Method for rapidly degrading new pollutants by utilizing active oxygen generated by photosynthetic bacteria

Abstract

The invention discloses a method for rapidly degrading new pollutants by utilizing active oxygen generated by photosynthetic bacteria, and belongs to the field of water treatment. The photosynthetic bacteria inoculated in the culture medium are subjected to growth metabolism through illumination, and the photosynthetic bacteria in the stable period are obtained; the method comprises the following steps: adding a stable-phase photosynthetic bacterium solution into a to-be-treated water body containing new pollutants, and providing a visible light source or a full-spectrum light source to efficiently generate active oxygen, thereby realizing rapid degradation of the new pollutants. The method for degrading the new pollutants by using the active oxygen generated by the photosynthetic bacteria has the advantages of high treatment efficiency, greenness, economy and simplicity in operation, and a new solution is provided for the problem of new pollutant residues in the water body.

Description

Technical Field

[0001] The invention belongs to the field of water treatment, and in particular relates to a method for rapidly degrading new pollutants by utilizing photosynthetic bacteria to generate active oxygen. Background Art

[0002] With the rapid development of the economy and society, a large number of toxic and hazardous chemicals are produced, used and discharged in industries such as industry, agriculture, medical care and social life, resulting in a wide variety of new pollutants being detected in water environments around the world. New pollutants, including persistent organic pollutants, endocrine disruptors and antibiotics, generally have the characteristics of environmental persistence, bioaccumulation, serious harm and hidden risks, thus continuously destroying the ecosystem, endangering biological safety and threatening human health.

[0003] Sewage treatment plants are centralized purification sites for anthropogenic sewage before it is discharged into environmental water bodies. The primary treatment and secondary treatment processes are mainly physical and biological methods, respectively. Physical separation methods are difficult to separate dissolved trace new pollutants. Various biological methods, represented by the activated sludge method, use the growth and metabolism of microorganisms to remove dissolved and colloidal biodegradable organic matter, nitrogen and phosphorus in sewage, but the removal effect of biologically inert new pollutants is poor. Therefore, various new pollutants often remain in the effluent of sewage treatment plants, and further use of appropriate deep treatment processes is required to effectively reduce the discharge of new pollutants into receiving water bodies. Advanced oxidation technology and other deep treatment processes can produce highly oxidizing free radicals, thereby achieving the removal of new pollutants in the effluent. However, the current mainstream advanced oxidation technologies, including photocatalytic oxidation, ozone oxidation, electrochemical oxidation, and Fenton oxidation, generally have the disadvantages of high treatment costs, large oxidant consumption, and secondary pollution, which reduces the feasibility of their practical application.

[0004] Therefore, in order to solve the problem of new pollutant residues in the water environment, finding an economical, green, environmentally friendly, simple and efficient treatment method is a technical problem that needs to be solved urgently by technical personnel in this field. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for rapidly degrading new pollutants by utilizing photosynthetic bacteria to produce active oxygen. The purpose is to utilize the characteristics of photosynthetic bacteria, such as low price, green and harmless, simple cultivation operation, and the ability to efficiently produce active oxygen with high reaction activity under visible light or sunlight irradiation, to effectively remove new pollutants in water treatment.

[0006] The method of the present invention for rapidly degrading new pollutants by utilizing photosynthetic bacteria to generate active oxygen comprises the following steps:

[0007] (1) Activate photosynthetic bacteria, inoculate the activated photosynthetic bacteria into the culture medium, and use a tungsten filament lamp to provide light to allow the photosynthetic bacteria to grow and metabolize until the stable period.

[0008] (2) Adding the photosynthetic bacterial solution in the stable phase obtained in step (1) above to the water body to be treated containing new pollutants, stimulating them to produce active oxygen by providing light, degrading the new pollutants, and monitoring the concentration changes of the new pollutants in the water body.

[0009] Furthermore, in step (1), the photosynthetic bacteria is Rhodopseudomonas palustris, which is purchased from Guangdong Microbial Culture Collection Center.

