Device and method for screening phosphorus accumulating organisms by alternating anoxic / oxic culture

Abstract

This invention discloses an apparatus and method for screening polyphosphate-accumulating bacteria through alternating anaerobic / aerobic culture. The apparatus includes a top cover module and a deep-well plate that can be sealed and disassembled. Solid phosphorus-containing culture medium is added to the wells of the deep-well plate. The top cover module has cover holes corresponding to the wells of the deep-well plate, and solid supplementary culture medium is added to these cover holes to provide additional nutrients for polyphosphate growth under anaerobic conditions. The solid supplementary culture medium in the cover holes can be removed and replaced. When the top cover module and the deep-well plate are sealed together, the solid supplementary culture medium and the solid phosphorus-containing culture medium come into contact to form a closed anaerobic environment. The method is a preliminary screening method based on high-throughput deep-well plate culture – alternating anaerobic / aerobic modular culture – BCIP colorimetric observation, which avoids the need for repeated inoculation of large numbers of strains under anaerobic / aerobic conditions, effectively reducing experimental intensity.

Description

Technical Field

[0001] This invention relates to the field of polyphosphate culture and domestication technology, specifically to an apparatus and method for screening polyphosphate bacteria through alternating anaerobic / aerobic culture. Background Technology

[0002] In recent years, biological phosphorus removal technology has shown broad application prospects due to its advantages such as safety, efficiency, and economy. The proportion of polyphosphate-accumulating bacteria in mixed bacteria directly determines the effectiveness of biological phosphorus removal, but challenges such as scarce strain resources and low efficiency still exist. Therefore, single-strain screening of polyphosphate-accumulating bacteria remains a key focus for future biological phosphorus removal efforts.

[0003] Traditional polyphosphate-accumulating bacteria (PABs) are typically acclimated and enriched in alternating anaerobic / anoxic and anaerobic / aerobic reactor processes. During this process, PPAs utilize the carbon source stored in the anaerobic zone and excessively absorb phosphorus in the aerobic / anoxic zone, thereby removing phosphorus from the environment. However, due to the complexity and high requirements of this alternating acclimation process, single-strain screening often involves direct purification and shake-flask verification after screening on enrichment media (such as LB or beef extract peptone), lacking specificity. Based on the growth characteristics of PPAs, simple and convenient methods are needed to artificially create alternating anaerobic / aerobic environments for single-strain screening in the laboratory.

[0004] Currently, anaerobic culture, which is mainly based on liquid shake flasks, generally requires the use of liquid paraffin or anaerobic bottles to construct an anaerobic environment. For example, Chinese patent application CN201410243620.8 discloses a high-throughput detection method for anaerobic degradation of dyes by microorganisms. A mixture of dye contaminants and bacterial cells is added to each well of a 96-well plate, sealed with liquid paraffin, and then sealed with high-transmittance transparent tape in a nitrogen atmosphere to form an anaerobic reaction system. The absorbance value of the dye is detected at a specific wavelength using a full-wavelength microplate reader for timed non-destructive monitoring. Furthermore, existing research requires repeated acclimatization in anaerobic phosphorus-deficient / aerobic phosphorus-rich media (Li Ao, Hu Yue, Sun Ling, Meng Na, Yu Nannan, Screening and Characterization Study of a Denitrifying Polyphosphate Bacterium. Guangzhou Chemical Industry, 2020, 48(12)). In practice, a large number of shake flask experiments are time-consuming and labor-intensive, resulting in low experimental efficiency. The 5-bromo-4-chloro-3-indolyl phosphate (BCIP) plate blue-white staining method can indirectly prove the polyphosphate accumulation (PPI) effect of polyphosphate-accumulating bacteria by indicating the presence of alkaline phosphatase, avoiding large-scale shake-flask experiments. The experimental operation is simple and convenient. However, creating anaerobic conditions for solid culture media is very complicated, requiring the use of anaerobic equipment such as anaerobic glove boxes or plate sandwich methods to isolate air (He Donglan, Zhan Rui, Wan Wenjie, Xue Zhiyun. Isolation, identification and polyphosphate accumulation characteristics of an anaerobic polyphosphate-accumulating bacteria [J]. Journal of South-Central University for Nationalities (Natural Science Edition), 2016, 35(02):23-25+50). When culturing and screening polyphosphate-accumulating bacteria, it is necessary to repeatedly place the plates under anaerobic / aerobic conditions, which has problems such as the complexity of creating anaerobic conditions, the large workload of alternating anaerobic / aerobic conditions, the single screening target, and the inconvenience of real-time verification. Therefore, a rapid and convenient method for screening and accurately verifying native polyphosphate-accumulating bacteria in the natural environment can help to further improve the efficiency of biological phosphorus removal. Summary of the Invention

[0005] This invention provides a device suitable for screening polyphosphate-accumulating bacteria through alternating anaerobic / aerobic culture. By fixing and removing the top module, the anaerobic and aerobic environments can be quickly switched, avoiding the complex operation of repeatedly transferring strains. This device simplifies the process and saves time. Furthermore, anaerobic culture of strains no longer requires specialized anaerobic equipment such as anaerobic glove boxes; culturing can be done in a regular incubator, reducing the cost and complexity of strain cultivation. This enables high-throughput, large-scale screening and provides a simple and convenient way to create the culture environment.

