use of fuel cell technology to directly convert lignocellulosic biomass or its fractionated components into electricity has become a focus of intense scientific research. Microbial fuel cells (MFCs) are innovative bioreactor technologies that directly convert the chemical energy contained in organic matter into electricity, based on microbial metabolic processes. In-depth research has revealed that the exoelectrogen bacteria (exoelectrogen) residing on the MFC anode are capable of degrading organic matter within the anode chamber and transferring the electrons generated during the degradation process to the anode, thereby achieving the dual purpose of consuming the organic carbon source in the lignocellulosic hydrolysate while simultaneously generating electricity. This demonstrates broad application prospects and scientific value.

[0003] The extracellular electron transfer process of electrogenic bacteria is mainly completed with the help of the electron transport chain. Its essence is that electrogenic bacteria transfer electrons generated by intracellular metabolism to extracellular electron acceptors through a series of redox reactions. At present, the extracellular electron transfer mechanisms of electrogenic bacteria that have been confirmed by the academic community are mainly divided into two types: direct electron transfer and indirect electron transfer. Direct electron transfer refers to the use of its own cytochrome proteins, outer membrane vesicles, conductive nanowires or electroactive biofilms to directly contact the anode, thereby efficiently transferring electrons to the anode; while indirect electron transfer refers to the use of electron shuttles (such as flavins, phenazines) and cofactors to carry electrons through diffusion and ultimately transfer them to the anode electrode.

[0004] Shewanella oneidensis MR-1 is a leading example of electroactive microorganisms. It is a facultative anaerobic bacterium that utilizes lactate as its optimal substrate for growth and metabolism. Its metabolic pathway primarily involves the catalytic conversion of lactate to pyruvate, followed by the conversion of pyruvate to acetyl-CoA. Most of the acetyl-CoA then enters the tricarboxylic acid cycle (TCA) to generate NADH and FADH2, which then enter the electron transport chain. A smaller amount of acetyl-CoA enters the acetate biosynthesis pathway. The optimal temperature for electroproduction for Shewanella oneidensis MR-1 is 30°C, and its optimal pH is approximately 7.0. Shewanella oneidensis MR-1 belongs to the Gram-negative Proteobacteria and is typically short rod-shaped, 2-3 μm long and 0.4-0.7 μm in diameter. Colonies are round, salmon-colored (pink-orange), with smooth, shiny edges. The genome sequencing results of Shewanella oneidensis MR-1 are as follows: the genome consists of a circular chromosome of 4,969,803 base pairs with 4,758 predicted protein-coding open reading frames. In addition, it contains a 161,613-base-pair iteron-type plasmid with 173 open reading frames. Its 16S rRNA sequence is available from the GenBank nucleotide database (AF005251.1). The whole genome sequencing results of the strain have been deposited in GenBank (the plasmid sequence is deposited in GenBank under accession number AE014300.1) and publicly available in the TIGR Comprehensive Microbial Resource database. The accession numbers for Shewanella oneidensis MR-1 are ATCC 700550 and CIP 106686.

[0005] The construction of artificial microbial consortia is a key research topic in fields such as synthetic biology, microbiology, and bioengineering. Its core principle is to adhere to the principle of "modular division of labor and collaborative optimization." By precisely designing the metabolic module division of labor and collaborative cooperation between microbial communities, microbial communities with specific functions and stable structures are artificially constructed. First, based on the diversity of microbial physiological functions, more functionally complete microbial communities are constructed through the rational combination of functionally complementary microorganisms. Second, by leveraging the synergistic metabolism between microorganisms and the complementary supply of nutrients and growth factors between strains, complex metabolic pathways that are difficult for a single strain to achieve independently are jointly completed, thereby improving the overall metabolic efficiency of the community. Third, by fully considering the differences in the ecological niches occupied by microorganisms within the community, microbial strains with different functional characteristics are organically integrated according to the niche differentiation strategy. Each strain leverages its unique functions to play its strengths, achieves complementary advantages, and ultimately achieves the predetermined target community function, opening up new paths to solving biotechnology challenges in multiple fields. Summary of the Invention

[0006] In order to broaden the spectrum of available carbon sources for Shewanella oneidensis MR-1 and achieve its goal of efficiently generating electricity from lignocellulose hydrolysate, the present invention discloses an artificial microbial community biohybrid system for efficiently generating electricity from lignocellulose hydrolysate. By constructing an artificial microbial community consisting of Shewanella oneidensis, Lactococcus lactis and Bacillus subtilis, and using synthetic biology methods, the global transcriptional regulatory factor CodY is overexpressed in Lactococcus lactis, the ribA gene is overexpressed in Bacillus subtilis, and the gene encoding the Saccharomyces cerevisiae gene is overexpressed in Shewanella oneidensis. cerevisiae and the outer membrane c-type cytochrome (cyc2 gene) encoding the enzyme were used to optimize the lactate metabolic pathway and enhance the EET efficiency. Carbon felt was used as the anode material and graphene oxide (GO) was added to finally construct an artificial microbial community biohybrid system S7L1B1@CF&GO. This system can generate electricity from lignocellulose hydrolysate, achieving a power output of ~739.40 mW / m -2 This achievement expands the range of carbon sources that Shewanella oneidensis can utilize, significantly improves the ability to efficiently generate electricity from lignocellulosic hydrolysate, and provides a new reference for the conversion of lignocellulosic biomass into clean energy.