[0010] Furthermore, in step (1), the activation step of the photosynthetic bacteria is as follows: the photosynthetic bacteria liquid is transferred to a freshly prepared yeast culture medium at a volume ratio of 5%-20%, the culture container is a sealed serum bottle with a 5% volume headspace, and the culture is carried out for 1.5 days to the logarithmic phase under the light intensity of 2000 lux provided by a 40W tungsten lamp, the culture temperature is 30°C, and the culture is manually inverted and shaken 3 times a day. The composition of the yeast culture medium is: 1 g / L K2HPO4, 0.5 g / L MgSO4, and 10 g / L yeast extract. After the above culture medium is prepared with ultrapure water in a blue-mouth bottle, it is sterilized at 121°C for 20 min using an autoclave, and it can be used after it is naturally cooled to room temperature.

[0011] Furthermore, in step (1), the photosynthetic bacteria inoculation step is as follows: the activated photosynthetic bacteria solution is divided into 50 mL sterile centrifuge tubes, centrifuged at 6000 rpm for 5 min, the supernatant is discarded, the bacteria are resuspended in culture medium and evenly dispersed by vortexing, centrifuged again at 6000 rpm for 5 min, and this is repeated three times to complete the washing. The washed photosynthetic bacteria are resuspended in culture medium to an initial OD of 600 = 0.35. The operation of adding and pouring liquid should be carried out in a clean bench. The composition of the culture medium is: 1 g / L sodium acetate, 1 g / L K2HPO4, 0.5 g / L MgSO4, 0.5 g / LNaCl, 1 g / L NH4Cl. Prepare the above culture medium with ultrapure water in a blue-mouth bottle, adjust the pH to 7.1 with hydrochloric acid, sterilize it at 121°C for 20 min in an autoclave, and use it after naturally cooling to room temperature.

[0012] Furthermore, in step (1), the culture conditions for the growth and metabolism of the photosynthetic bacteria are: the culture container is a sealed serum bottle with a 5% volume headspace, and the culture is carried out for 6 days to the stable period under the light intensity of 2000 lux provided by a 40 W tungsten filament lamp, the culture temperature is 30°C, and the culture is manually inverted and shaken 3 times a day.

[0013] Furthermore, in step (2), the dosage of the photosynthetic bacterial solution is based on OD 600 = 0.01-0.5.

[0014] Furthermore, in step (2), the new pollutants include cimetidine and sulfamethoxazole.

[0015] Furthermore, in step (2), the light source wavelength band of the illumination includes at least one or two of the ultraviolet light band (290-400 nm) and the visible light band (400-780 nm) in the full spectrum.

[0016] Furthermore, in step (2), the monitoring of the change in the concentration of the new pollutant is analyzed by high performance liquid chromatography equipped with an ultraviolet detector. The test method for cimetidine is: 5 mM KH2PO4 (pH = 3) and acetonitrile as the mobile phase, and the detection wavelength is 219 nm; the test method for sulfamethazine is: 1‰ formic acid and acetonitrile as the mobile phase, and the detection wavelength is 260nm.

[0017] In the existing technology, the mainstream idea of ​​using microorganisms to degrade pollutants is to transform and degrade pollutants through the growth and metabolism of microorganisms. The basic process is to obtain strains that can effectively use pollutants as nutrients through screening and strengthening, thereby achieving the transformation and degradation of pollutants. In this idea: (1) it is necessary to obtain specific functional strains through screening and strengthening to achieve the transformation and degradation of new pollutants with low bioavailability; (2) it is highly dependent on the metabolic activity of microorganisms, which will limit its effect in actual water bodies, because the conditions of actual water bodies are likely to be far from the optimal growth conditions of functional strains.