[0006] A device for screening polyphosphate-accumulating bacteria by alternating anaerobic / aerobic culture includes an upper cover module and a deep-well plate that can be sealed and disassembled.

[0007] Solid phosphorus-containing culture medium is added to the pores of the deep well plate;

[0008] The upper cover module has cover holes that correspond one-to-one with the holes in the deep well plate. Solid supplementary culture medium that can provide additional nutrients for the growth of polyphosphate-accumulating bacteria under anaerobic conditions is added into the cover holes. The solid supplementary culture medium in the cover holes can be demolded, separated and replaced.

[0009] When the upper cover module and the deep well plate are sealed together, the solid supplementary culture medium comes into contact with the solid phosphorus-containing culture medium to form a closed anaerobic environment.

[0010] The device of this invention uses a deep-well plate with a large pore volume and a surface area sufficient to hold the solid phosphorus-containing culture medium required for the growth of the strain. The upper space of the solid phosphorus-containing culture medium can accommodate the oxygen required for the growth of the strain.

[0011] The covering module in the device of the present invention has the following functions:

[0012] 1) Create a convenient anaerobic environment. The cap hole is coupled with the pores of the deep-well plate in the growth substrate layer to form a closed environment, thereby achieving anaerobic conditions. The target strain of polyphosphate-accumulating bacteria can be screened through anaerobic / aerobic alternation. Non-target strains cannot adapt to the anaerobic environment and do not grow or grow at a slower rate or even die in large numbers.

[0013] 2) Timely replenishment of nutrients. Adding a solid supplementary culture medium with a different composition from the growth substrate medium into the cap well can provide additional nutrients for the growth of the strain under anaerobic conditions, avoiding insufficient nutrients in the substrate layer caused by the strain growing in a solid phosphorus-containing medium for a long time.

[0014] The device of this invention allows the solid supplementary culture medium of the upper anaerobic isolation layer to be repeatedly replaced, continuously providing nutrients for the growth of the strains and avoiding problems such as insufficient nutrients in the substrate layer caused by the strains growing in the substrate layer (solid phosphorus-containing culture medium).

[0015] The deep-well plate used in this invention is a commercially available model, and those skilled in the art can select the appropriate plate size according to different experimental needs and sample sizes. It should be noted that the more wells in a deep-well plate, the smaller the volume of each well. During aerobic culture, the air content in the upper layer of the growth substrate may be insufficient. Therefore, the use of a plate with a throughput higher than 96 wells (such as a 384-well deep-well plate) should be carefully considered.

[0016] In a preferred embodiment, the device for screening polyphosphate-accumulating bacteria by alternating anaerobic / aerobic culture has an upper module made of high-temperature resistant soft silicone, which facilitates the replacement of solid supplementary culture medium and allows for reuse after high-temperature and high-pressure sterilization.

[0017] In a preferred embodiment, the device for screening polyphosphate-accumulating bacteria by alternating anaerobic / aerobic culture has a gap between adjacent cover holes for inserting the sidewall of the deep well plate. When the upper cover module and the deep well plate are sealed together, the sidewall of the cover hole is inserted into the inner sidewall of the hole of the deep well plate.

[0018] In a preferred embodiment, in the apparatus for screening polyphosphate-accumulating bacteria through alternating anaerobic / aerobic culture, the amount of solid phosphorus-containing culture medium in each well of the deep-well plate is such that it just completely covers the bottom surface of the well to no more than half the depth of the well. More preferably, the height of the solid phosphorus-containing culture medium within the well of the deep-well plate is 1 / 3 to 1 / 2 of the depth of the well.

[0019] The thickness of the solid supplementary culture medium can completely fill the entire cap hole, which is more conducive to ensuring that the growth substrate layer (solid phosphorus-containing culture medium) and the anaerobic isolation layer (solid supplementary culture medium) can be in full contact.

[0020] Preferably, the solid phosphorus-containing culture medium comprises: 3.20–3.30 g / L CH3COONa·3H2O, 0.03–0.04 g / L KH2PO4, 0.30–0.32 g / L NH4Cl, 0.09–0.10 g / L MgSO4·7H2O, 0.01–0.03 g / L K2SO4, 0.02–0.03 g / L CaCl2·2H2O, 0.8–1.2 mL / L trace element concentrate I, and 15–20 g / L agar powder; the pH of the trace element concentrate I is 6.0–6.5, and it comprises the following components: 0.4–0.6 g / L FeCl2·4H2O, 0.010–0.015 g / L MnCl2·4H2O, and 0.06–0.08 g / L... ZnCl2, 0.005–0.007 g / L; H3BO3, 0.035–0.040 g / L; Na2MoO4·2H2O, 0.010–0.015 g / L; NiCl2·6H2O, 0.001–0.003 g / L; CuCl2·2H2O. This formula aims to provide basic carbon sources, trace elements, and sufficient phosphorus for the aerobic growth stage of microorganisms to be absorbed into their bodies.