[0007] Based on the concept of "modular division of labor and collaborative optimization," the electrogenic microorganism Shewanella oneidensis MR-1 was subjected to synthetic biology modification and materials engineering optimization. First, a three-bacteria consortium consisting of Shewanella oneidensis, Lactococcus lactis, and Bacillus subtilis was constructed. In this consortium, the global transcriptional regulator codY was overexpressed in Lactococcus lactis, which acts on lactate dehydrogenase (LDH) to accelerate the conversion of glucose to lactate. The ribA gene, encoding 3,4-dihydroxy-2-butanone-4-phosphate synthase and GTP cyclohydrolase II, was overexpressed in Bacillus subtilis to accelerate riboflavin synthesis. Furthermore, the dld gene, encoding D-lactate dehydrogenase from Saccharomyces cerevisiae, and the cyc2 gene, encoding the outer membrane c-type cytochrome from Acidithiobacillus ferrooxidans, were overexpressed in Shewanella oneidensis MR-1 to improve lactate metabolism and extracellular electron transfer efficiency. Then, the carbon cloth anode was replaced with a carbon felt anode and a dispersion with a particle size greater than 500 nm and a final concentration of 0.2 mg mL was added. -1 Using GO, they constructed a three-dimensional microbial biohybrid system, S7L1B1@CF&GO, to increase the thickness of the electroactive biofilm at the anode and the content of electrogenic bacteria, effectively reducing internal resistance and significantly increasing power generation. Ultimately, this system achieved high electrical energy output and simultaneously achieved efficient degradation of lignocellulose hydrolysate and electrical energy recovery.

[0008] The technical solution of the present invention is summarized as follows:

[0009] The present invention provides a method for enhancing the consumption of lignocellulose hydrolyzate and recovery of electrical energy by Shewanella oneidensis MR-1 bacteria, comprising the following steps:

[0010] 1) Based on the concept of "modular division of labor," a three-bacteria microbial consortium consisting of Shewanella oneidensis, Lactococcus lactis, and Bacillus subtilis was gradually constructed. Lactococcus lactis metabolizes glucose to produce lactic acid, providing a carbon source and electron donor for the exoelectrogenic bacteria Shewanella oneidensis. Bacillus subtilis metabolizes glucose and xylose to synthesize riboflavin, continuously supplying Shewanella oneidensis with extracellular electron shuttles, thereby improving EET efficiency.

[0011] 2) Inoculate the three-bacteria microbial flora system in step 1) with OD 600 Optimization, the optimized microbial fuel cell has the most suitable inoculation OD 600 For Shewanella oneidensis:Lactococcus lactis:Bacillussubtilis=1:0.1:1;

[0012] 3) The fermentation bacteria Lactococcus lactis in step 1) was genetically modified, primers containing BamHI and HindIII restriction sites were designed, and the global transcriptional regulator codY (Gene ID: 89632301) was amplified using the genome as a template. The target gene and the pLEB124 plasmid were then double-digested with BamHI and HindIII enzymes and the fragments were recovered, leaving the same sticky ends. The target gene was ligated to the plasmid under the action of T4 ligase, and the resulting recombinant plasmid was electroporated. Import From the fermentation bacteria Lactococcus lactis , an engineered Lactococcus lactis strain L1 was obtained;

[0013] 4) The fermentation bacteria Bacillus subtilis of step 1) was genetically modified. First, the upstream homologous arm ribA-F of the ribA gene (Gene ID: 938946) was amplified using B. subtilis 168 genomic DNA as a template using primers, and the recombinant plasmid pSS-ribA-F was constructed by enzyme digestion and ligation; then, the genomic DNA of B. subtilis 168 was used as a template and the primers P 43 The strong promoter (SEQ ID No. 4) was amplified using primers to amplify the ribA gene (Gene ID: 938946), and then the two-fragment fusion PCR technique was used to convert the P 43 The fragment was fused with the ribA fragment to form a new PCR fragment P 43 -ribA was used as the downstream homology arm and inserted into the plasmid pSS-ribA-F by enzyme digestion and ligation to construct P 43 -ribA overexpression plasmid pSS-P43-ribA-FB;

[0014] Then the plasmid pSS-P43-ribA-FB was integrated into the chromosome of Bacillus subtilis BS77 by double crossover homologous recombination, and positive transformants were screened to obtain P 43 The strong promoter (SEQ ID No. 4) was site-specifically integrated into the ribA gene (Gene ID: 938946) to overexpress the target transformant, thereby obtaining the Bacillus subtilis engineered strain B1;

[0015] 5) The electrogenic bacterium Shewanella oneidensis of step 1) was genetically engineered, firstly designing a target gene dld containing NdeI and SpeI restriction sites at both ends, then using these two enzymes to perform double enzyme digestion on the target gene dld and the pYYDT plasmid, respectively, and then ligating the target gene to the plasmid under the action of T4 ligase to obtain a recombinant plasmid pYYDT-dld; then designing a target gene cyc2 containing XbaI and SbfI restriction sites at both ends, and cutting the target gene with these two enzymes, and simultaneously performing double enzyme digestion on the recombinant plasmid pYYDT-dld with SpeI and SbfI enzymes, XbaI and SpeI being isoenzymes, and ligating the target gene to the plasmid under the action of T4 ligase to obtain a recombinant plasmid pYYDT-dld-cyc2; the obtained recombinant plasmid was first transformed into Escherichia coli WM3064, and then introduced into the electrogenic bacterium Shewanella oneidensis by conjugation transformation to obtain Shewanella oneidensis engineered strain S7;

[0016] 6) Replace the carbon cloth anode with a 2.5 × 2.5 cm carbon felt anode and add a dispersion with a diameter greater than 500 nm and a final concentration of 0.2 mg mL -1 Graphene oxide was used to optimize the anode electroactive biofilm through material engineering, resulting in the artificial microbial community biohybrid system S7L1B1@CF&GO;

[0017] In step 6), the artificial microbial flora biohybrid system S7L1B1@CF&GO was inoculated into the MFC anode chamber, and the anode liquid was 140 mL M9 minimal medium, 0.5 mM IPTG and a carbon source, and the carbon source was diluted to a final concentration of 2 g L glucose. -1 The lignocellulose hydrolysate was prepared and 140 mL of traditional K3[Fe(CN)6] solution was added to the cathode chamber as a cathode electron acceptor to achieve effective utilization of the lignocellulose hydrolysate and high electrical energy recovery.

