A semi-artificial photosynthetic material enhanced by biologically active substances and its preparation method and application

Abstract

The present invention discloses a bioactive substance-reinforced semi-artificial photosynthetic material, its preparation method and application. In the present invention, a culture medium containing extracellular respiratory bacteria and water-soluble sulfur-containing compounds is anaerobically cultured; then a bioactive substance is added, and then a solution of a water-soluble Cd<supgt;2+< / supgt>-containing compound is added for anaerobic culture to obtain the semi-artificial photosynthetic material. The bioactive substance wrapped on the surface of bacteria and CdS in the present invention can inhibit the generation of CdS free radicals, thereby reducing the damage to bacteria. At the same time, the bioactive substance contains a variety of functional groups, which can effectively bind Cd<supgt;2+< / supgt; generated by the dissolution of CdS during the photocatalytic process and high-concentration heavy metal ions in the initial stage of the heavy metal wastewater treatment process, alleviating the toxic effect of heavy metal ions on cells. The maintenance of bacterial activity enables the semi-artificial photosynthetic material to have higher photocatalytic stability and a longer service life compared with conventional semi-artificial photosynthetic materials.

Description

Technical Field

[0001] The invention belongs to the technical field of water treatment, and in particular relates to a semi-artificial photosynthetic material enhanced by biologically active substances, and a preparation method and application thereof. Background Art

[0002] Chromium has a wide range of uses in industry, and with it comes the massive generation of chromium-containing wastewater. Commonly used physical and chemical treatment methods include adsorption, flocculation, ion exchange, and chemical oxidation-reduction, but they have the disadvantages of high cost and easy secondary pollution. Therefore, bioremediation is considered to be an effective alternative. For example, researchers have screened out some strains with natural resistance to heavy metals from natural samples and used them to reduce hexavalent chromium to trivalent chromium, which has weaker biological toxicity and mobility. However, traditional bioremediation technology is still limited by the large amount of organic matter (such as glycerol, lactic acid, etc.) added and the long repair time.

[0003] In response to these problems, researchers have recently proposed using semi-artificial photosynthetic materials composed of microorganisms and semiconductors to treat pollutants, including azo dyes and hexavalent chromium, under the irradiation of sunlight. Under the excitation of light of appropriate wavelength, semiconductors will generate electron-hole pairs. When the holes react with suitable scavengers (such as cysteine), the photogenerated electrons can be transferred to microorganisms. These electrons can enter the cell as reducing equivalents for respiration, or they can be ultimately transferred to pollutants through oxidoreductases on the surface of bacteria, accelerating the rate of reduction / degradation of pollutants. However, during the process of photocatalytic reduction, a large number of free radicals (including ·OH, ·O2 - and H2O2) will damage or even kill bacteria, seriously affecting the stability of the treatment effect of the biocomposite material; and in the process of using a semi-artificial photosynthetic system to treat heavy metals, the coexisting heavy metal ions will also have a negative impact on the activity of bacteria. Du et al. used Shewanella Oneida-semiconductor composites to remove azo dyes. After three consecutive cycles of operation, the removal efficiency dropped significantly. At the same time, the system needs to add more lactic acid as a microbial electron donor during operation, which increases the operating cost of the system (Du Y, Guo J, Hou YN, et al. A light-switch for efficient decolorization of azo dye wastewater based on bacteria–semiconductor interaction [J]. Environmental Science: Nano, 2022, 9: 1819-1830.). Therefore, there is still a lack of efficient and low-cost response methods for these two threats that bacteria may encounter during the treatment process. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to reduce the photochemical toxicity of the semi-artificial photosynthetic material itself, thereby improving the stability of its photocatalytic application, and to provide a semi-artificial photosynthetic material enhanced with biologically active substances and a preparation method and application thereof.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for preparing a semi-artificial photosynthetic material enhanced by biologically active substances comprises the following steps:

[0007] (1) anaerobically culturing a culture medium containing extracellular respiratory bacteria and a water-soluble sulfur-containing compound;

[0008] (2) Add the bioactive substance to the mixed solution obtained in step (1), and then add a water-soluble Cd-containing 2+ The solution of the compound is cultured anaerobically to obtain a semi-artificial photosynthetic material enhanced with biologically active substances.

[0009] Preferably, the concentration of the extracellular respiratory bacteria in step (1) is 1×10 7 ~10 8 CFU / mL (concentration before anaerobic culture); further preferably, the concentration of the extracellular respiratory bacteria is 5×10 7 CFU / mL;

[0010] Preferably, the extracellular respiratory bacteria in step (1) are selected from one or more of Shewanella Oneida and Geobacter sulfurreducens.