[0021] More preferably, the solid phosphorus-containing culture medium comprises: 3.23 g / L CH3COONa·3H2O, 0.035 g / L KH2PO4, 0.3055 g / L NH4Cl, 0.0913 g / L MgSO4·7H2O, 0.02 g / L K2SO4, 0.026 g / L CaCl2·2H2O, 1 mL / L trace element concentrate I, and 15–20 g / L agar powder; the pH of the trace element concentrate I is 6.0–6.5, and it comprises the following components: 0.5 g / L FeCl2·4H2O, 0.011 g / L MnCl2·4H2O, 0.07 g / L ZnCl2, 0.006 g / L H3BO3, 0.036 g / L Na2MoO4·2H2O, and 0.014 g / L... NiCl2·6H2O, 0.002g / LCuCl2·2H2O.

[0022] Preferably, the solid supplementary culture medium comprises: 3.20–3.30 g / L CH3COONa·3H2O, 0.01–0.02 g / L KH2PO4, 0.30–0.32 g / L NH4Cl, 0.09–0.10 g / L MgSO4·7H2O, 0.01–0.03 g / L K2SO4, 0.02–0.03 g / L CaCl2·2H2O, 0.8–1.2 mL / L trace element concentrate II, and 6–15 g / L agar powder (in a preferred embodiment, 10 g / L; the lower agar content facilitates demolding, separation, and replacement, and the slower solidification rate of the culture medium reduces the likelihood of air bubbles when poured into the cap hole of the upper module); the pH of the trace element concentrate II is 6.0–6.5, and it comprises the following components: 0.4–0.6 g / L The formula consists of FeCl2·4H2O, 0.010–0.015 g / L; MnCl2·4H2O, 0.06–0.08 g / L; ZnCl2, 0.005–0.007 g / L; H3BO3, 0.035–0.040 g / L; Na2MoO4·2H2O, 0.010–0.015 g / L; NiCl2·6H2O, 0.001–0.003 g / L; and CuCl2·2H2O. This formula provides supplementary nutrients for the anaerobic growth of microorganisms, preventing them from remaining in the substrate layer where nutrients are insufficient. Furthermore, the relatively low phosphorus concentration allows for anaerobic phosphorus release when polyphosphate-accumulating bacteria come into contact with the solid supplemented culture medium.

[0023] More preferably, the solid supplementary culture medium comprises: 3.23 g / L CH3COONa·3H2O, 0.015 g / L KH2PO4, 0.3055 g / L NH4Cl, 0.0913 g / L MgSO4·7H2O, 0.02 g / L K2SO4, 0.026 g / L CaCl2·2H2O, 1 mL / L trace element concentrate II, and 6–15 g / L agar powder; the pH of the trace element concentrate II is 6.0–6.5, and it comprises the following components: 0.5 g / L FeCl2·4H2O, 0.011 g / L MnCl2·4H2O, 0.07 g / L ZnCl2, 0.006 g / L H3BO3, 0.036 g / L Na2MoO4·2H2O, and 0.014 g / L... NiCl2·6H2O, 0.002g / LCuCl2·2H2O.

[0024] This invention employs an anaerobic / aerobic modular alternating culture screening method, fully utilizing the physiological characteristic of polyphosphate-accumulating bacteria (PABs) requiring alternating anaerobic / aerobic phosphorus release / uptake. This prevents or halts the growth of non-target bacteria under continuous anaerobic / aerobic alternating conditions, thereby achieving rapid screening of PPAs from environmental samples and improving the specificity of PPA screening. Simultaneously, the phosphorus concentration differs between the solid phosphorus-containing medium in the matrix layer and the solid supplementary medium in the anaerobic isolation layer. This is because PPAs undergo anaerobic phosphorus release and aerobic phosphorus uptake processes; therefore, a lower phosphorus concentration under anaerobic conditions and sufficient phosphorus under aerobic conditions are beneficial for PPA accumulation. The aforementioned medium's phosphorus concentration provides sufficient phosphorus for aerobic phosphorus uptake by PPAs in the matrix layer, while the relatively lower phosphorus concentration in the anaerobic isolation layer allows for anaerobic phosphorus release when PPAs come into contact with the supplementary medium, thus improving the specificity of PPA screening.

[0025] The present invention also provides a method for screening polyphosphate-accumulating bacteria by alternating anaerobic / aerobic culture, using the aforementioned device suitable for screening polyphosphate-accumulating bacteria by alternating anaerobic / aerobic culture;

[0026] The method includes the following steps:

[0027] S1. Enrichment and coating stage: Inoculate environmental samples into LB liquid medium for enrichment culture, dilute the enriched culture solution under aseptic conditions, and then coat it on LB solid medium.

[0028] S2, Screening stage of alternating anaerobic / aerobic culture of polyphosphate-accumulating bacteria:

[0029] S21. Select different single bacterial colony sites from LB solid medium and inoculate them onto the surface of solid phosphorus-containing medium in the wells of a deep-well plate.

[0030] S22. First anaerobic / aerobic alternating culture screening: The cover module is sealed with the deep well plate to form a closed anaerobic environment by contacting the solid supplemented culture medium with the solid phosphorus-containing culture medium. After incubation until signs of colony growth are observed on the solid phosphorus-containing culture medium, the cover module is removed, and the deep well plate is sealed to form a closed aerobic environment. Incubation continues until colony diameter increases or colony color darkens on the solid phosphorus-containing culture medium. Then, a second anaerobic / aerobic alternating culture screening is performed.

