A method for rapidly degrading new pollutants by using photosynthetic bacteria to produce active oxygen

Abstract

The present invention discloses a method for rapidly degrading new pollutants by utilizing photosynthetic bacteria to generate active oxygen, and belongs to the field of water treatment. The present invention causes photosynthetic bacteria inoculated in a culture medium to grow and metabolize through illumination, thereby obtaining photosynthetic bacteria in a stable phase; a photosynthetic bacterial solution in a stable phase is added to a water body to be treated containing new pollutants, and a visible light source or a full-spectrum light source is provided to enable the efficient generation of active oxygen, thereby achieving rapid degradation of the new pollutants. The present invention's method for degrading new pollutants by utilizing photosynthetic bacteria to generate active oxygen has the advantages of high treatment efficiency, green economy, and simple operation, and provides a new solution to the problem of residual new pollutants in water bodies.

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 being produced, used, and discharged in industries such as industry, agriculture, healthcare, and social life. This has led to the detection of a wide variety of new pollutants in water environments around the world. These new pollutants, including persistent organic pollutants, endocrine disruptors, and antibiotics, are generally characterized by environmental persistence, bioaccumulation, severe harm, and hidden risks. Consequently, they continue to damage ecosystems, endanger biosafety, and threaten human health.

[0003] Wastewater treatment plants are centralized purification sites for anthropogenic wastewater before it is discharged into the environment. Primary and secondary treatment processes are primarily physical and biological, respectively. Physical separation methods struggle to isolate trace amounts of new pollutants in dissolved form. Various biological methods, such as the activated sludge process, utilize microbial growth and metabolism to remove dissolved and colloidal biodegradable organic matter, nitrogen, and phosphorus from wastewater. However, they are ineffective in removing biologically inert new pollutants. Consequently, various new pollutants often remain in sewage treatment plant effluent, necessitating the use of appropriate advanced treatment processes to effectively reduce the release of new pollutants into receiving waters. Advanced treatment processes, such as advanced oxidation technologies, can generate highly oxidizing free radicals, thereby removing new pollutants from the effluent. However, currently mainstream advanced oxidation technologies, including photocatalytic oxidation, ozone oxidation, electrochemical oxidation, and Fenton oxidation, suffer from high treatment costs, high oxidant consumption, and secondary pollution, reducing their practical feasibility.

[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 technicians in this field. Summary of the Invention

[0005] The present invention addresses the problems existing in the prior art and provides a method for rapidly degrading new pollutants by utilizing photosynthetic bacteria to generate active oxygen. The method aims to utilize the characteristics of photosynthetic bacteria, such as low price, green and harmless, simple cultivation operation, and the ability to efficiently generate highly reactive active oxygen under visible light or sunlight, to achieve effective removal of 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 Provincial Microbial Culture Collection Center.

[0010] Furthermore, in step (1), the activation of the photosynthetic bacteria is as follows: transferring the photosynthetic bacteria solution to a freshly prepared yeast culture medium at a volume ratio of 5%-20%, the culture container being a sealed serum bottle with a 5% headspace, culturing for 1.5 days to the logarithmic phase under illumination of 2000 lux provided by a 40W tungsten filament lamp, the culture temperature being 30°C, and manually shaking the culture medium three 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 preparing the above culture medium with ultrapure water in a blue-mouthed bottle, sterilize it in an autoclave at 121°C for 20 minutes and allow it to cool naturally to room temperature before use.

[0011] Furthermore, in step (1), the steps of inoculating the 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, and 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 make the initial OD 600 = 0.35. Addition and decanting should be performed in a clean bench. The culture medium consists of: 1 g / L sodium acetate, 1 g / L K2HPO4, 0.5 g / L MgSO4, 0.5 g / L NaCl, and 1 g / L NH4Cl. Prepare the culture medium in a blue-mouth bottle with ultrapure water. Adjust the pH to 7.1 with hydrochloric acid. Sterilize in an autoclave at 121°C for 20 minutes. Allow to cool naturally to room temperature before use.

[0012] Furthermore, in step (1), the culture conditions for the growth and metabolism of the photosynthetic bacteria are as follows: the culture container is a sealed serum bottle with a 5% headspace, the culture is carried out under illumination with a light intensity of 2000 lux provided by a 40 W tungsten filament lamp for 6 days until the stable period, 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 bacteria 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 performed 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 260 nm.

[0017] In the existing technology, the mainstream approach to 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 utilize pollutants as nutrients through screening and strengthening, thereby achieving the transformation and degradation of pollutants. In this approach: (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 in actual water bodies are likely to be far from the optimal growth conditions of functional strains.

[0018] The present invention differs from the above approach. It is the first to examine the photochemical activity of Rhodopseudomonas palustris bacterial broth and discover that its singlet oxygen quantum yield is as high as 0.4, an order of magnitude higher than the singlet oxygen quantum yields reported for various microbial extracellular secretions. This demonstrates photochemical activity comparable to that of many synthetic photocatalytic materials, enabling highly efficient pollutant removal.

[0019] In addition, the present invention plays a major role in 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, and thus can efficiently achieve the degradation and removal of pollutants 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 inexpensive, 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 uses the visible light with higher penetrability in sunlight to drive the process operation, without the need for additional instruments and energy input.

[0025] 4. Compared with the chemicals required in current advanced oxidation technologies, 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 chemical 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 the photosynthetic bacteria solution in Example 1-2 of the present invention producing active oxygen under dark conditions, full spectrum illumination conditions, and visible light illumination conditions are shown. 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, 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 This 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 a degradation kinetic diagram of sulfamethazine in Example 2, wherein the darkness + bacterial solution group and the light-only 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. Furthermore, 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-first-order degradation rate of the new pollutant in Examples 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 sulfamethazine 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 lifetime of singlet oxygen, and the +sodium azide group was used to quench hydroxyl radicals and singlet oxygen simultaneously to illustrate the role of singlet oxygen in the degradation of new pollutants by photosynthetic bacteria. DETAILED DESCRIPTION

[0032] To more clearly describe the technical solution of the present invention, preferred embodiments are described in detail with reference to the accompanying drawings. Obviously, the technical solution of the present invention is not limited to the specific embodiments listed below. All other embodiments obtained by persons of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 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 the stable period.

