A method for simultaneously treating wastewater, degrading antibiotics and recovering protein by using purple photosynthetic bacteria

Abstract

The present application relates to a kind of method for simultaneously treating wastewater, degrading antibiotic and recycling protein by purple photosynthetic bacteria, belong to wastewater treatment technical field.The present application provides a kind of method for simultaneously treating wastewater, degrading antibiotic and recycling protein, comprising the following steps: adding purple photosynthetic bacteria seed liquid to wastewater, to obtain mixed solution, the mixed solution is cultured under 1000-3000Lux light intensity and 300-900 nm wavelength for 5-9 days, to obtain culture;The obtained culture is solid-liquid separation, the obtained solid is protein, and the obtained liquid is treated wastewater.The present application utilizes the method for simultaneously treating wastewater, degrading antibiotic and recycling protein by purple photosynthetic bacteria, wherein under light, photosynthetic bacteria use light as energy, absorb and assimilate organic pollutants in wastewater, and convert into own bacterial biomass, realize the purpose of " recycling " protein from wastewater.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, and in particular to a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins using purple photosynthetic bacteria. Background Technology

[0002] In recent years, with the development of large-scale and intensive aquaculture technologies, my country's aquaculture industry has grown rapidly, making it the world's largest producer of aquatic products. While this rapid development has boosted economic growth, the subsequent treatment of aquaculture wastewater has become a challenge. Purple photosynthetic bacteria can grow under anaerobic conditions under light, decomposing and utilizing various organic matter, thus making them suitable for wastewater treatment. Furthermore, photosynthetic bacteria are rich in various bioactive substances such as proteins and photosynthetic pigments, giving them a dual advantage in treating aquaculture wastewater and making them more competitive in the wastewater treatment field.

[0003] Currently, my country has limited methods for treating aquaculture wastewater. Most treatments still rely on traditional physical, chemical, and biological processes to reduce pollutants such as chemical oxygen demand (COD), ammonia nitrogen, and total phosphorus, aiming to achieve standards for recycling. However, due to the large volume and high content of nutrients like nitrogen and phosphorus in aquaculture wastewater, current treatment methods are not ideal. Furthermore, nutrients in the wastewater are often converted into sludge during treatment, making recycling difficult and resulting in resource waste. Therefore, there is an urgent need for novel aquaculture wastewater treatment technologies that combine wastewater treatment with resource recovery.

[0004] Modern aquaculture widely uses antibiotics to prevent and treat diseases in order to improve economic efficiency. Only a small portion of these antibiotics are absorbed and utilized; the remainder is discharged into aquaculture wastewater. Under antibiotic stress, antibiotic-resistant genes in drug-resistant bacteria spread among microorganisms, increasing the risk of superbugs. Furthermore, antibiotics have complex structures and are difficult to degrade, ultimately accumulating in the human body through the food chain and impacting human health. Therefore, effectively removing antibiotics from aquaculture wastewater, while simultaneously treating, disposing of, and recycling it, is of great significance for environmental protection and human health.

[0005] Currently, common wastewater treatment methods only treat wastewater and recover proteins, or treat wastewater and degrade antibiotics. There is no integrated wastewater treatment method that can treat wastewater, degrade antibiotics, and recover proteins simultaneously. Therefore, there is an urgent need to develop a method that can treat wastewater, degrade antibiotics, and recover proteins at the same time. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins using purple photosynthetic bacteria.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins using purple photosynthetic bacteria, comprising the following steps: (1) Add purple photosynthetic bacteria seed solution to wastewater to obtain a mixture. Incubate the mixture at 25-35℃, 20-100rpm, 1000-3000 Lux light intensity and 300-900 nm light wavelength for 5-9 days to obtain a culture. The purple photosynthetic bacteria seed solution contains volatile solid substances. (2) The culture obtained in step (1) was allowed to settle for 1.5-2.5 h, and the solid and liquid were separated. The solid obtained was protein, and the liquid obtained was treated wastewater.