[0018] The solution of the present invention is different from the above ideas. The present invention is the first to pay attention to the photochemical activity of the bacterial solution of Rhodopseudomonas palustris, and found that its singlet oxygen quantum yield is as high as 0.4, which is one order of magnitude higher than the singlet oxygen quantum yield of various microbial extracellular secretions that have been reported. It has a photochemical activity similar to that of many artificially synthesized photocatalytic materials, so it can efficiently remove pollutants.

[0019] In addition, the main role in the present invention is played by the extracellular secretions secreted into the solution by Rhodopseudomonas palustris, and even after the bacterial cells in the bacterial solution are removed, the same pollutant removal effect is still achieved.

[0020] Therefore, the scheme of the present invention does not need to consider the metabolic activity of Rhodopseudomonas palustris in the water to be treated, so that the degradation and removal of pollutants can be efficiently achieved in water environments with various water quality conditions.

[0021] The beneficial effects of the present invention are embodied in:

[0022] 1. The photosynthetic bacteria in the present invention have a singlet oxygen quantum yield of up to 0.4, have a photochemical activity similar to that of artificially synthesized photocatalytic materials, and can efficiently remove new pollutants.

[0023] 2. Compared with the catalysts in current advanced oxidation technologies, the photosynthetic bacteria in the present invention are low-priced, simple to culture and operate, and are suitable for large-scale production and application.

[0024] 3. Compared with the current photocatalytic oxidation method, ozone oxidation method, electrochemical oxidation method and other technologies, the present invention only needs to use the visible light with higher penetration in sunlight to drive the process operation, without the need for additional instruments and energy input.

[0025] 4. Compared with the chemicals required in the current advanced oxidation technology, the photosynthetic bacteria used in the present invention are green and harmless, and will not cause secondary pollution problems such as iron sludge formation and reagent residues in the Fenton oxidation process.

[0026] 5. The main strong oxidizing active species used in the present invention is selective singlet oxygen, which can reduce the interference of water quality compared with the non-selective hydroxyl radicals relied on in methods such as Fenton oxidation and ozone oxidation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the growth curve of the photosynthetic bacteria in the culture medium in Example 1-2 of the present invention.

[0028] Figure 2 The electron paramagnetic resonance (EPR) spectra of active oxygen produced by the photosynthetic bacterial liquid in Example 1-2 of the present invention under dark conditions, full-spectrum illumination conditions, and visible light illumination conditions are shown in FIG. Figure 2 (a) is the EPR spectrum of singlet oxygen. Figure 2 (b) is the EPR spectrum of hydroxyl radicals.

[0029] Figure 3 OD in Example 1-2 of the present invention 600 = 0.1 photosynthetic bacteria liquid under full spectrum illumination conditions and visible light conditions, the steady-state concentration and quantum yield of active oxygen produced, Figure 3 (a) is the steady-state concentration and quantum yield of singlet oxygen. Figure 3 (b) shows the steady-state concentration and quantum yield of hydroxyl radicals.

[0030] Figure 4 is a degradation kinetic diagram of the new pollutant in Example 1-2 of the present invention, Figure 4 (a) is the degradation kinetics diagram of cimetidine in Example 1, Figure 4(b) is the degradation kinetics diagram of sulfamethoxazole in Example 2, wherein the darkness + bacterial solution group and the light alone group are control groups, and the light + bacterial solution group is the experimental group to illustrate the effects of both photosynthetic bacterial solution and light. Further, the light + bacterial solution group is the control group, and the light + bacterial solution supernatant group is the experimental group to illustrate the role of the supernatant in the degradation of new pollutants by photosynthetic bacterial solution.