[0018] Electroactive microorganisms are core microorganisms in microbial fuel cells (MFCs), playing a crucial role in energy conversion and power generation applications using renewable biomass resources. The currently discovered electrogenic microorganism, Shewanella oneidensis, suffers from a narrow carbon spectrum, low power output, and weak electricity production, limiting its practical application in power generation from lignocellulosic hydrolysates. Therefore, using synthetic biology approaches to modify and optimize these electrogenic microorganisms to broaden their carbon spectrum and enhance their EET capabilities is a key task. This study, considering that Shewanella oneidensis MR-1 lacks the ability to metabolize glucose and xylose or produce the electron shuttle riboflavin, optimized Shewanella oneidensis MR-1 using synthetic biology and materials engineering to reduce the metabolic burden of a single strain while achieving efficient electricity generation from lignocellulosic hydrolysate. Adhering to the principle of "modular division of labor and collaborative optimization," a substrate cascade reaction between the microbial communities was carefully designed to construct a three-dimensional microbial biohybrid system, S7L1B1@CF&GO, capable of directly generating electricity from lignocellulosic hydrolysate. The global transcriptional regulator codY was overexpressed in Lactococcus lactis, which acts on lactate dehydrogenase (LDH) to accelerate the conversion of glucose to lactate; the ribA gene encoding 3,4-dihydroxy-2-butanone-4-phosphate synthase and GTP cyclohydrolase II was overexpressed in Bacillus subtilis to accelerate riboflavin synthesis; at the same time, the dld gene encoding D-lactate dehydrogenase from Saccharomyces cerevisiae and the cyc2 gene encoding the outer membrane c-type cytochrome from Acidithiobacillus ferrooxidans were overexpressed in Shewanella oneidensis MR-1 to improve lactate metabolism and extracellular electron transfer efficiency. Then, the carbon cloth anode was replaced with a carbon felt anode and a dispersion with a diameter greater than 500 nm and a final concentration of 0.2 mg mL was added. -1 GO was added to further optimize the electricity generation and conductivity of the anode electroactive biofilm.

[0019] The data showed that the system performed well in a real environment, with a maximum power density of about 739.40 mWm when using lignocellulose hydrolysate as an electron donor. -2 , is the maximum power density of SLB in the control group (about 100.60mWm -2) times; the slope of the polarization curve shows that the internal resistance of the system is greatly reduced; the glucose in the MFC is quickly consumed within 24 hours; most of the xylose is consumed; the lactic acid concentration shows a rapid upward trend, stabilizes after reaching about 48.00mM, and then enters a slow decline stage. After the power generation process is completed, the lactic acid is completely converted; the riboflavin concentration shows a trend of first rapidly increasing and then stabilizing, which meets the needs of extracellular electron transfer.

[0020] In summary, based on the concept of "modular division of labor and collaborative optimization", synthetic biology and materials engineering methods were used to rationally and directionally transform the Shewanella oneidensis MR-1 strain to construct a three-dimensional microbial community biohybrid system S7L1B1@CF&GO, which has good lignocellulose hydrolysate degradation and biopower generation capabilities. This has opened up a new path for the high-value utilization of lignocellulose resources, provided a solid theoretical basis and practical guidance, and promoted the application and development of MFC technology in the field of lignocellulose resource conversion. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the recombinant plasmid structure containing target genes dld and cyc2;

[0022] Figure 2 This is the SEM characterization of the anode biofilm of the S7L1B1@CF&GO system;

[0023] Figure 3 This is the voltage-time curve of the S7L1B1@CF&GO system;

[0024] Figure 4 This is the power density curve of the S7L1B1@CF&GO system;

[0025] Figure 5 is the polarization curve of the S7L1B1@CF&GO system;

[0026] Figure 6 is the glucose consumption curve of the S7L1B1@CF&GO system;

[0027] Figure 7 is the xylose consumption curve of the S7L1B1@CF&GO system;

[0028] Figure 8 This is the lactic acid concentration change curve of the S7L1B1@CF&GO system;

[0029] Figure 9 This is the riboflavin concentration change curve of the S7L1B1@CF&GO system;

[0030] Figure 10It is a technical flow chart of the present invention. DETAILED DESCRIPTION

[0031] Example 1

[0032] With Shewanella oneidensis MR-1 as the core bacteria, a three-dimensional microbial community biohybrid system S7L1B1@CF&GO was constructed, which has good lignocellulose hydrolysate degradation and bio-power generation capabilities. In order to allow Shewanella oneidensis MR-1 to directly consume lignocellulose hydrolysate while efficiently generating electricity, the present invention constructs an artificial microbial community system consisting of Shewanella oneidensis, Lactococcus lactis and Bacillus subtilis. Using synthetic biology methods, the global transcriptional regulatory factor CodY is overexpressed in Lactococcus lactis, the ribA gene is overexpressed in Bacillus subtilis, and the gene encoding the gene from Saccharomyces is overexpressed in Shewanella oneidensis. cerevisiae and the outer membrane c-type cytochrome (cyc2 gene) encoding the enzyme were used to optimize the lactate metabolic pathway and enhance the EET efficiency. Carbon felt was used as the anode material and graphene oxide (GO) was added to finally construct an artificial microbial community biohybrid system S7L1B1@CF&GO. This system can generate electricity from lignocellulose hydrolysate, achieving a power output of ~739.40 mW / m -2 This invention expands the range of carbon sources that Shewanella oneidensis MR-1 can utilize, significantly improving its ability to efficiently generate electricity from lignocellulosic hydrolysate, and providing a new reference for the conversion of lignocellulosic biomass into clean energy.

[0033] (1) Acquisition and sequence optimization of target genes:

[0034] First, the sequence information of the D-lactate dehydrogenase gene dld, which is related to lactate utilization, and the heterologous outer membrane c-type cytochrome gene cyc2, which is related to extracellular direct electron transport, of Shewanella oneidensis MR-1 were searched on NCBI (https: / / www.ncbi.nlm.nih.gov / ). ,and Using Jcat( http: / / www.jcat.de / ) Codon optimization and site-directed mutagenesis of the coding sequence were performed to remove the required restriction sites. Optimization was performed in Shewanella oneidensis MR-1, avoiding the restriction sites NdeI, XbaI, SpeI, and SbfI required for future experiments. The optimized sequence was obtained:

[0035] The nucleotide sequence of gene dld is SEQ ID NO. 1;

[0036] The nucleotide sequence of gene cyc2 is SEQ ID NO.2.