[0031] S23. Second Anaerobic / Aerobic Alternating Culture Screening: Remove the solid supplementary medium used in the previous anaerobic culture and wash the overlying module. After sterilization, add new solid supplementary medium. The second anaerobic / aerobic alternating culture screening process is the same as the first anaerobic / aerobic alternating culture screening process, except that the culture time is adjusted according to the colony growth. Once the colonies on the solid phosphorus-containing medium have grown into single colonies of moderate size and obvious morphology, proceed to the BCIP colorimetric observation stage. If the colonies on the solid phosphorus-containing medium are still growing, repeat the anaerobic / aerobic alternating culture screening process as described in the second anaerobic / aerobic alternating culture screening until the colonies on the solid phosphorus-containing medium have grown into single colonies of moderate size and obvious morphology.

[0032] S3, BCIP colorimetric observation stage: Using sterile coating beads, the BCIP colorimetric agent is evenly coated onto the surface of the solid phosphorus-containing medium to obtain the initial screening polyphosphate strains that grow well on the solid phosphorus-containing medium and show obvious blue colony turning reaction.

[0033] In step S1, the environmental sample may be soil, water (including sewage from sewage treatment plants), sediment, etc.

[0034] In step S1, the LB liquid culture medium has a pH of 7.0–7.2 and a temperature of 28–37°C, and its components include: 10 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract. The LB solid culture medium is prepared by adding 15 g / L agar powder to the LB liquid culture medium.

[0035] In step S3, the BCIP chromogenic agent can be an N,N-dimethylformamide solution with a BCIP mass concentration of 0.1% to 2% (0.5% in a preferred example), and the coating amount of the BCIP chromogenic agent can be 2 to 5 μL / well, 3 μL / well in a preferred example. In step S3, the strains in the wells have undergone multiple alternating anaerobic / aerobic cultures, and non-polyphosphate bacteria are basically unable to grow. The blue-colored polyphosphate colonies can be screened out by utilizing the principle of BCIP hydrolysis to generate insoluble blue BCIP dimer particles.

[0036] In a preferred embodiment, in step S3, 5 to 8 coating beads with a diameter of 0.4 mm to 0.8 mm are placed in each well, and the BCIP colorimetric agent is evenly coated on the surface of the solid phosphorus-containing culture medium by a figure-eight shaking method.

[0037] This invention combines anaerobic / aerobic modular alternating culture screening with BCIP colorimetric observation, using the BCIP blue-white colorimetric reaction to intuitively and conveniently indicate the screening results of polyphosphate-accumulating bacteria. Simultaneously, this invention organically integrates BCIP colorimetric observation for initial screening and high-throughput absorbance measurement with an ELISA reader for secondary screening, achieving high-throughput screening of polyphosphate-accumulating bacteria. The experimental operation is simple and convenient, further ensuring the accuracy of the screening results.

[0038] Preferably, the method further includes the step of:

[0039] S4. Colony transfer: The pre-screened polyphosphate strains are transferred to a deep-well plate containing liquid phosphorus-containing medium, sealed, and cultured on a shaker at 28–37°C.

[0040] S5. Determination of total phosphorus degradation capacity: Centrifuge the deep-well plate from step S4 to remove the bacterial cells, take the supernatant and add colorimetric reagent, screen out the strain with the lowest absorbance, which is the strain with the strongest ability to degrade total phosphorus, and identify the strain.

[0041] Preferably, in step S4, the liquid phosphorus-containing culture medium comprises: 3.2–3.3 g / L CH3COONa·3H2O, 0.03–0.04 g / L KH2PO4, 0.30–0.32 g / L NH4Cl, 0.09–0.10 g / L MgSO4·7H2O, 0.01–0.03 g / L K2SO4, 0.02–0.03 g / L CaCl2·2H2O, and 0.8–1.2 mL / L trace element concentrate IIII; the pH value of the trace element concentrate IIII is 6.0–6.5, and it comprises the following components: 0.4–0.6 g / L FeCl2·4H2O, 0.010–0.015 g / L MnCl2·4H2O, and 0.06–0.08 g / L ZnCl2, 0.005~0.007g / L H3BO3, 0.035~0.040g / LNa2MoO4·2H2O, 0.010~0.015g / L NiCl2·6H2O, 0.001~0.003g / L CuCl2·2H2O.

[0042] Further preferably, in step S4, the liquid phosphorus-containing culture medium comprises: 3.23 g / L CH3COONa·3H2O, 0.035 g / L KH2PO4, 0.3055 g / L NH4Cl, 0.0913 g / L MgSO4·7H2O, 0.02 g / L K2SO4, 0.026 g / L CaCl2·2H2O, and 1 mL / L trace element concentrate III; the pH value of the trace element concentrate III is 6.0-6.5, and it comprises the following components: 0.5 g / L FeCl2·4H2O, 0.011 g / L MnCl2·4H2O, 0.07 g / L ZnCl2, 0.006 g / L H3BO3, 0.036 g / L Na2MoO4·2H2O, and 0.014 g / L... NiCl2·6H2O, 0.002g / LCuCl2·2H2O.

