Construction and application of shewanella hydrogengens-CdS nanoparticle hybrid system based on hydAdds gene
By constructing a hybrid system of recombinant plasmid pYYDT-hydAdds and CdS nanoparticles in the Shewanella oneidensis MR-1 strain, the problem of low hydrogen production efficiency in the existing technology was solved, and the effect of efficient photocatalytic hydrogen production was achieved.
Patent Information
- Application Number
- CN202510431385.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-09-23
AI Technical Summary
In existing technologies, semiconductor-microorganism hybrid systems have deficiencies in hydrogen production efficiency and hydrogenase activity improvement, making it difficult to efficiently utilize clean solar energy to produce hydrogen.
A recombinant plasmid pYYDT-hydAdds was constructed and introduced into the Shewanella oneidensis MR-1 strain. It was combined with CdS nanoparticles to form a hybrid system. The hydrogenase HydAdds was used to enhance the proton reduction ability and photoelectron transfer, thereby improving the hydrogen production efficiency.
The hydrogen production efficiency is significantly improved, and efficient photocatalytic hydrogen production is achieved. The cost is low and the method is simple, and the target genes can be independently regulated.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of bioenergy technology and specifically relates to a method based on hydA dds Construction and application of a hybrid system of Shewanella oxytoca and CdS nanoparticles Background Art
[0002] As energy and environmental crises become a global concern, the development and utilization of clean, renewable energy has garnered widespread attention worldwide. Hydrogen energy, with its high energy density and clean combustion products, has broad application prospects in transportation, industry, power generation, and other fields, and has garnered widespread attention. It is projected that by 2050, hydrogen energy will occupy an indispensable position in the global energy system, with demand potentially reaching 24% of total global energy consumption.
[0003] Hydrogen can be categorized into three types based on production methods: gray hydrogen, blue hydrogen, and green hydrogen. Gray hydrogen is extracted from fossil fuels through steam methane reforming; blue hydrogen is produced from fossil fuels using carbon capture and storage (CCS); and green hydrogen is produced through water electrolysis powered by renewable energy. Green hydrogen, due to its sustainability and environmental friendliness, is increasingly being considered as a clean energy alternative with the greatest potential for development, particularly as biohydrogen production technology gains increasing attention.
[0004] Shewanella is one of the most intensively studied electrogenic microorganisms, with its metabolic pathways and extracellular electron transfer pathways relatively well understood. Shewanella oneidensis MR-1 (abbreviated as Shewanella MR-1) is the most extensively studied strain within the genus in terms of its genetic characteristics and electron transfer mechanisms.
[0005] Cadmium sulfide (CdS) is a direct bandgap semiconductor (bandgap approximately 2.4 eV) with high visible light absorption efficiency, simple preparation process, low cost, and strong practical application value. It also has high electron mobility and good interface properties. In addition, CdS has stable chemical properties, corrosion resistance, and adaptability to high-temperature processes. Its nanostructure can also adjust the bandgap through quantum confinement effect to expand its applications.
[0006] Semiconductor-microbe biohybrid systems utilize microorganisms as biocatalytic platforms, harnessing photoelectrons generated by semiconductor materials to provide energy and reducing power for microbial metabolism. This system combines the excellent photon conversion capabilities of semiconductors with the metabolic specificity of microorganisms. By capturing clean solar energy to produce energy or high-value-added chemicals while simultaneously degrading pollutants, biohybrid systems offer promising scientific solutions to environmental challenges. Summary of the Invention
[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a hydrogen-producing Shewanella engineered strain and a cadmium sulfide-Shewanella engineered bacteria hybrid system with high hydrogen production efficiency;
[0008] The second object of the present invention is to provide a recombinant plasmid that can enhance the hydrogenase activity of engineered bacteria to improve hydrogen production efficiency;
[0009] The third object of the present invention is to provide a method for preparing a semiconductor-microorganism hybrid system using the recombinant Shewanella, so that it has higher hydrogen production performance.
[0010] The technical solution of the present invention is summarized as follows:
[0011] A recombinant plasmid is constructed by connecting a basic vector plasmid containing at least one promoter and the hydA gene encoding hydrogenase from Desulfovibrio desulfuricans, and connecting the target gene to the vector pYYDT to obtain the recombinant plasmid pYYDT-hydA. dds .
[0012] The recombinant plasmid was transformed into Escherichia coli WM3064 strain (abbreviated as WM3064) using conventional chemical transformation methods in the art. Shewanella oneidensis MR-1 was then selected as the final host strain. The recombinant plasmid was transferred into Shewanella oneidensis MR-1 by conjugative transfer to obtain the hydrogen-producing Shewanella engineered strain SED. Colony PCR verification experiments were then performed. Agarose gel results showed the presence of positive clones, confirming that the target gene was successfully amplified in the host strain.