[0008] This invention utilizes purple photosynthetic bacteria to simultaneously treat aquaculture wastewater, degrade antibiotics, and recover proteins. Under light, the photosynthetic bacteria use light as energy to absorb and assimilate organic pollutants (carbon and nitrogen sources) in the wastewater, converting them into their own biomass. Since the bacteria themselves are rich in protein, this process achieves the goal of "recovering" protein from the wastewater. Furthermore, light can induce excited transitions of dissolved organic matter to the singlet state, which is then rapidly converted to the triplet excited state via intersystem crossing. This triplet then reacts with dissolved oxygen to generate reactive oxygen species, such as singlet oxygen (¹O₂), hydroxyl radicals (•OH), and superoxide radicals (•O₂). - These reactive oxygen species possess extremely strong oxidizing properties, capable of indiscriminately attacking and oxidizing specific structures (such as benzene rings, double bonds, and amino groups) in antibiotic molecules, causing them to open rings, break bonds, mineralize into CO2 and H2O, or transform into less toxic and more easily biodegradable intermediates. Furthermore, some hydrophobic antibiotics or contaminants can be adsorbed onto the cell membrane or extracellular polymeric surfaces of photosynthetic bacteria. Subsequently, these adsorbed contaminants may be absorbed by the cell and enzymatically degraded within the cell.

[0009] As a preferred embodiment of the method of the present invention, in step (1), the wastewater includes, but is not limited to, poultry and livestock breeding wastewater and / or aquaculture wastewater.

[0010] In a preferred embodiment of the method described in this invention, in step (1), the total chemical oxygen demand in the wastewater is ≥30 mg / L.

[0011] In a preferred embodiment of the method described in this invention, in step (1), the total chemical oxygen demand in the wastewater is 31-3000 mg / L.

[0012] As a preferred embodiment of the method of the present invention, in step (1), the amount of purple photosynthetic bacteria seed liquid added is 1 g of volatile solids per 2-5 g of total chemical oxygen demand.

[0013] As a preferred embodiment of the method of the present invention, in step (1), the 300-900 nm light wavelength includes at least one of white light with a wavelength of 380-700 nm, red light with a wavelength of 620-780 nm, and near-infrared light with a wavelength of 780-900 nm.

[0014] As a preferred embodiment of the method of the present invention, in step (1), the 300-900nm light wavelength includes red light with a wavelength of 620-780 nm and / or near-infrared light with a wavelength of 780-900 nm.

[0015] In a preferred embodiment of the method described in this invention, the light wavelength is 650 nm red light.

[0016] As a preferred embodiment of the method described in this invention, the light wavelength is near-infrared light of 850 nm.

[0017] In a preferred embodiment of the method described in this invention, in step (1), the OD of the purple photosynthetic bacteria seed solution is... 660 =1.5-1.9.

[0018] As a preferred embodiment of the method described in this invention, in step (1), the purple photosynthetic bacteria are deposited at the China Center for Type Culture Collection, with accession number CCTCC AB 2018220.

[0019] In a preferred embodiment of the method described in this invention, the median particle size of the purple photosynthetic bacteria seed solution is 55-60 μm.

[0020] In a preferred embodiment of the method described in this invention, the settling time in step (2) is 2 h.

[0021] As a preferred embodiment of the method of the present invention, in step (2), the solid-liquid separation includes, but is not limited to, at least one of filtration and centrifugation.

[0022] Secondly, the present invention provides the application of the above method in the preparation of livestock feed.

[0023] The method described in this invention can isolate a high-protein purple photosynthetic bacterial product, which can be added to livestock feed to increase the protein intake of poultry and livestock, providing a stable local protein source for the livestock industry, reducing dependence on imported fishmeal and soybeans, and helping to address the challenges of global protein feed shortages and price fluctuations.

[0024] Thirdly, the present invention provides a livestock feed comprising the solid obtained in step (2) of the above method.

[0025] As a preferred embodiment of the aquaculture feed described in this invention, the aquaculture feed includes poultry and livestock feed and / or aquatic feed.

[0026] In a preferred embodiment of the aquaculture feed of the present invention, the aquaculture feed further includes feed additives acceptable to aquaculture feed.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method of the present invention is simple and fast, and can enrich samples on a large scale. The enriched samples can be used directly without additional pretreatment.

[0028] (2) Compared with the traditional activated sludge process which requires a lot of aeration, the purple photosynthetic bacteria selected in this invention are photoautotrophic microorganisms whose energy comes directly from sunlight or artificial light. They require little or no aeration, have low energy consumption, and significantly reduce carbon emissions in the treatment process. In addition, the photosynthetic bacteria system has a low sludge yield because it converts more organic matter in wastewater into bacterial cells rather than unusable residual sludge, thus reducing the amount of sludge at the source and lowering the risk of secondary pollution.