[0043] This invention provides a method for initial screening based on high-throughput culture in deep-well plates, alternating anaerobic / aerobic modular culture, and BCIP colorimetric observation. Following deep-well plate proliferation culture, high-throughput secondary screening using a microplate reader is completed, improving overall screening efficiency and avoiding the need for repeated inoculation of large numbers of strains under anaerobic / aerobic conditions, effectively reducing experimental intensity. It also enables simultaneous determination of the total phosphorus degradation effect of batches of strains, improving accuracy compared to existing screening methods. The polyphosphate-accumulating bacteria obtained by this invention can be widely used to address current environmental problems of excessive phosphorus levels in sites, providing technical support for ecological restoration applications.

[0044] Compared with the prior art, the beneficial effects of this invention are as follows:

[0045] 1. This invention provides an improved double-layer culture device for screening polyphosphate-accumulating bacteria through alternating anaerobic / aerobic culture. It utilizes an upper cover module that is fully compatible with a deep-well plate to create an anaerobic environment at the contact surface. The multi-well plate is then used as a culture platform to achieve high-throughput large-scale culture and high-throughput screening. This prevents non-target bacteria from growing under repeated alternating anaerobic / aerobic conditions, thereby enabling targeted screening of polyphosphate-accumulating bacteria.

[0046] 2. This invention enables rapid switching between anaerobic and aerobic environments through the fixing and removal of the top cover module, avoiding the complex operation of repeatedly transferring strains. Cultivating strains does not require specialized anaerobic equipment such as anaerobic glove boxes; it can be done in a regular incubator, reducing the cost and difficulty of strain cultivation.

[0047] 3. Compared with the traditional anaerobic culture method—the plate sandwich method—this invention avoids pouring unsolidified culture medium onto the inoculated bacteria, further protecting the strains from death due to high temperatures in the culture medium. Compared with the traditional anaerobic culture method—the paraffin sealing method—this invention avoids liquid paraffin adhering to the inner wall of the well plate, saving the time of cleaning the well plate, and making the transfer culture of strains more convenient.

[0048] 4. The method for screening polyphosphate-accumulating bacteria by alternating anaerobic / aerobic culture provided by this invention combines the growth characteristics of polyphosphate-accumulating bacteria for targeted screening, and utilizes a multi-well plate screening platform to greatly improve screening efficiency; strains do not need to be repeatedly inoculated, the anaerobic environment is easy to create, large-scale shake-flask experiments are avoided, and the method is easy to implement; the use of an enzyme-linked immunosorbent assay (ELISA) reader to measure absorbance further ensures the accuracy of screening results.

[0049] 5. From the perspective of cultivation and screening methods, the difference in component content between the solid phosphorus-containing medium in the growth substrate layer and the solid supplementary medium in the anaerobic isolation layer can be used to meet the nutrient requirements of polyphosphate-accumulating bacteria during cultivation, thereby achieving the screening and domestication of polyphosphate-accumulating bacteria. In the aerobic environment, individual bacteria can fully absorb phosphorus from the growth substrate layer; the anaerobic environment provides a solid supplementary medium, allowing for anaerobic phosphorus release in a low-phosphorus environment while also utilizing the carbon source in the supplementary medium, thus avoiding the situation where strains continuously grow in the substrate layer where the substrate layer is nutrient-deficient.

[0050] 6. In terms of initial screening results, using solid culture medium to screen instead of traditional large-scale anaerobic / aerobic alternating culture in shake flasks allows for the simultaneous screening of multiple strains, avoiding the complex operations of repeatedly inoculating large numbers of samples; at the same time, using multi-well plates for initial screening avoids single culture on plates, greatly reducing workload and making it suitable for rapid, large-scale, and high-throughput screening.

[0051] 7. From the perspective of the overall culture pathway, this method organically integrates microbial cell culture and degradation capacity assay. Absorbance is measured using a microplate reader on a multi-well plate to further verify the total phosphorus degradation effect. Compared to traditional spectrophotometry, all chromogenic reagents are added to the well plate, further reducing reagent usage and experimental costs. Simultaneously, it avoids the need for a separate digestion tube and stoppered tube for each strain in traditional methods, saving time spent on repeated tube washing. This approach improves screening accuracy while enhancing the convenience of initial screening, enabling high-throughput and simultaneous screening of target strains and ensuring timely screening. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the structure of the overlying module (a), the deep-well plate (b), and the assembled device (c) suitable for screening polyphosphate-accumulating bacteria through alternating anaerobic / aerobic culture.

[0053] Figure 2 The image shows a photo of the initially screened strains that were successfully selected in a specific implementation method.

[0054] Figure 3 This is a graph showing the results of the total phosphorus degradation effect measurement in a specific implementation method. Figure 3 (a) is a photo showing the color rendering effect. Figure 3 (b) The absorbance results measured by the ELISA reader. Figure 3(c) represents the average total phosphorus degradation efficiency of the strain.

[0055] Figure 4 This is a colony morphology diagram of the strain of this invention.

[0056] Figure 5 This is a phylogenetic tree diagram of the strain of this invention and its closely related strains.