[0037] (2) Using the double enzyme digestion method, construct the recombinant plasmid:

[0038] First, an NdeI restriction site was added to the 5' end of the optimized dld target gene fragment, and an SpeI restriction site was added to the 3' end. The target gene dld and the pYYDT plasmid were then double-digested with these two enzymes. The target gene was then ligated to the plasmid under the action of T4 ligase to obtain the recombinant plasmid pYYDT-dld.

[0039] Then, an XbaI restriction site was added to the 5' end of the optimized cyc2 target gene fragment, and an SbfI restriction site was added to the 3' end. The target gene was cut with these two enzymes. At the same time, the recombinant plasmid pYYDT-dld was double-digested with SpeI and SbfI enzymes. XbaI and SpeI are homozygous enzymes. The target gene was ligated to the plasmid under the action of T4 ligase to obtain the recombinant plasmid pYYDT-dld-cyc2 (SEQ ID NO. 3);

[0040] The construction of the recombinant plasmid was completed in sequence according to the above steps. The obtained recombinant plasmid map is as follows Figure 1 As shown, the recombinant plasmid sequence is based on the pYYDT plasmid sequence, with the two optimized target gene sequences superimposed. Since pYYDT, as a basic plasmid vector, contains the kanamycin resistance gene sequence, it can be used for targeted screening of recombinant strains in the later stage.

[0041] (3) Construction of recombinant Shewanella strains that efficiently utilize lactate and optimize direct electron transfer

[0042] ① Transformation: The recombinant plasmid obtained above was introduced into E. coli WM3064 by physical transformation method for the subsequent construction of Shewanella oneidensis recombinant strain S7.

[0043] Remove 50 μL of competent E. coli WM3064 cells from a -80°C freezer, thaw naturally in an ice box, and then add 25 μL of the recombinant plasmid pYYDT-dld-cyc. Incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then incubate on ice again for 2 minutes. Add 1 mL of LB+DPA liquid medium to the EP tube containing the competent cells and recombinant plasmid and reconstitute the cells in a shaker at 37°C, 200 rpm, for 1 hour. After centrifugation, spread the culture onto a solid LB+DPA+Kan plate and invert the plate to incubate overnight at 37°C in a constant temperature incubator. Select positive colonies and preserve the strains. Take 750 μL of the overnight cultured positive colonies and 250 μL of 80% glycerol, add them to a culture tube, and store at -80°C.

[0044] ② Conjugation transformation: The recombinant plasmid in the positive clone obtained above was transferred into Shewanella oneidensis through conjugation to obtain a recombinant strain.

[0045] E. coli WM3064 containing the recombinant plasmid was inoculated into 5 mL of LB+DPA+Kan liquid medium and grown at 37°C, 220 rpm, for 10-12 hours. Simultaneously, Shewanella oneidensis MR-1 was inoculated into 5 mL of LB liquid medium and grown at 30°C, 200 rpm, for 10-12 hours. 500 μL of each of the resulting E. coli and Shewanella oneidensis MR-1 cultures was combined in a sterile 1.5 mL EP tube, mixed thoroughly, centrifuged at 4500 rpm for 10 minutes, and the supernatant discarded. The culture was resuspended in 1 mL of LB+DPA liquid medium and allowed to stand in a 30°C incubator for 2 hours for conjugation and transformation. After mixing thoroughly, 50 μL of the culture was inoculated onto a solid LB+Kan plate, which was then inverted and incubated in a 30°C incubator for at least 12 hours to obtain engineered Shewanella oneidensis strains.

[0046] Verification by colony PCR: After distinct single colonies (approximately 0.5-1 mm in diameter) have grown on the plate coated during conjugation, perform a liquid culture PCR to verify successful plasmid transfer into Shewanella oneidensis. Prepare LB+Kan plates and mark the grids. Prepare the PCR system and aliquot into EP tubes for liquid culture PCR. Prepare 2-5 more systems than needed to prevent loss due to solution contamination of the pipette tip. Prepare a sufficient number of sterilized toothpicks. In a laminar flow hood, use one toothpick to pick a single colony and place it into an EP tube. After selecting each colony, each toothpick streaks into the same numbered well on the gridded plate and discards the toothpick. Once all wells have been streaked, seal the 96-well plate tightly with a plate sealer, place in the PCR instrument, set the parameters, and start the run. Place the plate in a 30°C incubator (grow for at least 10 hours, depending on the colony's growth. Once the colony has reached a certain size, wrap it in plastic wrap and store at 4°C). Single PCR reaction system configuration (trans fastTaq): ddH2O 33.5μL; upstream and downstream primers 2.5μL each; 5×Q5 buffer 10μL; dNTPs 1.0μL; Q5 DNA Polymerase 0.5μL; a total of 50μL per reaction. PCR reaction conditions: 95℃ pre-denaturation for 5min, 95℃ denaturation for 30s, 53℃ annealing for 30s, 72℃ extension for 2 kb min -1 , sufficient time is required), 30-35 cycles, finally extended at 72℃ for 7min, and kept warm at 16℃.

[0047] Agarose gel electrophoresis: Prepare the gel at least 1 hour in advance, add 100 mL of 1× TAE buffer, 1.0 g of agarose, and dissolve it in a microwave oven. After cooling appropriately, add 5 μL of nucleic acid dye, shake well, pour into the assembled gel tank, and wait for solidification.

[0048] Run the gel: After PCR, add DNA Loading Buffer and apply 10 μL to each well. Finally, apply DNA maker and run the gel for 8–12 minutes. Observe the target band at the corresponding position using the DNA maker and record the corresponding number.