[0031] Figure 5 is the pseudo-primary degradation rate of the new pollutant in Example 1-2 of the present invention, Figure 5 (a) is the pseudo-first order degradation rate of cimetidine in Example 1, Figure 5 (b) is the pseudo-first-order degradation rate of sulfamethoxazole in Example 2, in which only photosynthetic bacterial culture was added to the control group. On this basis, the +isopropanol group was used to quench hydroxyl radicals, the +heavy water group was used to extend the life of singlet oxygen, and the +sodium azide group was used to quench hydroxyl radicals and singlet oxygen at the same time, so as to illustrate the role of singlet oxygen in the degradation of new pollutants by photosynthetic bacteria. DETAILED DESCRIPTION

[0032] In order to more clearly describe the technical solution of the present invention, a preferred embodiment is described in detail with the accompanying drawings. Obviously, the technical solution of the present invention is not limited to the specific embodiments listed below, and all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Embodiment 1:

[0034] 1. Activate photosynthetic bacteria, inoculate the activated photosynthetic bacteria into a culture medium, and use a tungsten filament lamp to provide light to allow the photosynthetic bacteria to grow and metabolize until a stable period.

[0035] In step 1, the photosynthetic bacteria is Rhodopseudomonas palustris, purchased from Guangdong Microbiological Culture Collection Center, with a collection number of GDMCC NO: 1.167.

[0036] In step 1, the activation step of the photosynthetic bacteria is as follows: the photosynthetic bacteria solution is transferred to a freshly prepared yeast culture medium at a volume ratio of 10%, the culture container is a closed serum bottle with a 5% volume headspace, and the light intensity provided by a 40 W tungsten lamp is 2000 lux. The culture is cultured for 1.5 days to the logarithmic phase, the culture temperature is 30 ° C, and the yeast culture medium is manually inverted and shaken 3 times a day. The composition of the yeast culture medium is: 1 g / L K2HPO4, 0.5 g / L MgSO4, 10 g / L yeast extract. After the above culture medium is prepared with ultrapure water in a blue-mouth bottle, it is sterilized at 121 ° C for 20 min using an autoclave, and it can be used after naturally cooling to room temperature.

[0037] In step 1, the steps of inoculating photosynthetic bacteria are as follows: the activated photosynthetic bacteria solution is divided into 50 mL sterile centrifuge tubes, centrifuged at 6000 rpm for 5 min, the supernatant is discarded, the bacteria are resuspended in culture medium and evenly dispersed by vortexing, centrifuged again at 6000 rpm for 5 min, and this is repeated three times to complete the washing. The washed photosynthetic bacteria are resuspended in culture medium to an initial OD of 600 = 0.35. The operation of adding and pouring liquid should be carried out in a clean bench. The composition of the culture medium is: 1 g / L sodium acetate, 1 g / L K2HPO4, 0.5 g / L MgSO4, 0.5 g / L NaCl, 1 g / L NH4Cl. Prepare the above culture medium with ultrapure water in a blue-mouth bottle, adjust the pH to 7.1 with hydrochloric acid, sterilize it at 121°C for 20 min in an autoclave, and use it after naturally cooling to room temperature.

[0038] In step 1, the culture conditions for the growth and metabolism of the photosynthetic bacteria are: the culture container is a sealed serum bottle with a 5% headspace, and the culture is carried out under the light intensity of 2000 lux provided by a 40 W tungsten filament lamp for 6 days until the stable period (see Figure 1 ), the culture temperature was 30°C, and the culture medium was manually shaken three times a day.

[0039] In step 1, the obtained photosynthetic bacterial culture in the stable phase does not produce reactive oxygen in the dark, but can produce reactive oxygen including singlet oxygen and hydroxyl radicals under full spectrum or visible light illumination. The production of reactive oxygen under different conditions is qualitatively detected using EPR technology (see Figure 2 ). The illumination is provided by a 500 W xenon lamp, which is equipped with a 420 nm or 290 nm cut-off filter to obtain a visible light source or a full-spectrum light source.

[0040] In step 1, the obtained photosynthetic bacterial solution in the stable phase is at a concentration of OD 600 = 0.1, the steady-state concentration can be 3.4×10 -12 M and 1.3×10 -12 M of singlet oxygen, and the steady-state concentration is 4.3×10 -17 M and 5.2×10 -18 M hydroxyl radical (see Figure 3). The illumination is provided by a 500 W xenon lamp, and a visible light source or a full-spectrum light source is obtained by equipping a 420 nm or 290 nm cutoff filter, respectively. The specific quantitative method of the steady-state concentration of active oxygen is referred to the literature "The Role of Dissolved Organic Matter Composition in Determining Photochemical Reactivity at the Molecular Level".