[0057] Figure 6 This is a microscopic image (Gram staining) of the strain of this invention.

[0058] Figure 7 The figures show the phosphorus removal effect and growth curves of the strain of this invention under different growth conditions. Figure 7 (a) Phosphorus removal effect and growth curve of bacteria at different pH levels; Figure 7 (b) Phosphorus removal effect and growth curve of bacteria under different salinity conditions; Figure 7 (c) Phosphorus removal effect and growth curve of bacteria under different carbon sources; Figure 7 (d) shows the phosphorus removal effect and growth curve of bacteria under different C / P ratios.

[0059] Figure 8 The figures show the phosphorus removal effect and growth curve of the strain of this invention in actual river water (alkaline conditions). Figure 8 (a) is a growth curve of ZS1D-28 in actual river water; Figure 8 (b) is a diagram showing the nitrogen and phosphorus removal effect of ZS1D-28. Detailed Implementation

[0060] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0061] like Figure 1As shown, the device used in this embodiment for screening polyphosphate-accumulating bacteria through alternating anaerobic / aerobic culture includes a top cover module that can be sealed and disassembled, and a deep-well plate. Solid phosphorus-containing culture medium 1 is added to the wells 4 of the deep-well plate. The top cover module has cap holes 3 corresponding one-to-one with the wells 4 of the deep-well plate, and solid supplementary culture medium 5, which provides additional nutrients for polyphosphate-accumulating bacteria growth under anaerobic conditions, is added to the cap holes 3. The solid supplementary culture medium 5 in the cap holes 3 can be removed and replaced. When the top cover module and the deep-well plate are sealed together, the solid supplementary culture medium 5 and the solid phosphorus-containing culture medium 1 form a closed anaerobic environment. A gap is left between adjacent cap holes 3 for the insertion of the sidewall of the well 4 of the deep-well plate. When the top cover module and the deep-well plate are sealed together, the sidewall of the cap hole 3 is inserted into the inner sidewall of the well 4 of the deep-well plate. The inoculated strain 2 is located at the interface between the solid supplementary culture medium 5 and the solid phosphorus-containing culture medium 1. The height of the solid phosphorus-containing culture medium 1 within the well 4 of the deep well plate should preferably be 1 / 3 to 1 / 2 of the depth of the well 4.

[0062] The solid phosphorus-containing culture medium 1 consists of: 3.23 g / L CH3COONa·3H2O, 0.03–0.04 g / L KH2PO4, 0.3055 g / L NH4Cl, 0.0913 g / L MgSO4·7H2O, 0.02 g / L K2SO4, 0.026 g / L CaCl2·2H2O, 1 mL / L trace element concentrate I, and 15–20 g / L agar powder; the pH of the trace element concentrate I is 6.0–6.5, and its components are: 0.5 g / L FeCl2·4H2O, 0.011 g / L MnCl2·4H2O, 0.07 g / L ZnCl2, 0.006 g / L H3BO3, 0.036 g / L Na2MoO4·2H2O, and 0.014 g / L... NiCl2·6H2O, 0.002g / L CuCl2·2H2O.

[0063] The composition of solid supplementary culture medium 5 is as follows: 3.23 g / L CH3COONa·3H2O, 0.01–0.02 g / L KH2PO4, 0.3055 g / L NH4Cl, 0.0913 g / L MgSO4·7H2O, 0.02 g / L K2SO4, 0.026 g / L CaCl2·2H2O, 1 mL / L trace element concentrate II, and 8–15 g / L agar powder; the pH value of trace element concentrate II is 6.0–6.5, and its components are: 0.5 g / L FeCl2·4H2O, 0.011 g / L MnCl2·4H2O, 0.07 g / L ZnCl2, 0.006 g / L H3BO3, 0.036 g / L Na2MoO4·2H2O, and 0.014 g / L... NiCl2·6H2O, 0.002g / L CuCl2·2H2O.

[0064] Environmental samples were selected from Gou Shan River in Zhoushan City and Hu Hai Tang in Jinhua City, Zhejiang Province. The high-throughput culture and screening steps for polyphosphate-accumulating bacteria are as follows:

[0065] (1) Sample collection: Surface water and riverbed silt were collected from Zhoushan City and Jinhua City, Zhejiang Province. 100 mL of water sample and 500 mL of riverbed silt sample were collected. The Zhoushan sample was designated ZS1(D), and the Jinhua sample was designated JH1. D indicates the bottom sediment sample.

[0066] (2) Enrichment Coating: 50 mL of water sample or diluted sludge sample was filtered through a 0.22 μm filter membrane. The filter membrane was then added to a 250 mL Erlenmeyer flask containing 50 mL of sterilized fresh LB liquid medium and incubated at 30 °C and 160 r / min for 24 h. The enriched bacterial solution was then diluted to 10⁻⁶ using a serial dilution method. -7 The concentration was evenly spread onto LB solid medium plates using a spiral inoculator and incubated at 30°C for 48 hours. The LB liquid medium had a pH of 7.0–7.2 and a temperature of 28–37°C, and consisted of 10 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract. The LB solid medium was prepared by adding 15 g / L agar powder to the LB liquid medium.