[0011] Preferably, the water-soluble sulfur-containing compound in step (1) is selected from one or more of thiosulfate, sulfite and sulfide salt; and the concentration of the water-soluble sulfur-containing compound in the culture medium is 5 to 15 mmol / L.

[0012] Preferably, the bioactive substance in step (1) is a solution of the bioactive substance.

[0013] Preferably, the culture medium in step (1) is a sterile anaerobic culture medium.

[0014] Preferably, the anaerobic culture time in step (1) is 24 to 36 hours.

[0015] Preferably, the temperature of the anaerobic culture in step (1) is 20-35° C.; the rotation speed of the anaerobic culture is 100-200 rpm.

[0016] Preferably, the bioactive substance in step (2) is produced by Shewanella Oneida.

[0017] Further preferably, the bioactive substance is extracted after culturing Oneida Shewanella; specifically, Oneida Shewanella is cultured and resuspended to obtain a bacterial solution, then shaken and centrifuged to obtain a supernatant, and dialyzed to obtain the bioactive substance.

[0018] Preferably, the amount of the bioactive substance added in step (2) is 0.5 to 2 mg / mL.

[0019] Preferably, the water-soluble Cd-containing 2+ The compound is selected from one or more of cadmium chloride and cadmium nitrate; the water-soluble Cd-containing 2+ The amount of compound added was 0.5 to 2 mmol / L;

[0020] Preferably, the anaerobic culture time in step (2) is 36 to 48 hours.

[0021] Preferably, the temperature of the anaerobic culture in step (2) is 20-35°C; the rotation speed of the anaerobic culture is 100-200 rpm.

[0022] The semi-artificial photosynthetic material enhanced with bioactive substances prepared by the above-mentioned preparation method; excessive bioactive substances are distributed on the cell surface of the extracellular respiratory bacteria, and CdS nanoparticles are loaded in the bioactive substances.

[0023] The application of the above-mentioned semi-artificial photosynthetic material enhanced by bioactive substances in the treatment of heavy metal-containing wastewater.

[0024] Preferably, it is carried out under the action of light.

[0025] Preferably, the heavy metal-containing wastewater includes hexavalent chromium ions.

[0026] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0027] (1) The bioactive substances used in the present invention are natural biomacromolecules, and the extraction method used is simple, effective, green and safe, and does not require other pretreatment and subsequent processing; 2+ The previous method of injecting bioactive substances into the culture system minimized costs.

[0028] (2) The bioactive substances wrapped on the surface of bacteria and CdS can inhibit the generation of CdS free radicals, thereby reducing their damage to bacteria. At the same time, the bioactive substances contain a variety of functional groups that can effectively bind to the CdS produced by the dissolution of CdS during the photocatalytic process. 2+ High concentration of Cr in the initial stage of heavy metal wastewater treatment 6+The maintenance of bacterial activity makes the material have higher photocatalytic stability and longer service life compared with conventional semi-artificial photosynthetic materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are SEM images of the microorganism-CdS composite material and the microorganism-bioactive substance-CdS composite material in Example 2 of the present invention.

[0030] Figure 2 1 and 1 are XRD diagrams of the microorganism-CdS composite material and the microorganism-bioactive substance-CdS composite material in Example 2 of the present invention.

[0031] Figure 3 1 is the tauc plot of the microorganism-CdS composite material and the microorganism-bioactive substance-CdS composite material in Example 2 of the present invention.

[0032] Figure 4 This is a graph showing the experimental kinetic data of photocatalytic reduction of hexavalent chromium by the microorganism-CdS system under different conditions in Comparative Example 2 of the present invention.

[0033] Figure 5 This is a graph showing the experimental kinetic data of the photocatalytic reduction of hexavalent chromium by the microorganism-CdS system and the microorganism-bioactive substance-CdS system in Example 3 of the present invention. DETAILED DESCRIPTION

[0034] The specific method, materials and application of using bioactive substances to improve the photocatalytic stability of semi-artificial photosynthetic materials in the present invention are described in detail below in conjunction with the examples, which are not intended to limit the present invention.

[0035] It should be noted that, if the experimental operation in the following examples is not particularly described in detail, reference can be made to conventional methods in the field. The reagents used in the examples can all be purchased from the market.

[0036] Example 1

[0037] The biologically active substances are obtained by the following operations:

[0038] (1) Dip a loop of thawed Oneida Shewanella (ATCC_700550) cryopreservation solution with an inoculating loop and culture on LB agar solid plate (30°C, 24 h), then pick a single Oneida Shewanella colony from the solid agar plate and culture it in LB liquid medium with constant temperature shaking (30°C, 150 rpm) for 48 h to obtain a bacterial suspension;

[0039] (2) Centrifuge the bacterial suspension (5000 g, 4°C, 10 min), discard the supernatant, and then add an equal volume of sterile 0.9% NaCl solution to resuspend the bacteria;

[0040] (3) The bacterial solution was vortexed for 2 minutes and centrifuged again (5000g, 4°C, 20 minutes). The supernatant obtained was the bioactive substance. The supernatant was placed in a sealed 1000da dialysis bag and dialyzed in deionized water for 24 hours. The dialyzed solution was then freeze-dried to obtain the powder of the bioactive substance, which was sealed and stored at 4°C.