[0041] In step 1, the singlet oxygen quantum yield of the photosynthetic bacterial culture obtained in the stable phase under full spectrum and visible light illumination conditions was quantitatively detected to be 0.41 and 0.40, respectively, and the hydroxyl radical quantum yield was quantitatively detected to be 1.4×10 -5 and 6.3×10 -6 (See Figure 3 ). The illumination is provided by a 500 W xenon lamp, and a visible light source or a full-spectrum light source is obtained by equipping a 420 nm or 290 nm cutoff filter, respectively. The specific quantitative method of the active oxygen quantum yield is referred to the literature "The Role of Dissolved Organic Matter Composition in Determining Photochemical Reactivity at the Molecular Level".

[0042] 2. Add the photosynthetic bacterial solution obtained in the above step 1 to the water to be treated containing 2 mg / L cimetidine, and degrade cimetidine by providing visible light to stimulate the production of reactive oxygen species, and monitor the concentration change of cimetidine in the water (see Figure 4 (a)).

[0043] In step 2, the dosage of the photosynthetic bacteria solution is based on OD 600 = 0.1.

[0044] In step 2, during the degradation of cimetidine, the pH of the system was controlled to 7.0 by adding 5 mM phosphate buffer.

[0045] In step 2, the visible light is provided by a 500 W xenon lamp equipped with a 420 nm cut-off filter to simulate the visible light in sunlight.

[0046] In step 2, the degradation of cimetidine in the water body reached 0.0%, 2.2%, 50.2%, and 51.3% in the dark + bacterial solution group, the light alone group, the light + bacterial solution group, and the light + bacterial solution supernatant group after 6 h of treatment, respectively (see Figure 4(a)), indicating that the addition of photosynthetic bacterial solution can greatly promote the degradation of cimetidine in water under light conditions, and the main role in this process is played by the extracellular secretions of photosynthetic bacteria in the supernatant of the bacterial solution. The monitoring of the change in cimetidine concentration is analyzed by high performance liquid chromatography equipped with an ultraviolet detector. The specific test method is: 5 mM KH2PO4 (pH = 3) and acetonitrile as mobile phases, and the detection wavelength is 219 nm. In the system of the present invention, prolonging the time can continue degradation and improve the degradation rate; increasing the amount of bacterial solution added can also accelerate degradation. Our research group has conducted relevant experiments. If the bacterial solution concentration changes from OD 600 = 0.1 increased to 0.5, 100% removal of cimetidine could be achieved in 3 hours.

[0047] In step 2, the photosynthetic bacteria-mediated cimetidine degradation mechanism was further analyzed by adding different reactive oxygen quenchers or life extenders (see Figure 5 (a)), the degradation rate of cimetidine did not change significantly after adding 1‰ isopropanol to quench hydroxyl radicals, the degradation rate of cimetidine increased significantly after adding 50% heavy water to extend the lifetime of singlet oxygen, and the degradation rate of cimetidine decreased significantly after adding 5 mM sodium azide to quench both hydroxyl radicals and singlet oxygen, indicating the key role of singlet oxygen in the degradation of cimetidine by photosynthetic bacteria.

[0048] Embodiment 2:

[0049] 1. Same as step 1 of Example 1.

[0050] 2. Add the photosynthetic bacterial solution obtained in the above step 1 to the water to be treated containing 2 mg / L sulfamethoxazole, and degrade sulfamethoxazole by providing visible light to stimulate the production of active oxygen, and monitor the concentration change of sulfamethoxazole in the water (see Figure 4 (b)).

[0051] In step 2, the dosage of the photosynthetic bacteria solution is based on OD 600 = 0.1.