[0029] (3) The method of the present invention can produce high-value single-cell protein. The purple photosynthetic bacteria are rich in protein, providing a stable local protein source for the aquaculture industry, reducing dependence on imported fishmeal and soybeans, and helping to cope with the challenges of global protein feed shortage and price fluctuations. Attached Figure Description

[0030] Figure 1 The results of characterization of the purple photosynthetic bacteria seed liquid used in the embodiments of the present invention are shown in Figure 1, where a represents the determination results of chlorophyll and carotenoids, and b represents the determination results of the median particle size of the bacteria. Figure 2 This is a flowchart illustrating the operation of the method described in this invention. Figure 3 The removal capacity of purple photosynthetic bacteria on pollutants and antibiotics in aquaculture wastewater in Example 1 of the present invention is shown, where a is the pollutant removal capacity and b is the antibiotic removal capacity. Figure 4 The detection results of free radicals in aquaculture wastewater (liquid phase) in Example 1 of the present invention; Figure 5 Example 2 of the present invention shows the removal capacity of purple photosynthetic bacteria for pollutants and antibiotics in aquaculture wastewater, where a represents the pollutant removal capacity and b represents the antibiotic removal capacity.

[0031] In the above figure, TCOD represents Total Chemical Oxygen Demand, SCOD represents Dissolved Chemical Oxygen Demand, TP represents Total Phosphorus, and NH4+ represents... + Represented as ammonium ion; In the same indicator, different lowercase letters between the two groups indicate a significant difference between the two groups. p <0.05), the presence of the same lowercase letter between the two groups indicates that there is no significant difference between the two groups. p >0.05). Detailed Implementation

[0032] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0033] Unless otherwise specified, all other materials and reagents used in the examples, comparative examples, and effect examples are commercially available.

[0034] The pollutant removal performance indicators in the following examples, comparative examples, and effect examples include lincomycin, total chemical oxygen demand, dissolved chemical oxygen demand, total phosphorus, ammonium, and protein; the indicators for characterizing purple photosynthetic bacteria include optical density, total solids, volatile solids, chlorophyll, carotenoids, and median particle size.

[0035] The extraction and determination of ciprofloxacin were performed according to the following literature, and the total solids and volatile solids were determined according to the following literature: Xiao K, Abbtbraun G, Borowska E, et al. Solid–liquid distribution of ciprofloxacin during sludge dewatering after Fe(II)-activated peroxymonosulfate treatment: Focusing on the role of dissolved organic components [J]. ACS EST&Engineering, 2022, 2(5):863-873. The total chemical oxygen demand, dissolved chemical oxygen demand, total phosphorus, and ammonium were determined with reference to the following literature: Eaton A D, Clesceri LS, Greenberg AE, et al. Standard methods for the examination of water and wastewater [J]. Am J Public Health Nations Health, 1966, 56(3):387-388. The protein content was determined with reference to the following literature: WMJ Van Gelder. Conversion factor from nitrogen to protein for potato tuber protein. Potato Research. 1981, 24: 423-425. The optical density, chlorophyll, carotenoids, and median particle size were determined with reference to the following literature: Kuo FS, Chien YH, Chen CJ. Effects of light sources on growth and carotenoid content of photosynthetic bacteria. Rhodopseudomonas palustris .[J].Bioresource Technology, 2012, 113(none):315-318.; The singlet oxygen (¹O2), hydroxyl radical (•OH), and superoxide radical (•O2) - The determination of ) was performed using the same instrument as described in the following literature: Jiang YZ, Nabi M, Luo X, Ding W, Zhou Y, Xiao K K. Relationship between electron donor-acceptor capacity of organic components and sludgedewatering performance during pre-oxidation. [J]. Chemical EngineeringJournal, 2025, 522: 167181. The purple photosynthetic bacteria in the following examples and comparative examples were enriched on a laboratory scale. The bacterial strains were purchased from the China Center for Type Culture Collection (CCTCC), accession number CCTCC AB 2018220. Enrichment was performed according to the culture methods provided by the CTCC, and the bacteria were cultured for a total of 9 days to achieve an OD of [missing information - likely referring to OD values]. 660 Approximately 1.78, total solids 2.72 g / L, volatile solids 2.37 g / L, chlorophyll 112.83 mg / L, carotenoids 26.96 mg / L, median particle size 56.8 μm (see...). Figure 1 ).

[0036] The wastewater used in the following examples and comparative examples was aquaculture wastewater, taken from Lingquan Aquaculture Ecological Co., Ltd., Haojiang District, Shantou City. The initial parameters were as follows: Total Chemical Oxygen Demand (COD) 2079.5 mg / L, Dissolved COD 1425.6 mg / L, Total Phosphorus 28.0 mg / L, Ammonium 827.7 mg / L, and Dissolved Protein 543.9 mg / L. The aquaculture wastewater was pre-screened through a 100-mesh sieve to remove large particulate pollutants and stored in a 4°C freezer for later use.