[0067] (3) Strains Culture and Initial Screening: After colony formation, colonies with different morphologies and colors were selected from LB solid medium and inoculated into the growth substrate layer of the deep-well plate. The anaerobic isolation layer was immediately placed on the substrate layer to create an anaerobic environment for each colony. A total of 10 strains were inoculated, with 3 replicates for each strain. After 12 hours of incubation in a biochemical incubator, the anaerobic isolation layer was removed, the deep-well plate was covered with a film, and then placed in an incubator for constant temperature aerobic incubation to allow the colonies to enter the aerobic phosphorus uptake stage. The culture was then carried out at 30°C for another 12 hours. The above process was recorded as one anaerobic-aerobic culture process. After repeating the anaerobic-aerobic culture twice, a total of 10 strains were observed to have grown into single colonies. The BCIP chromogenic agent was evenly coated onto each well using sterilized coating beads. Five to eight beads with a diameter of 0.4 mm to 0.8 mm were placed in each well. The BCIP chromogenic agent was evenly coated onto the surface of the solid phosphorus-containing medium using a figure-eight mixing method. After standing in an incubator for 14 hours, a total of 9 single bacterial strains showed a blue color change. The BCIP chromogenic agent was a 0.5% N,N-dimethylformamide solution, and the coating amount was 3 μL per well. Figure 2 Photos of the successfully screened strains are shown.

[0068] (4) Colony inoculation: Nine strains with obvious color change reaction were selected as the initial screening strains. At the same time, an equal amount of bacteria without color change reaction (JH1-5) and sterile water were selected as experimental control and blank control (CK), respectively. They were picked into 48-well deep-well plates containing liquid phosphorus medium with sterile inoculation loops. Six wells were left unselected as blank control. Three replicate wells were set for each strain, and three parallel wells were set for each well plate. Blank control was also set. After sealing, the plates were placed in a microplate shaker at 30°C for 2 days. The liquid phosphorus-containing culture medium consists of: 3.23 g / L CH3COONa·3H2O, 0.035 g / L KH2PO4, 0.3055 g / L NH4Cl, 0.0913 g / L MgSO4·7H2O, 0.02 g / L K2SO4, 0.026 g / L CaCl2·2H2O, and 1 mL / L trace element concentrate III. The pH of the trace element concentrate III is 6.0-6.5, and its components are: 0.5 g / L FeCl2·4H2O, 0.011 g / L MnCl2·4H2O, 0.07 g / L ZnCl2, 0.006 g / L H3BO3, 0.036 g / L Na2MoO4·2H2O, 0.014 g / L NiCl2·6H2O, and 0.002 g / L CuCl2·2H2O.

[0069] (5) Total phosphorus degradation capacity determination: Starting 2 days after inoculation, the well plates were placed in a microplate centrifuge. After removing the bacterial cells, the supernatant was added sequentially with colorimetric reagents (ammonium molybdate and ascorbic acid) for observation. Figure 3As shown in (a), the color of 9 out of 10 experimental groups was lower than that of the blank control group, with strain ZS1D-28 (from Zhoushan sediment) completely fading, demonstrating that ZS1D-28 has good polyphosphate accumulation effect. The ability of the strain to degrade total phosphorus was detected using a microplate reader, such as... Figure 3 As shown, the nine initial screening strains can effectively reduce the total phosphorus concentration. When comparing the total phosphorus degradation capacity of the experimental group and the blank control group, the removal effect of the blank control group was minimal, while the average removal rate of ZS1D-28 in the experimental group reached 86.89%.

[0070] (6) The isolated ZS1D-28 polyphosphate-accumulating bacteria were subjected to bacterial culture PCR to amplify its 16S rDNA. The amplified product was sent for analysis, and the sequencing results (SEQ ID NO: 1) were compared with the NCBI database. It was found that the homology with a Rhodococcus pyridinivorans strain reached 99.93%. The above-mentioned isolated ZS1D-28 polyphosphate-accumulating bacteria is deposited at the China Center for Type Culture Collection (CCTCC) on June 12, 2023. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the strain accession number is CCTCC NO: M 2023986.

[0071] Based on morphological characteristics, physiological and biochemical features, and 16S rRNA sequence analysis, this invention classifies the isolated polyphosphate-accumulating bacteria ZS1D-28 (named *Rhodococcus sp. ZS1D-28*) as a novel strain within the genus *Rhodococcus*. Biological characteristic analysis was conducted to determine the optimal growth conditions for this strain, providing technical support for its ecological restoration applications. A phylogenetic tree of *Rhodococcus sp. ZS1D-28* and its closely related strains can be found [link to phylogenetic tree]. Figure 5 .

[0072] The following is a detailed description of Rhodococcus sp. ZS1D-28:

[0073] 1) Morphological characteristics: Colonies grow rapidly on LB agar plates, forming round colonies with a raised center, smooth surface, and even edges. They are pink, opaque, and glossy. See the example for the colony morphology of Rhodococcus sp. ZS1D-28. Figure 4 .