[0052] In step 2, during the degradation of sulfamethoxazole, the pH of the system was controlled to 7.0 by adding 5 mM phosphate buffer.

[0053] In step 2, the visible light is provided by a 500 W xenon lamp equipped with a 420 nm cut-off filter to simulate the visible light in sunlight.

[0054] In step 2, the degradation of sulfamethoxazole in the water body reached 0.0%, 20.7%, 33.5%, and 33.1% in the dark + bacterial solution group, the light alone group, the light + bacterial solution group, and the light + bacterial solution supernatant group after 12 h of treatment, respectively (see Figure 4 (b)), indicating that the addition of photosynthetic bacterial culture can greatly promote the degradation of sulfamethoxazole in water under light conditions, and the main role in this process is played by the extracellular secretions of photosynthetic bacteria in the supernatant of the culture. The monitoring of the change in the concentration of sulfamethoxazole is analyzed by high performance liquid chromatography equipped with an ultraviolet detector. The specific test method is: 1‰ formic acid and acetonitrile are used as the mobile phase, and the detection wavelength is 260 nm.

[0055] In step 2, the mechanism of sulfadiazine degradation mediated by the photosynthetic bacteria was further analyzed by adding different reactive oxygen species quenchers or lifespan extenders (see Figure 5 (b)), the degradation rate of sulfamethoxazole did not change significantly after adding 1‰ isopropanol to quench hydroxyl radicals, the degradation rate of sulfamethoxazole increased significantly after adding 50% heavy water to extend the lifetime of singlet oxygen, and the degradation rate of sulfamethoxazole decreased significantly after adding 5 mM sodium azide to quench hydroxyl radicals and singlet oxygen at the same time, indicating the key role of singlet oxygen in the degradation of sulfamethoxazole by photosynthetic bacteria.

Claims

1. A method for rapidly degrading new pollutants by using photosynthetic bacteria to produce active oxygen, characterized in that The steps include: (1) Activating photosynthetic bacteria, inoculating the activated photosynthetic bacteria into a culture medium, and using light to allow the photosynthetic bacteria to grow and metabolize until a stable period; (2) Adding the photosynthetic bacterial solution in the stable phase obtained in step (1) to the water body to be treated containing new pollutants, and stimulating them to produce active oxygen by providing light to degrade the new pollutants.

2. The method according to claim 1, characterized in that: In step (1), the photosynthetic bacteria is Rhodopseudomonas palustris.

3. The method according to claim 1, characterized in that: In step (1), the activation step of the photosynthetic bacteria is as follows: transferring the photosynthetic bacteria liquid into a freshly prepared yeast culture medium at a volume ratio of 5%-20%, and then culturing under light to the logarithmic phase.

4. The method according to claim 3, characterized in that: The yeast culture medium comprises the following components: 1 g / L K2HPO4, 0.5 g / L MgSO4, 10 g / L yeast extract, and is sterilized at 121°C.

5. The method according to claim 1 or 3, characterized in that: In step (1), the photosynthetic bacteria inoculation step is: the activated photosynthetic bacteria are washed three times with culture medium, and then resuspended in culture medium to make the initial OD 600 = 0.

35.

6. The method according to claim 5, characterized in that: The culture medium comprises: 1 g / L sodium acetate, 1 g / L K2HPO4, 0.5 g / L MgSO4, 0.5 g / L NaCl, 1 g / L NH4Cl, the pH is adjusted to 7.1, and sterilized at 121°C.

7. The method according to claim 1, characterized in that: In step (2), the dosage of the photosynthetic bacterial solution is based on the initial OD 600 = 0.01-0.

5.

8. The method according to claim 1, characterized in that: In step (2), the new pollutant includes one or more of cimetidine and sulfamethoxazole.

9. The method according to claim 1, characterized in that: In step (2), the light source wavelength band of the illumination includes at least one or two of the ultraviolet light band and the visible light band in the full spectrum.

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