[0037] Example 1 This embodiment provides a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins using purple photosynthetic bacteria. The operation flow of the method is described below. Figure 2 This includes the following steps: S1. Add OD to a sealed bioreactor containing aquaculture wastewater. 660 =1.78 purple photosynthetic bacteria seed solution, 100 mL of purple photosynthetic bacteria seed solution was added to every 900 mL of aquaculture wastewater to obtain a mixture. The mixture was cultured for 7 days at 25℃, 100 rpm, 2000 Lux and 650 nm light wavelength red light to obtain the culture. S2. The culture obtained in step S1 is allowed to settle for 2 hours, and the solid and liquid are separated to obtain the solid and the treated wastewater.

[0038] Example 2 This embodiment provides a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins. The method is similar to that of Embodiment 1, except that: In step (1), the light wavelength is adjusted to near-infrared light with a wavelength of 850 nm, while the remaining steps and parameters remain unchanged.

[0039] Example 3 This embodiment provides a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins. The method is similar to that of Embodiment 1, except that: In step (1), the light wavelength is adjusted to white light with a wavelength of 380-700 nm, while the other steps and parameters remain unchanged.

[0040] Example 4 This embodiment provides a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins. The method is similar to that of Embodiment 1, except that: In step (1), the culture temperature is adjusted to 30°C, the light intensity is adjusted to 1000 Lux, the culture time is adjusted to 9 days, and the remaining steps and parameters remain unchanged.

[0041] Example 5 This embodiment provides a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins. The method is similar to that of Embodiment 1, except that: In step (1), the culture temperature is adjusted to 35°C, the light intensity is adjusted to 3000 Lux, the culture time is adjusted to 5 days, and the remaining steps and parameters remain unchanged.

[0042] Example 6 This embodiment provides a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins, the method comprising the following steps: S1. Add OD to an open bioreactor (racetrack reactor) containing aquaculture wastewater. 660 =1.78 Purple photosynthetic bacteria seed solution, 135 L of aquaculture wastewater were mixed with 15 L of purple photosynthetic bacteria seed solution to obtain a mixture. The mixture was cultured for 7 days at 25℃, 20 rpm, 2000 Lux and 850 nm light wavelength near infrared light. The near infrared light source was 30 cm away from the liquid surface to obtain the culture. S2. The culture obtained in step S1 is allowed to settle for 2 hours, and the solid and liquid are separated to obtain the solid and the treated wastewater.

[0043] Comparative Example 1 This comparative example provides a method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins. The method is similar to that of Example 3, except that: In step (1), the purple photosynthetic bacteria are replaced with capsular red bacteria, while the remaining steps and parameters remain unchanged.

[0044] The capsular rhodopseudomonas strain was purchased from Shanghai Preservation Microbiology Co., Ltd., with strain preservation number SHMCCD11389. It was enriched using the activation and culture methods for Rhodopseudomonas palustris. All operations were performed in a clean bench. Specific procedures are as follows: Use a grinding wheel to make two grooves at the tip of the lyophilized tube. Wipe the grooves with alcohol and then break off the tube tip. Inoculate the lyophilized powder into a screw-capped tube (50 mL) containing culture medium, filling it to the cap level. Then place it in a constant temperature shaker and incubate at 100 rpm and 25°C until OD reaches the specified level. 660 Harvest and expand the culture when the value reaches 0.8-1, so that the OD of capsular red bacteria can be increased. 660 The concentration of total solids was approximately 1.36 g / L, total solids were 2.07 g / L, and volatile solids were 1.57 g / L.

[0045] Example 1 The wastewater before treatment, and the treated wastewater (liquid phase) and solid (solid phase) obtained in Examples 1-3 and Comparative Example 1 were characterized, and the results are shown in the figure. Figure 3And Table 1. Meanwhile, the singlet oxygen (¹O2), hydroxyl radicals (•OH), and superoxide radicals (•O2) were measured in the wastewater treated according to the method of Example 1 under conditions without white light, red light, and near-infrared light irradiation. - The measurements were performed, and the results are shown below. Figure 4 .

[0046] Table 1. Characterization results of the treated wastewater and solids obtained in Examples 1-3 and Comparative Example 1.