[0041] Example 2

[0042] The composite material of Shewanella oneidensis MR-1 loaded with CdS was selected as a control semi-artificial photosynthetic material, and bioactive substances were introduced at an appropriate time. The resulting bacteria-bioactive substances-CdS hybrid was observed, characterized and performance tested.

[0043] The preparation method of the microorganism-bioactive substance-CdS composite material comprises the following steps:

[0044] (1) Preparation of Shewanella seed culture medium: A single colony of Shewanella Oneida was picked from a solid agar plate and placed in 100 mL of LB liquid culture medium (containing 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride) and cultured at a constant temperature (30°C, 150 rpm) for 12 h to obtain a bacterial suspension. After centrifugation, the sediment was resuspended in sterile saline. This operation was repeated at least twice. The OD value of the bacterial suspension was finally calculated. 600 The value dilution is 2.0.

[0045] (2) Preparation of anaerobic culture medium: Dissolve 4.5 g sodium lactate, 1.6 g sodium thiosulfate, and 0.6 g cysteine ​​in 950 mL LB culture medium (containing 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride), adjust the pH value of the solution to 7.2, and finally make up to 1000 mL. Dispense the culture medium into 100 mL serum bottles (50 mL per bottle), fully aerate and deoxygenate with nitrogen (15-20 min), seal and sterilize (121°C, 20 min).

[0046] (3) Preparation of bacteria-bioactive substance-CdS composite material: The Shewanella seed culture medium was added into the sterilized anaerobic culture medium by syringe to a final concentration of 5×10 7CFU / mL; after culturing at constant temperature and shaking (30°C, 150rpm) for 24h, add 100mg / mL of the bioactive substance solution of Example 1 that has been filtered and sterilized (final concentration is 2mg / mL), and oscillate at constant temperature (30°C, 150rpm) for 30min; finally, add sterilized 25mmol / L CdCl2 mother solution (final concentration is 0.5mmol / L), and culturing at constant temperature and shaking (30°C, 150rpm) for 36h to obtain a composite material.

[0047] For conventional systems without adding biologically active substances, the step of adding biologically active substances is omitted.

[0048] The bacteria-bioactive substance-CdS composite material prepared in this example was characterized by materials science:

[0049] Figure 1 is the SEM image of microorganism-CdS composite material and microorganism-bioactive substance-CdS composite material. Figure 1 It can be seen that more round nanoparticles are loaded on the surface of microorganisms with added bioactive substances.

[0050] Figure 2 2 are XRD patterns of the microorganism-CdS composite material and the microorganism-bioactive substance-CdS composite material. Compared with the standard card (JADE6 PDF#75-1546), it can be seen that the characteristic peaks of the composite material synthesized in Example 1 at 2θ of 26.7°, 43.8° and 51.6° correspond to the characteristic peaks of CdS at the (111), (220) and (311) crystal planes, respectively, confirming that the mineral phase composition of the obtained two composite materials is mainly CdS.

[0051] Figure 3 is the tauc plot of microorganism-CdS composite material and microorganism-bioactive substance-CdS composite material. Figure 3 It can be seen that the band gap of the microorganism-CdS composite material is 2.53ev, while the band gap of the microorganism-bioactive substance-CdS composite material is 2.55ev. This shows that compared with the conventional microorganism-CdS composite material, the CdS photoelectric conversion efficiency in the microorganism-bioactive substance-CdS composite material is slightly lower, which may lead to fewer photoelectrons and free radicals generated during the photocatalytic process, thereby reducing its damage to bacteria.

[0052] Comparative Example 1

[0053] Based on a single factor control experiment, the microorganism-CdS composite material synthesized in Example 1 was used to treat Cr-containing 6+ Wastewater, explore the role of each component of the composite material.

[0054] (1) 50 mL of the synthesized composite material solution was centrifuged (3000 g, 10 min), washed once with sterile saline, and then washed with 20 mmol / L HEPES buffer (4-hydroxyethylpiperazineethanesulfonic acid), 1 mmol / L cysteine ​​and 50 mg / L Cr 6+ The solution was resuspended to the same volume (i.e. 50 mL) to obtain a working solution; the above operations were carried out in the dark.

[0055] (2) The working solution was dispensed into 50 mL serum bottles (20 mL per bottle), which were then sealed and placed under a 400 ± 5 nm LED light strip (3.07 ± 0.14 mW / cm 2 ) and maintain the temperature in the range of 25-35°C. Samples were taken at specific time points to determine the Cr content in the solution. 6+ Residual amount.