[0013] A method for preparing a semiconductor-microorganism hybrid system, wherein CdS nanoparticles are introduced into the intracellular and extracellular membranes of a hydrogen-producing Shewanella engineered strain.
[0014] The engineered bacteria SED were cultured in 100 mL of LB liquid medium with the corresponding resistance and inducer until the stable phase, the cells were collected by centrifugation, and washed three times with 20 mM HEPES solution. The cells were resuspended in 100 mL of anaerobic mineral medium containing 1 mM Na2S2O3 and 20 mM sodium lactate to an initial OD of 600 The concentration of CdS nanoparticles was 0.1, and the cells were incubated at 30°C and 200 rpm for 24 hours, 1 mM CdCl2 was added, and the cells were incubated for another 24 hours to form CdS nanoparticles. The entire incubation process needed to be carried out under anaerobic conditions.
[0015] The use of the above-mentioned hydrogen-producing semiconductor-microorganism hybrid system CdS / SED for photocatalytic hydrogen production.
[0016] The washed CdS / SED hybrid system was transferred to hydrogen production medium and photocatalytic hydrogen production was carried out under anaerobic conditions. 2 The reaction was carried out under visible light conditions, and the temperature and oxygen-free conditions were maintained. Samples were taken every 12 hours and hydrogen production was monitored using a gas chromatograph.
[0017] The advantages of the present invention are as follows:
[0018] A recombinant plasmid constructed by the present invention is pYYDT-hydA dds , which can make the engineered Shewanella SED introduced with the recombinant plasmid heterologously express the highly active hydrogenase HydA from Desulfovibrio desulfuricans dds , enhance the proton reduction ability of the engineered bacteria, thereby enhancing the utilization efficiency of intracellular reducing force and improving the hydrogen production efficiency of the semiconductor-microorganism hybrid system.
[0019] The semiconductor-microorganism hybrid system constructed by the present invention is CdS / SED. CdS nanoparticles are excited by light to generate photoelectrons, which are transported by hydrogenase HydA located in the periplasm through a direct electron transfer pathway. dds Uptake, thereby enhancing the hydrogen production efficiency of the hybrid system.
[0020] The semiconductor / engineered bacteria hybrid system constructed by the present invention has the advantages of simple construction method, low raw material cost, independent regulation of target genes, and high hydrogen production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:
[0021] Figure 1 Microscopic diagram of the constructed semiconductor-microorganism hybrid system
[0022] Figure 2 SEM image of CdS / SED hybrid system
[0023] Figure 3 TEM image of CdS / SED hybrid system
[0024] Figure 4 The structure of the recombinant plasmid
[0025] Figure 5 The hydrogen production ratio of the hybrid system CdS / MR-1 constructed for Shewanella and the hybrid system CdS / SED constructed for the hydrogen-producing recombinant strain of Shewanella.
[0026] Figure 6 Intracellular reducing power equivalents NADH / NAD of the hybrid system CdS / MR-1 constructed for Shewanella and the hybrid system CdS / SED constructed for the hydrogen-producing recombinant strain of Shewanella + Comparison Chart DETAILED DESCRIPTION
[0027] The original strain, wild-type Shewanella oneidensis MR-1, referred to as wild-type Shewanella MR-1, was purchased from ATCC (USA, https: / / www.atcc.org / ) in September 2010 as ATCC 700550 strain.
[0028] The present invention will be further described below with reference to specific embodiments.
[0029] Example 1: Construction of hydrogen-producing Shewanella engineered strain SED
[0030] A method for constructing a hydrogen-producing Shewanella recombinant strain comprises the following steps: constructing a recombinant plasmid pYYDT-hydA dds The above plasmid was transformed into wild-type Shewanella MR-1 to obtain the hydrogen-producing Shewanella engineered strain SED.
[0031] The specific construction method includes the following steps:
[0032] 1. Recombinant plasmid pYYDT-hydA dds Build:
[0033] Desulfovibrio desulfuricans encoding HydA dds Hydrogenase gene hydA dds Connect to the vector pYYDT to obtain the recombinant plasmid pYYDT-hydA dds .
[0034] The gene hydA dds The nucleotide sequence is shown as SEQ ID NO.2;
[0035] The recombinant plasmid pYYDT-hydA dds The nucleotide sequence is shown as SEQ ID NO.1.