[0047] like Figure 3 As shown in Table 1, the methods in Examples 1-3 can simultaneously treat wastewater, degrade antibiotics, and recover proteins. Red light and near-infrared light showed better wastewater treatment effects than white light, with near-infrared light exhibiting the best effects in both antibiotic degradation and protein recovery. Based on the above data, selecting near-infrared light as the light source can further enhance the effectiveness of purple photosynthetic bacteria in treating wastewater, degrading antibiotics, and recovering proteins. Figure 4 The results of the free radical determination indicate that the system contains singlet oxygen (¹O₂), hydroxyl radicals (•OH), and superoxide radicals (•O₂). - ),in Figure 4 The term "no light" refers to treatment performed under conditions without white light, red light, or near-infrared light, as described in Example 1. These reactive oxygen species possess extremely strong oxidizing properties and can indiscriminately attack and oxidize specific structures (such as benzene rings, double bonds, amino groups, etc.) in antibiotic molecules, causing them to open rings, break bonds, mineralize into CO2 and H2O, or transform into less toxic and more easily biodegradable intermediates.

[0048] To investigate the superiority of *Rhodopseudomonas palustris*, we used another photosynthetic bacterium—*Rhodopseudomonas capsulatum* (Comparative Example 1). As shown in Table 1, under near-infrared light irradiation, the wastewater treatment effect of *Rhodopseudomonas capsulatum* was far lower than that of *Rhodopseudomonas palustris*. Although *Rhodopseudomonas capsulatum* accumulates more protein, its solid phase contains a large amount of antibiotics. Antibiotics in the wastewater are transferred to the solid-phase bacteria and are difficult to remove, which significantly impacts the subsequent utilization of the photosynthetic bacteria. Overall, under the same treatment conditions, *Rhodopseudomonas palustris* showed better performance in removing pollutants, degrading antibiotics, and recovering proteins from wastewater.

[0049] Example 2 The treated wastewater (liquid phase) and solid (solid phase) obtained in Example 6 were characterized, and the results are shown in [Figure 6]. Figure 5 As shown in Table 2, the calculation formulas for the relevant indicators are the same as those in Example 1.

[0050] Table 2. Characterization results of the treated wastewater and solids obtained in Example 6. As shown in Table 2 and Figure 5 As shown, compared with treatment in a closed photobioreactor, treatment in an open reactor can further improve the wastewater treatment effect. In addition to the removal rate of dissolved chemical oxygen demand, the removal effect of other pollutants in the wastewater is better, especially the removal effect of ammonium ions is more significant. At the same time, the protein recovery effect of purple photosynthetic bacteria is also much higher than that of closed photobioreactor. This indicates that the use of an open reactor can achieve better pollutant removal and protein recovery effects.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for simultaneously treating wastewater, degrading antibiotics, and recovering proteins using purple photosynthetic bacteria, characterized in that, Includes the following steps: (1) Add purple photosynthetic bacteria seed solution to wastewater to obtain a mixture. Incubate the mixture at 25-35℃, 20-100rpm, 1000-3000 Lux light intensity and 300-900 nm light wavelength for 5-9 days to obtain a culture. The purple photosynthetic bacteria seed solution contains volatile solid substances. (2) The culture obtained in step (1) was allowed to settle for 1.5-2.5 h, and the solid and liquid were separated. The solid obtained was protein, and the liquid obtained was treated wastewater.

2. The method as described in claim 1, characterized in that, In step (1), the total chemical oxygen demand in the wastewater is ≥30 mg / L.

3. The method as described in claim 1, characterized in that, In step (1), the amount of purple photosynthetic bacteria seed liquid added is 1 g of volatile solids per 2-5 g of total chemical oxygen demand.

4. The method as described in claim 1, characterized in that, In step (1), the 300-900 nm light wavelength includes at least one of white light with a wavelength of 380-700 nm, red light with a wavelength of 620-780 nm, and near-infrared light with a wavelength of 780-900 nm.

5. The method as described in claim 4, characterized in that, In step (1), the 300-900 nm light wavelength includes red light with a wavelength of 620-780 nm and / or near-infrared light with a wavelength of 780-900 nm.

6. The method as described in claim 1, characterized in that, In step (1), the OD of the purple photosynthetic bacteria seed solution 660 =1.5-1.

9.

7. The method as described in claim 1, characterized in that, In step (1), the purple photosynthetic bacteria are deposited at the China Center for Type Culture Collection, with accession number CCTCC AB 2018220.

8. The application of the method according to any one of claims 1-7 in the preparation of animal feed.

9. A type of livestock feed, characterized in that, Includes the solid obtained in step (2) of the method described in claim 1.

10. The aquaculture feed as described in claim 9, characterized in that, The aquaculture feed includes poultry and livestock feed and / or aquatic feed.

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