[0056] Figure 4 This is the experimental kinetic data diagram of the photocatalytic reduction of hexavalent chromium by microorganism-CdS composite materials under various conditions. Figure 4 It can be seen that the complete composite material has the highest efficiency in photoreduction of hexavalent chromium under light conditions, while the composite material that has been sterilized at high temperature (bacteria die) can only reduce 80% of hexavalent chromium under the same conditions, and the reaction rate is about 1 / 3 of the former. Under dark conditions, the efficiency of composite materials in reducing hexavalent chromium is very low. At the same time, the reduction effect of bacteria on hexavalent chromium is also very limited regardless of light or dark conditions, which may be related to the fact that we did not add electron donors to the system. The above results show that light is the main driving factor for the reduction of hexavalent chromium in this material, and bacteria and CdS may have a synergistic effect in the photoreduction of hexavalent chromium.

[0057] Example 3

[0058] The microorganism-CdS composite material and the microorganism-bioactive substance-CdS composite material synthesized in Example 1 were used to treat Cr-containing 6+ The working solution preparation process and treatment conditions were the same as those in Comparative Example 1.

[0059] Figure 5 The experimental kinetic data of photocatalytic reduction of hexavalent chromium by microorganism-CdS composite material and microorganism-bioactive substance-CdS composite material are shown in Figure 2. Figure 5 It can be seen that in the first two cycles, the Cr 6+ The reduction rate is slightly faster than that of the microorganism-bioactive substance-CdS system. However, starting from the fourth cycle, the Cr reduction rate of the bioactive substance-CdS within 1.5 h 6+The reduction degree can only reach about 75%, while the microorganism-bioactive substance-CdS system can still reach more than 99%. In the 5-cycle experiment lasting 7.5 hours, the Cr 6+ The total concentration removed was about 210 mg / L, while that of the microorganism-bioactive substance-CdS system was 250 mg / L; the total level of the five rounds of treatment increased to 1.2 times the original level. This shows that although the increase in bioactive substances will slightly slow down the reaction rate, its treatment effect in the later stage of the reaction cycle is significantly better than that of general microorganism-CdS materials, enabling the material to reduce more Cr under the same conditions. 6+ , proving that the microorganism-bioactive substance-CdS semi-artificial photosynthetic material has better stability and sustainability.

[0060] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of a semi-artificial photosynthetic material enhanced by biologically active substances in the treatment of heavy metal-containing wastewater, characterized in that: The heavy metal is hexavalent chromium ion; The preparation method of the semi-artificial photosynthetic material enhanced by the biologically active substance comprises the following steps: (1) using an inoculating loop to dip a loop of thawed ATCC_700550 Oneida Shewanella cryopreservation solution and culture it on a LB agar solid plate at 30°C for 24 hours, then picking a single Oneida Shewanella colony from the solid agar plate and culture it in LB liquid culture medium at 30°C and 150 rpm for 48 hours to obtain a bacterial suspension; (2) Centrifuge the bacterial suspension at 5000 g and 4°C for 10 min, discard the supernatant, and then add an equal volume of sterile 0.9% NaCl solution to resuspend the bacteria; (3) The resuspended cells were vortexed for 2 minutes, centrifuged again at 5000 g and 4°C for 20 minutes, the supernatant was placed in a sealed 1000 Da dialysis bag, dialyzed in deionized water for 24 h, and then the dialyzed solution was freeze-dried to obtain the bioactive substance; (4) 4.5 g of sodium lactate, 1.6 g of sodium thiosulfate, and 0.6 g of cysteine ​​were dissolved in 950 mL of LB culture medium containing 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, and the pH value of the solution was adjusted to 7.

2. The volume was finally fixed to 1000 mL; nitrogen aeration was used for deoxygenation for 15 to 20 min, and the culture medium was sealed and sterilized at 121° C. for 20 min to obtain an anaerobic culture medium; (5) OD 600 The Shewanella seed suspension with a value of 2.0 was added to the sterilized anaerobic medium by syringe to a final concentration of 5×10 7 CFU / mL; after culturing at a constant temperature of 30°C and 150rpm for 24h, add a 100mg / mL solution of the bioactive substance in step (3) that has been filtered and sterilized, and the final concentration of the bioactive substance is 2mg / mL, and oscillate at a constant temperature of 30°C and 150rpm for 30min; finally, add a sterilized 25mmol / L CdCl2 mother solution, and the final concentration of CdCl2 is 0.5mmol / L, and culturing at a constant temperature of 30°C and 150rpm for 36h to obtain a semi-artificial photosynthetic material enhanced with bioactive substances.

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