[0036] The obtained recombinant plasmid pYYDT-hydA dds Transform into E. coli WM3064 competent cells. The production process of WM3064 competent cells is as follows:
[0037] 1. Take WM3064 glycerol culture and streak inoculate on LD solid medium (LB medium supplemented with 0.3M / mL 2,6-diaminopimelate);
[0038] 2. Place the solid culture medium in a 37°C incubator for 24 hours;
[0039] 3. Use an inoculating loop to pick a single colony on the solid culture medium and transfer it to a test tube containing LD antibiotic-free culture medium for primary inoculation culture. Place the test tube in a shaker at 37°C and 220 rpm for 12 hours.
[0040] 4. Take out 1mL of bacterial solution from the test tube and transfer it to a flask containing LD antibiotic-free medium for secondary culture. Place the flask in a shaker at 37℃ and 220rpm and culture until OD 600 The value is 0.6
[0041] 5. Place the culture medium in an ice bath for 20 minutes, and centrifuge the bacterial solution at 5000 rpm and 4°C for 10 minutes.
[0042] 6. Pour off the supernatant, resuspend the pellet with competent preparation reagent, aliquot the resuspension, and store in a -80℃ refrigerator.
[0043] The specific steps of WM3064 chemical transformation are as follows:
[0044] 1. Take 100 μL of WM3064 competent cells from the -80°C refrigerator and place on ice for 10 minutes;
[0045] 2. Insert pYYDT-hydA dds The plasmid and WM3064 competent cells were mixed in a 1.5 mL centrifuge tube and placed on ice for 30 min;
[0046] 3. Place the centrifuge tube after ice bath in a 42℃ water bath for 90 seconds, and then ice bath again for 2 minutes;
[0047] 4. Add 1 mL of LD medium to the above system and incubate at 37°C, 200 rpm in a shaker for 1 hour;
[0048] 5. Place the centrifuge tube after shaking culture in a centrifuge and centrifuge at 5000 rpm for 5 minutes. Pour off the supernatant and resuspend the bacterial pellet in about 100 μL of LB culture medium. Spread the resuspended liquid on an LB plate (containing 1 mM / mL kanamycin) and incubate at 30°C for 24 hours.
[0049] 6. Pick a single colony for PCR verification to verify the correct WM3064 and Shewanella MR-1 conjugation transfer.
[0050] The specific steps for conjugative transfer of WM3064 and Shewanella MR-1 are as follows:
[0051] 1. Pick a single WM3064 colony that has been verified to be correct on the solid culture medium, inoculate it into LD medium (containing 1 mM / mL kanamycin), and perform primary culture at 37°C and 220 rpm;
[0052] 2. Take out the MR-1 glycerol culture, spread it on LB solid medium, and culture it in a 30°C incubator for 24 hours. Then pick a single colony on the solid medium, inoculate it into LB medium, and carry out primary culture at 30°C and 220 rpm;
[0053] 3. Take 500 μL each of the WM3064 and MR-1 primary bacterial suspensions mentioned above and place them into a 1.5 mL centrifuge tube. Mix thoroughly and centrifuge at 5000 rpm for 5 minutes. Discard the supernatant and resuspend in 1 mL of anti-LD-free medium. Then, place the tube in a 30°C incubator for 2 hours.
[0054] 4. After standing, centrifuge the tube containing the bacterial solution at 5000 rpm for 5 minutes, discard the supernatant, resuspend the culture in 1 mL of antibiotic-free LB medium, spread 100 μL of the bacterial solution on an LB plate (containing 1 mM / mL kanamycin), and incubate at 30°C for 24 hours.
[0055] 5. Pick a single colony for PCR verification to verify that the correct Shewanella is the target hydrogen-producing Shewanella engineered strain.
[0056] Example 2: CdS Self-Assembly of Hydrogen-Producing Shewanella Engineered Strain SED
[0057] 1. SED strain activation
[0058] The hydrogen-producing Shewanella engineered strain SED was taken out of the -80°C refrigerator, streaked on LB solid medium (containing 1 mM / mL kanamycin), and placed in a 30°C incubator for 24 hours for activation. After that, a single colony was picked from the solid medium and cultured overnight in LB medium (containing 1 mM / mL kanamycin) at 30°C and 220 rpm.
[0059] 2. Construction of CdS / SED hybrid system
[0060] The SED strain was cultured in LB medium at 30°C with shaking at 200 rpm for 20 hours until it reached the stationary phase. The cells were collected by centrifugation at 6000 rpm for 5 minutes at 4°C and washed twice with 20 mM HEPES solution. Then, the cells were incubated in 100 mL of anaerobic mineral medium containing 1 mM Na2S2O3 and 20 mM sodium lactate at 30°C and 200 rpm. The initial OD 600After incubation for 24 hours, 1 mM CdCl2 was added and incubated for another 24 hours to form CdS nanoparticles, thus obtaining a CdS / SED hybrid system.