[0049] Preservation: Prepare the successfully validated engineered Shewanella oneidensis strain S7 for future use. Take 750 μL of the overnight recombinant bacterial culture and 250 μL of 80% glycerol, add them to a preservative tube, and store at -80°C for long-term storage.

[0050] (4) The fermentation bacteria Lactococcus lactis of step 1) was genetically engineered, and primers containing BamHI and HindIII restriction sites were designed. The global transcriptional regulatory factor codY was amplified using the genome as a template. The target gene and the pLEB124 plasmid were then double-digested with BamHI and HindIII enzymes and the fragments were recovered, leaving the same sticky ends. The target gene was ligated to the plasmid under the action of T4 ligase. The resulting recombinant plasmid was introduced into the fermentation bacteria Lactococcus lactis by electroporation to obtain the Lactococcus lactis engineered strain L1. The detailed operation is as follows:

[0051] ① Construction of recombinant plasmid

[0052] The glycerol-preserved strain L. lactis was inoculated into 50 mL of seed culture medium and activated for three generations. The third generation of bacteria in the logarithmic growth phase was taken and the genome was extracted using a Tiangen kit. Primers containing BamHI and HindIII restriction sites were designed, and the extracted genome was used as a template to amplify and recover the target gene fragment containing the restriction sites. At the same time, the pLEB124 plasmid was extracted from the cultured E. coli. The target gene and plasmid were double-digested and fragments were recovered using BamHI and HindIII enzymes, respectively, leaving the same sticky ends. The target gene was then ligated to the plasmid under the action of T4 ligase to obtain a recombinant plasmid.

[0053] ② Preparation of competent Lactococcus lactis

[0054] Take out the Lactococcus lactis from the -80℃ refrigerator and streak it for activation. Place it in a 30℃ constant temperature incubator and culture it until a single colony grows. Pick the colony and transfer it to the first-level seed liquid. Then, transfer the seed liquid to 100mL liquid culture medium at a ratio of 2% v / v and place it in a 30℃ constant temperature incubator and culture it for 6h (OD 600 =0.5~0.6). Add 50mg mL -1 Add 40 μL of ampicillin to weaken the cell walls and continue to let it stand for 1 hour. In a clean bench, divide 100 mL of bacterial solution into 4 centrifuge tubes, 25 mL per tube, centrifuge at 4°C, 4500 rpm, and discard the supernatant, retaining the bacteria. Add 25 mL of electroporation wash solution to fully suspend the bacteria, repeat the centrifugation, and discard the supernatant. Then add 12.5 mL and 6.25 mL of electroporation wash solution, repeat the centrifugation, and discard the supernatant. Finally, add 250 μL of pre-chilled Lactococcus lactis electroporation wash solution to each centrifuge tube, suspend the bacteria, divide into 100 μL per tube, and quickly store in a -80°C refrigerator.

[0055] ③Electroporation of the recombinant plasmid into competent Lactococcus lactis

[0056] For electroporation of Lactococcus lactis, use a 0.2 mm cuvette. Remove the cuvette from the clean bench, sterilize it with 75% ethanol, air dry it, and cool it thoroughly on ice. In the clean bench, pipette 5 μL of the target recombinant plasmid into 50 μL of competent lactic acid bacteria. Incubate on ice for 30 minutes. Simultaneously, start the electroporator to preheat. Then, pipette the entire mixture of recombinant plasmid and competent cells into the cuvette. Wipe any water droplets off the cuvette's exterior and place it in the electroporator. Electroporation is performed for 5.5 ms.

[0057] ④ Add 1 mL of liquid culture medium to the electroporation cuvette and pipette to mix thoroughly. Transfer the culture to a 1.5 mL sterile centrifuge tube and incubate at 30°C for 2 hours to allow the cells to recover. Centrifuge at 4500 rpm for 1 minute, reserving approximately 100 μL of liquid. Resuspend the pellet and spread it evenly on solid culture medium containing chloramphenicol-resistant Lactococcus lactis using sterile glass beads. Incubate for 36–48 hours. Once distinct colonies have grown, single colonies should be selected for colony PCR verification to identify positive clones.

[0058] (5) The fermentation bacteria Bacillus subtilis in step 1) was genetically modified. First, P 43 -ribA overexpression plasmid pSS-P43-ribA-FB, then integrate the plasmid pSS-P43-ribA-FB into the chromosome of Bacillus subtilis BS77 by double crossover homologous recombination, first screen positive transformants, then select positive transformants from the positive screening for negative screening, obtain the target transformants with a strong promoter site-specifically integrated into the front of the ribA gene for overexpression, and obtain Bacillus subtilis engineered strain B1;

[0059] ①P 43 -ribA overexpression plasmid pSS-P 43 - Construction of ribA-FB

[0060] Genomic DNA from B. subtilis 168 was extracted and used as a template. First, PCR was performed using primers ribA-F, the upstream homology arm of the ribA gene. RibA-F was inserted into the plasmid pSS via the restriction sites NdeI and Xhol. The successfully connected plasmid was named pSS-ribA-F.

[0061] Then, PCR amplification was performed using primers P 43 The promoter was expressed. The ribA gene was amplified by PCR using primers. The two-fragment fusion PCR technique was used to amplify the P 43 The fragment was fused with the ribA fragment to form a new PCR fragment P 43 -ribA serves as the downstream homology arm.

[0062] P 43 -ribA was inserted into the plasmid pSS-ribA-F through the restriction sites Pstl and BamHI, and the successfully connected plasmid was named pSS-P 43 -ribA-FB. PCR amplified upstream homology arms ribA-F and downstream homology arms P 43 -ribA all contain the same DR region, and the ribA fragment is introduced into the RBS sequence commonly used in Bacillus subtilis (SEQ ID No. 5) AAAGGAGGAATTCAAA through ribA-BU.

[0063] ② Construction of engineered strains overexpressing ribA gene

[0064] Plasmid pSS-P 43 -ribA-FB was integrated into the BS77 chromosome via double crossover homologous recombination. After plating, wait for distinct single colonies to grow and perform positive transformant screening.