[0035] In step 1, the photosynthetic bacteria is Rhodopseudomonas palustris, purchased from Guangdong Provincial Microbial 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 liquid is transferred to a freshly prepared yeast culture medium at a volume ratio of 10%. The culture container is a sealed serum bottle with a 5% headspace. The culture is cultured for 1.5 days to the logarithmic phase under a 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 three 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 preparing the above culture medium with ultrapure water in a blue-mouth bottle, sterilize it in an autoclave at 121°C for 20 minutes and let it cool naturally to room temperature before use.

[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, and 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. Addition and decanting should be performed in a clean bench. The culture medium consists of: 1 g / L sodium acetate, 1 g / L K2HPO4, 0.5 g / L MgSO4, 0.5 g / L NaCl, and 1 g / L NH4Cl. Prepare the culture medium in a blue-mouthed bottle with ultrapure water. Adjust the pH to 7.1 with hydrochloric acid. Sterilize in an autoclave at 121°C for 20 minutes and allow to cool naturally to room temperature before use.

[0038] In step 1, the culture conditions for the growth and metabolism of the photosynthetic bacteria are as follows: the culture container is a sealed serum bottle with a 5% headspace, and the culture is carried out under a 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℃, and the culture medium was manually shaken three times a day.

[0039] In step 1, the obtained photosynthetic bacterial culture in the stationary phase does not produce reactive oxygen species in the dark, but can produce reactive oxygen species including singlet oxygen and hydroxyl radicals under full spectrum or visible light illumination. The production of reactive oxygen species under different conditions was 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 3The illumination was provided by a 500 W xenon lamp equipped with a 420 nm or 290 nm cutoff filter, respectively, to provide a visible light source or a full-spectrum light source. The specific method for quantifying the steady-state concentration of reactive oxygen species is referenced in 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 was provided by a 500 W xenon lamp equipped with a 420 nm or 290 nm cutoff filter, respectively, to provide a visible light source or a full-spectrum light source. The specific quantitative method for the reactive oxygen species quantum yield is referenced in the literature "The Role of Dissolved Organic Matter Composition in Determining Photochemical Reactivity at the Molecular Level."

[0042] 2. Add the photosynthetic bacteria 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 at 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 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 the concentration of cimetidine is analyzed by high-performance liquid chromatography equipped with a UV 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 can be achieved in 3 hours.

[0047] In step 2, the mechanism of cimetidine degradation mediated by the photosynthetic bacteria was analyzed by further adding different reactive oxygen species quenchers or lifespan 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 was significantly increased after adding 50% heavy water to extend the lifetime of singlet oxygen. The degradation rate of cimetidine was significantly reduced 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] Example 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 sulfamethazine, and degrade sulfamethazine by providing visible light to stimulate the production of active oxygen, and monitor the concentration change of sulfamethazine 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 sulfamethazine, the pH of the system was controlled at 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 sulfamethazine in the water body reached 0.0%, 20.7%, 33.5%, and 33.1% in the dark + bacterial solution group, light alone group, light + bacterial solution group, and light + bacterial solution supernatant group after 12 h treatment, respectively (see Figure 4 (b)), indicating that the addition of photosynthetic bacterial culture can significantly promote the degradation of sulfamethazine 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 culture. The monitoring of the change in the concentration of sulfamethazine was carried out 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 sulfamethazine degradation mediated by the photosynthetic bacteria was analyzed by further adding different reactive oxygen species quenchers or lifespan extenders (see Figure 5 (b)), the degradation rate of sulfamethazine did not change significantly after adding 1‰ isopropanol to quench hydroxyl radicals, the degradation rate of sulfamethazine was significantly increased after adding 50% heavy water to extend the lifetime of singlet oxygen, and the degradation rate of sulfamethazine was significantly decreased 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 sulfamethazine 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) Activate photosynthetic bacteria, inoculate the activated photosynthetic bacteria into the culture medium, and use light to make the photosynthetic bacteria grow and metabolize until the stable stage; (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 the production of active oxygen by providing light to degrade the new pollutants; The new pollutants include one or more of persistent organic pollutants, endocrine disruptors and antibiotics; The photosynthetic bacteria is Rhodopseudomonas palustris, which was purchased from Guangdong Provincial Microbial Culture Collection Center with a collection number of GDMCCNO: 1.

167.

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

3. The method according to claim 2, wherein: The yeast culture medium comprises the following components: 1 g / L K2HPO4, 0.5 g / L MgSO4, and 10 g / L yeast extract, and is sterilized at 121°C.

4. The method according to claim 1 or 2, characterized in that: In step (1), the photosynthetic bacteria inoculation step is as follows: 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.

5. The method according to claim 4, characterized in that: The culture medium comprises the following components: 1 g / L sodium acetate, 1 g / L K2HPO4, 0.5 g / L MgSO4, 0.5 g / L NaCl, and 1 g / L NH4Cl. The pH is adjusted to 7.1 and sterilized at 121°C.

6. The method according to claim 1, wherein: In step (2), the dosage of the photosynthetic bacteria solution is based on the initial OD 600 = 0.01-0.

5.

7. The method according to claim 1, wherein: In step (2), the new pollutants include one or more of cimetidine and sulfamethazine.

8. The method according to claim 1, wherein: 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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