[0061] Anaerobic mineral medium contained 5.85 g / L NaCl, 0.3 g / L NaOH, 1.498 g / L NH4Cl, 0.097 g / L KCl, 11.91 g / L HEPES, 0.658 g / L β-glycerophosphate disodium and 1 mL trace mineral solution, and was purged with pure N2 for 30 min to completely remove oxygen.
[0062] 3. Results
[0063] The scanning electron microscopy (SEM) results of the hybrid system CdS / SED are as follows Figure 2 As shown in Figure 2, a large number of nanoparticles are distributed on its surface. Transmission electron microscopy (TEM) images are shown in Figure 2. Figure 3 As shown, the formed nanoparticles are mostly located in the extracellular space and tend to attach to the cell outer membrane. This confirms that the self-assembled CdS nanoparticles are mainly distributed in the extracellular space of S. oneidensis.
[0064] Example 3: Hydrogen production from a CdS / SED hybrid system
[0065] 1. System construction of photocatalytic hydrogen production
[0066] After CdS was synthesized on the cell surface, the nanoparticle hybrid was washed three times with 20mM HEPES to remove sodium thiosulfate and cadmium chloride adsorbed on the cell surface. Afterwards, the washed hybrid system was injected into the reactor for biological hydrogen production. The hydrogen production medium contained 50mM HEPES, 50mM NaCl and 20mM sodium lactate with a pH of 7.0. The volume of the reaction solution was 30mL and the headspace was 55mL. A 300W xenon lamp was used to emit visible light, and a filter was used to filter ultraviolet light below 420nm. The sample was irradiated at a distance of 20cm, and the light intensity was controlled at 1000W / m 2 The reaction system was rotated at 200 rpm, and the reaction temperature was controlled at 30°C using a circulating water bath. The hydrogen production was monitored by gas chromatography every 12 hours.
[0067] 2. Determination of intracellular reducing power level in CdS / SED hybrid system
[0068] Reducing power level was determined using Beyotime NAD + / NADH detection kit (WST-8 method). The CdS / SED hybrid system after the light reaction was centrifuged at 6000 rpm for 5 minutes, then the culture medium was aspirated and pre-cooled NADH / NADH was added. +The cells were then centrifuged at 6000 rpm for 5 minutes at 4°C, and the supernatant was used as the sample to be tested. + To determine the content of NADH, heat the sample in a 60°C water bath for 30 minutes. Add 20μL of standard or sample to a transparent 96-well plate, along with 90μL of alcohol dehydrogenase working solution. Incubate in a 37°C incubator in the dark for 10 minutes. Add 10μL of colorimetric solution and incubate in a 37°C incubator in the dark for 40 minutes. Measure the absorbance at 450nm using a microplate reader.
[0069] 3. Results
[0070] Photocatalytic hydrogen production Figure 5 As shown, after 96 hours of photocatalytic hydrogen production reaction, the hydrogen production capacity of the hybrid system CdS / SED reached 414.0±17.7μmol / mg, which is much higher than the 221.6±14.9μmol / mg of the CdS / MR-1 system; In addition, Figure 6 The intracellular reducing power equivalent (NADH / NAD + ) is 2.72, much higher than 2.05 of CdS / MR-1.
[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention. Sequence Listing 1 7022 DNA Artificial Sequence 1 2 1126 DNA Artificial Sequence 2
Claims
1. A recombinant plasmid, characterized in that: The basic vector plasmid contains at least one promoter and HydA from Desulfovibrio desulfuricans. dds The gene expression element composed of the hydrogenase synthesis gene has a sequence shown in SEQ ID NO.
2.
2. The recombinant plasmid according to claim 1, characterized in that The basic plasmid vector is pYYDT.
3. The recombinant plasmid according to claim 1-2 is introduced into the wild-type Shewanella oneidensis MR-1 to obtain the hydrogen-producing Shewanella engineered strain SED.
4. A semiconductor nanoparticle, characterized in that Na2S2O3 and CdCl2 were added to Shewanella oneidensis successively, and CdS nanoparticles were self-synthesized through intracellular metabolism and attached to the bacterial outer membrane and periplasm.
5. Use of the hydrogen-producing Shewanella engineered strain according to claims 1-4 and CdS nanoparticles in photocatalytic hydrogen production in a semiconductor-microorganism hybrid system.