[0065] (6) Construction of a three-dimensional microbial biohybrid system for efficient electricity generation

[0066] Carbon felt belongs to the category of non-woven fabrics. Its fibers are arranged more closely. Compared with carbon cloth, carbon felt can carry more biomass. On the other hand, the study found that bacterial cells can be captured by GO nanosheets through a "fishing" process. Subsequently, the GO nanosheets are reduced to reduced graphene oxide (rGO) and self-assembled to form a three-dimensional macroporous network structure. Therefore, this part of the study is based on the artificial microbial flora system S7L1B1 described above. 2.5cm×2.5cm carbon felt is used instead of carbon cloth, and GO with a dispersion diameter greater than 500nm is added to a final concentration of 0.2mg mL -1 , and constructed a three-dimensional microbial consortium biohybrid system S7L1B1@CF&GO.

[0067] (7) Electrical energy output of a three-dimensional microbial biohybrid system with high efficiency

[0068] Thus, the present invention has obtained a three-dimensional microbial consortium biohybrid system S7L1B1@CF&GO for efficient electricity generation. To verify that this system has broadened the carbon spectrum available to Shewanella oneidensis and can efficiently generate electricity using lignocellulose hydrolysate as a carbon source, the electrophysiological properties of the system were characterized:

[0069] ① Remove the glycerol stocks of the three recombinant strains from the -80°C freezer and activate them by streaking onto LB plates. Inoculate the activated bacteria into 50 mL shake tubes containing LB liquid medium or Lactococcus lactis liquid medium. Incubate Shewanella oneidensis S7 at 200 rpm and 30°C, Lactococcus lactis L1 in a static incubator at 30°C, and Bacillus subtilis B1 at 220 rpm and 37°C for 10-12 hours to obtain the primary seed solution.

[0070] ② The first-level seed liquid of Shewanella oneidensis S7 was transferred to a conical flask containing 100 mL LB liquid medium containing 0.5 mM IPTG at a 2% v / v inoculation volume, and cultured at 200 rpm and 30°C; the first-level seed liquid of Lactococcus lactis L1 was transferred to a conical flask containing 100 mL Lactococcus lactis liquid medium at a 2% v / v inoculation volume, and cultured at 30°C; the first-level seed liquid of Bacillus subtilis B1 was transferred to a conical flask containing 100 mL Lactococcus lactis liquid medium at a 4% v / v inoculation volume -1 The cells were cultured in a conical flask containing 100 mL of sterile glucose-containing LB liquid medium at 220 rpm and 37°C for 10-12 hours to obtain a secondary fermentation broth.

[0071] ③ Start the dual-chamber microbial fuel cell: The dual-chamber microbial fuel cell structure is a bipolar chamber H-type with a volume of 140 mL per chamber; the anode uses carbon felt as the electrode (2.5 cm × 2.5 cm), and the cathode uses pretreated carbon cloth as the electrode (2.5 cm × 3 cm); the anode chamber and the cathode chamber are separated by a DuPont Nafion117 proton exchange membrane; 5 mL of sterile anolyte (composition see Table 1) are used to resuspend the collected bacterial pellets of the three bacteria in turn and inoculate them into the anode chamber, and the inoculation OD is 0. 600 The ratio of Shewanella oneidensis:Lactococcus lactis:Bacillus subtilis is 1:0.1:1, and the dispersion piece diameter is greater than 500nm, and the final concentration is 0.2mg mL -1 The graphene oxide was prepared by diluting the volume to 140 mL with sterile anolyte; 140 mL of freshly prepared catholyte (composition see Table 2) was added to the cathode chamber; the external circuit was connected with a copper wire and a 2 kΩ resistor to form a closed loop, and the mixture was placed in a 30°C incubator for static culture.

[0072] Table 1 Composition of anolyte (1 L): the balance is sterile water

[0073]

[0074] Table 2 Catholyte composition (1 L): the balance is distilled water

[0075] composition <![CDATA[K3[Fe(CN)6]]]> <![CDATA[K2HPO4·3H2O]]> <![CDATA[KH2PO4]]> Content (g) 16.463 11.411 6.805

[0076] Additional notes:

[0077] The reagents required for this experiment are as follows:

[0078] ① Luria-Bertani (LB) liquid medium: 10 g L -1 NaCl, 10 g L -1 Peptone and 5g L -1 Yeast extract, adjust pH to 7.2, and sterilize at 121°C for 20 minutes. If antibiotics and IPTG are required, add them and shake well before use.

[0079] ② Luria-Bertani (LB) solid medium: 10 g L -1 NaCl, 10 g L -1 Peptone and 5g L -1 Yeast extract, 15-20 g L -1 Adjust the pH of agar powder to 7.2 and sterilize at 121°C for 20 minutes. Pour the mixture into a plate before solidification.

[0080] ③LB+DPA liquid medium: 10 g L -1 NaCl, 10 g L -1 Peptone and 5g L -1 Yeast extract, 0.059gL -1 2,6-Diaminopimelate. If antibiotics and IPTG are required, they can be added before use.

[0081] ④LB+DPA+Kan solid plate: 10 g L -1 NaCl, 10 g L -1 Peptone and 5g L -1 Yeast extract, 0.059 g L -1 2,6-diaminopimelate, 15-20 g L -1 Agar powder. Add kanamycin stock solution (1:2000) before pouring the plate.

[0082] ⑤ Kanamycin mother solution (Kan, 50 mg mL -1 ): Weigh 0.5 g Kan, dilute to 10 mL with ddH2O, sterilize with a 0.22 μm filter, aliquot into 1 mL tubes, and store at -20°C.

[0083] ⑥ 1M IPTG solution: Weigh 2.383g IPTG and dissolve it in ddH2O to make up to 10mL. Filter through a 0.22μm sterile water filter and aliquot into 1.5mL sterile EP tubes. Store at -20°C.

[0084] ⑦5×M9 mother liquor: 2.5 g L -1 NaCl, 5.0 g L -1 NH4Cl, 15.0 g L -1 KH2PO4, 34.0g L -1 Anhydrous Na2HPO4 was dissolved in an appropriate amount of ddH2O, and then the volume was adjusted to 1 L. After sterilization, the volume was cooled to room temperature and stored in a refrigerator at 4°C.

[0085] ⑧1MMgSO4: 24.6g MgSO4·7H2O is diluted to 100mL with deionized water and sterilized at 121℃ for 20 minutes.

[0086] ⑨0.1M CaCl2: Dilute 1.11g of anhydrous CaCl2 to 100mL with deionized water and sterilize at 121℃ for 20 minutes.

[0087] ⑩Mineral mix: 1.5g L -1 nitrilotriacetic acid (NTA), 0.1g L -1 MnCl2·4H2O, 0.3 g L -1 FeSO4·7H2O, 0.17 g L -1 CoCl2·6H2O, 0.1 g L -1 ZnCl2, 0.04 g L -1 CuSO4·5H2O, 0.005 g L -1 AlK(SO4)2·12H2O, 0.005 g L -1 H3BO3, 0.09 g L -1 Na2MoO4, 0.12g L -1 NiCl2, 0.02 g L -1 NaWO4·2H2O. Sterilize by filtration using a 0.2 μm sterile filter membrane.

[0088] Vitamin mix:0.004g L -1 Vitamin H, 0.004 g L -1 Folic acid, 0.04g L -1 Vitamin B6, 0.01gL -1 Thiamine, 0.01 g L -1 Niacin, 0.01 g L-1 D-calcium pantothenate, 0.0002g L -1 Vitamin B12, 0.01g L -1 p-Aminobenzoic acid, 0.01 g L -1 Lipoic acid was sterilized by filtration using a 0.2 μm sterile filter membrane.

[0089] Lactis liquid culture medium: 1.5 g L -1 NaCl, 0.15 g L -1 MgSO4·7H2O, 20g L -1 KH2PO4, 4g L -1 Glucose, 15 g L -1 Yeast extract, 15g L -1 Peptone. Adjust pH to 7.2-7.4. When preparing, glucose needs to be prepared into a high concentration mother solution (4g mL -1 ), sterilize separately at 115℃ for 30 minutes, cool to room temperature, and then add according to the amount.

[0090] L. lactis competent cell preparation electroporation wash solution: 68.46 g L -1 Dissolve sucrose and 10% v / v pure glycerol in deionized water, sterilize at 121°C for 20 minutes, and store in a refrigerator at 4°C.

[0091] B. subtilis fermentation medium: 20 g L -1 Glucose, 30 g L -1 Yeast extract, 0.5 g L -1 MgSO4·7H2O, 0.5g L -1 K2HPO4, 0.5g L -1 KH2PO4. Adjust pH to 7.2-7.4. When preparing, glucose is prepared to 4gmL -1 Sterilize the mother liquor separately at 115°C for 30 minutes. Sterilize the remaining ingredients at 121°C for 20 minutes. Cool to room temperature and then mix according to the amount.

[0092] The engineering bacteria selected for recombinant plasmid amplification were the auxotrophic strain E.coilWM3064 (commercially available, ColiGenetic Stock Center http: / / cgsc.biology.yale.edu / ); the initial strain Lactococcus lactis, CGMCC7.52; the initial strain Bacillus subtili 168Δupp ribC*ribO*yhcF*yvrH*ywaA*; and the initial strain S.oneidensis MR-1, with the deposit numbers ATCC 700550 and CIP 106686.

[0093] The restriction endonucleases, DNA ligases, and molecular biology reagents used above were purchased from Thermo Scientific (http: / / www.thermoscientificbio.com / fermentas); other biochemical reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ), Beijing Dingguo Changsheng Biotechnology Co., Ltd. (www.dingguo.com), and Tianjin Yuanli Chemical Co., Ltd. (https: / / yuanlihuagong.cn.chemnet.com).

[0094] ④ Connect the above-mentioned starting battery to the data logger and record the voltage (such as Figure 3 The data shows that the S7L1B1@CF&GO voltage peak remains stable above 0.5V for extended periods, with a higher voltage than the SLB for most of the time. The voltage-time curve directly reflects the stability of power generation, which in turn reflects the stability of the microbial community during continuous operation. This demonstrates that the S7L1B1@CF&GO has strong voltage output and sustained power generation capabilities.

[0095] ⑤ LSV (Linear Sweep Voltammetry) measurement of battery structure: When the voltage reaches the peak and remains constant, the electrochemical performance of the battery is measured using linear sweep voltammetry. The power density curve of the microbial fuel cell is obtained as follows: Figure 4 The polarization curves are shown as Figure 5 When scanning the LSV of the battery, the initial potential of the scan is set to -0.87 to -0.1, and the scan rate is set to 0.1mV s- 1 The data show that the maximum power density of the system (such as Figure 4 As shown) is approximately 739.40mWm -2 , is the maximum power density of SLB (about 100.60mWm -2) by 7.3 times, and the power generation capacity is greatly improved. The linear part of the polarization curve reflects the ohmic loss. The smaller the slope of the linear part, the smaller the internal resistance. Therefore, the three-dimensional microbial consortium biohybrid system S7L1B1@CF&GO is much smaller than the control group (such as Figure 5 This shows that the system's power loss is reduced and power generation efficiency is improved.

[0096] (8) Consumption of lignocellulose hydrolysate in a three-dimensional microbial biohybrid system with high efficiency in electricity production

[0097] Studies have shown that during the electrochemical reaction process of MFC technology, the artificial microbial community system can broaden the available carbon spectrum of the electrogenic bacteria Shewanella oneidensis. Combined with synthetic biology and materials engineering optimization, it can be used to efficiently generate electricity from lignocellulose hydrolysate, thereby achieving the efficient utilization of lignocellulose hydrolysate and the simultaneous progress of the biopower generation process.

[0098] Construct the MFC described in (7), connect the above-mentioned activated battery to the data acquisition device for operation, take samples from the sampling port at regular intervals (every 4 hours in the initial stage, every 6 hours and 8 hours in the middle, and every 12 hours in the final stage), store the samples at -20°C, and finally process them centrally for HPLC analysis. The samples from the MFC were characterized by high performance liquid chromatography (HPLC) equipped with a differential detector and a UV-vis detector (Waters, Corp). All samples were centrifuged at 12000 rpm for 1 min and filtered (0.22 μm, PES) before injection. Quantitative analysis of glucose, xylose and lactic acid: Aminex HPX-87H chromatographic column (300 mm × 7.8 mm, Bio-Rad) was used, the column temperature was 65°C, 5 mM sulfuric acid was used as the mobile phase, and the flow rate was 0.3 mL min -1 , using a differential detector, injection volume 10 μL, each sample 40 min. Quantitative analysis of riboflavin: using a C18 reverse phase column (5.0 μm, 4.6 mm × 250 mm, Thermo scientific), temperature 35 ° C, mobile phase 0.1% NaH2PO4 aqueous solution (1 g L -1 ): pure methanol = 70%: 30%, flow rate 1ml min -1 The UV-vis detector was used, with a test wavelength of λ = 444 nm, a sample volume of 10 μL, and a 25 min per sample. The glucose consumption curve of the lignocellulose hydrolyzate in the anode chamber was obtained as shown in FIG. Figure 6 As shown in Figure 2, the glucose in the MFC was rapidly consumed within 24 hours, and the glucose consumption rate of the constructed three-dimensional microbial community biohybrid system S7L1B1@CF&GO was significantly higher than that of the control group. Figure 7As shown in Figure 2, both S7L1B1@CF&GO and the control group showed the ability to consume xylose, but S7L1B1@CF&GO was significantly superior in xylose consumption. Figure 8 As shown in Figure 2, the lactate concentrations in both the SLB and S7L1B1@CF&GO systems showed a rapid upward trend, reaching approximately 48.00 mM before stabilizing and then entering a slow decline phase. The lactate concentration in the S7L1B1@CF&GO system decreased at a faster rate. After the power generation process was completed, lactate was completely converted. The concentration change curve of the electron shuttle riboflavin is shown in Figure 2. Figure 9 The riboflavin concentrations in both SLB and S7L1B1@CF&GO showed a trend of first rapidly increasing and then stabilizing. In the entire process, the riboflavin concentration in the S7L1B1@CF&GO system was always higher than that in the SLB system.

[0099] like Figure 10 As shown in the figure, based on the concept of "modular division of labor and collaborative optimization", the present invention utilizes synthetic biology and materials engineering methods to rationally and directional transform the Shewanella oneidensis MR-1 strain and construct a three-dimensional microbial community biohybrid system S7L1B1@CF&GO, which has good lignocellulose hydrolysate degradation and biopower generation capabilities, opens up a new path for the high-value utilization of lignocellulose resources, provides a solid theoretical basis and practical guidance, and promotes the application and development of MFC technology in the field of lignocellulose resource conversion.

[0100] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A microbial flora, characterized in that including Lactococcus lactis, Bacillus subtilis, and Shewanella oneidensis MR-1; Among them, the Lactococcus lactis overexpresses the global transcriptional regulator codY, the Bacillus subtilis overexpresses genes encoding 3,4-dihydroxy-2-butanone-4-phosphate synthase and GTP cyclohydrolase II, and the Shewanella oneidensis MR-1 overexpresses genes encoding D-lactate dehydrogenase and genes encoding outer membrane c-type cytochrome.

2. The microbial flora according to claim 1, characterized in that The gene encoding D-lactate dehydrogenase includes the dld gene, and the nucleotide sequence of the dld gene is SEQ ID NO.1; The gene encoding the outer membrane c-type cytochrome gene includes the cyc2 gene, and the nucleotide sequence of the cyc2 gene is SEQ ID NO.

2.

3. The microbial flora according to claim 1, characterized in that The GeneID of the global transcriptional regulator codY is: 89632301.

4. The microbial flora according to claim 1, characterized in that The genes encoding 3,4-dihydroxy-2-butanone-4-phosphate synthase and GTP cyclohydrolase II include the ribA gene with Gene ID: 938946.

5. The method for constructing a microbial flora according to any one of claims 1 to 4, characterized in that: Including Shewanellaoneidensis MR-1: Lactococcus lactis: Bacillus subtilis according to OD 600 =1:0.1:1 ratio mix; Among them, the Lactococcus lactis overexpresses the global transcriptional regulator codY, the Bacillus subtilis overexpresses genes encoding 3,4-dihydroxy-2-butanone-4-phosphate synthase and GTP cyclohydrolase II, and the Shewanella oneidensis MR-1 overexpresses genes encoding D-lactate dehydrogenase and genes encoding outer membrane c-type cytochrome.

6. Use of the microbial consortium according to any one of claims 1 to 4 as an anode electrochemical biofilm.

7. An artificial microbial flora biohybrid system, characterized in that: Use carbon felt as anode and add dispersion with a diameter greater than 500 nm and a final concentration of 0.2 mg mL -1 The graphene oxide is prepared by using the microbial flora described in any one of claims 1 to 4 as the anode electroactive biofilm to obtain an artificial microbial flora biohybrid system.

8. The artificial microbial flora biohybrid system according to claim 7, characterized in that: The artificial microbial flora biohybrid system was inoculated into the MFC anode chamber. The anode liquid contained 140 mL of M9 minimal medium, 0.5 mM IPTG, and a carbon source. The carbon source was diluted to a final concentration of 2 g L glucose. -1 of lignocellulose hydrolyzate, and 140 mL of K3[Fe(CN)6] solution was added to the cathode chamber as a cathode electron acceptor.

9. Use of the artificial microbial flora biohybrid system according to claim 7 or 8 in biopower generation.

10. A microbial fuel cell constructed using the artificial microbial flora biohybrid system according to claim 7 or 8.

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