[0074] 2) Physiological and biochemical characteristics: The bacterial cells are Gram-positive (see Gram staining results for details). Figure 6The bacteria appear spherical or rod-shaped under a microscope, and their morphology is related to the growth environment and growth stage. Staining with poly-P Albert staining and PHB Sudan Black staining revealed typical granular structures within the bacteria. They lack flagella, capsules, and spores, are oxidase-negative, and are facultative anaerobic. Physiological and biochemical characteristics are shown in Table 1.

[0075] Based on 16S rRNA sequence sequencing analysis, the bacterium belongs to the genus Rhodococcus, with the Latin scientific name Rhodococcus sp. ZS1D-28.

[0076] Table 1 Physiological and biochemical characteristics of Rhodococcus sp. ZS1D-28

[0077] Test Project Measurement results Test Project Measurement results Gram staining +, spherical Oxidase test - flagella staining - Gelatin liquefaction - Albert staining +, Contains dye particles Nitrate reduction + PHB Sudan Black staining +, Contains dye particles Hydrogen sulfide test - MR test - Lysine decarboxylase test - VP test - / /

[0078] "+" indicates a positive test result, and "-" indicates a negative test result.

[0079] The total phosphorus-degrading strain Rhodococcus sp. ZS1D-28 provided by this invention, when added to a phosphate-containing synthetic wastewater culture medium and reacted at 30-37°C for at least 72 hours, exhibits an aerobic phosphorus uptake of 400 mg / L and an anaerobic phosphorus release of 54.5 mg / L.

[0080] Further, the culture time was specified as 36 hours of aerobic treatment followed by 36 hours of anaerobic culture. The composition of the synthetic wastewater culture medium includes: 1.06 g / L CH3COONa·3H2O, 0.03–0.04 g / L KH2PO4, 0.3055 g / L NH4Cl, 0.0913 g / L MgSO4·7H2O, 0.02 g / L K2SO4, 0.026 g / L CaCl2·2H2O, and 1 mL / L trace element concentrate III; the pH value of the trace element concentrate III is 6.0–6.5, and it includes the following components: 0.5 g / L FeCl2·4H2O, 0.011 g / L MnCl2·4H2O, 0.07 g / L ZnCl2, 0.006 g / L H3BO3, 0.036 g / L Na2MoO4·2H2O, 0.014 g / L NiCl2·6H2O, and 0.002 g / L... CuCl2·2H2O.

[0081] Figure 7 The paper presents graphs and growth curves showing the phosphorus removal effect of the Rhodococcus sp. ZS1D-28 strain of this invention under different growth conditions (30–37°C) in the aforementioned phosphate-containing synthetic wastewater culture medium. Figure 7 (a) Phosphorus removal effect and growth curve of bacteria at different pH levels; Figure 7(b) Phosphorus removal effect and growth curve of bacteria under different salinity conditions;

[0082] Figure 7 (c) Phosphorus removal effect and growth curve of bacteria under different carbon sources; Figure 7 (d) shows the phosphorus removal effect and growth curve of bacteria under different C / P ratios.

[0083] Therefore, the optimal growth and phosphorus removal conditions for Rhodococcus sp. ZS1D-28 strain are as follows: the optimal initial pH of the wastewater is 10.0 (adaptation range 5.0–10.0), the optimal salinity is 0–2.5% (adaptation range 0.0%–3.5%), the optimal carbon source is sodium citrate, and the optimal initial C / P ratio is 500 / 16.

[0084] This invention provides a method for applying Rhodococcus sp. ZS1D-28 strain in the treatment of alkaline river water.

[0085] A phosphorus removal test was conducted using 1000 mL of water from the Goushan River in Zhoushan City. The specific steps are as follows:

[0086] To demonstrate the phosphorus removal effect of Rhodococcus sp. ZS1D-28 in high-alkali environments, KH2PO4 was added to the river water to achieve a total phosphorus concentration of 8 mg / L, and KNO3 was added to achieve an inorganic nitrogen concentration of 50 mg / L. COD was appropriately adjusted to ensure a C / P ratio of approximately 100:1, and the pH was adjusted to 10. 15 mL of bacterial suspension with an OD600 of 0.50 ± 0.02 was added to 285 mL of wastewater at an inoculation rate of 5% (bacterial suspension volume / (bacterial suspension + total wastewater volume)). A blank control group was also set up, in which 15 mL of sterile water was added to 285 mL of wastewater at an inoculation rate of 5%.

[0087] The reactor temperature was set at 25℃, and the shaker speed was 140 r / min for incubation. Samples were taken every 12 hours to determine the concentrations of TN, TP, and COD in the supernatant. The results are as follows: Figure 8 As shown.

[0088] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A device suitable for screening of phosphorus accumulating bacteria by alternating anoxic / oxic culture, characterized in that, This includes a top cover module and a deep-hole plate that can be sealed and disassembled. Solid phosphorus-containing culture medium is added to the pores of the deep well plate; The upper cover module has cover holes that correspond one-to-one with the holes in the deep well plate. Solid supplementary culture medium that can provide additional nutrients for the growth of polyphosphate-accumulating bacteria under anaerobic conditions is added into the cover holes. The solid supplementary culture medium in the cover holes can be demolded, separated and replaced. When the upper cover module and the deep well plate are sealed together, the solid supplemented culture medium comes into contact with the solid phosphorus-containing culture medium to form a closed anaerobic environment; the anaerobic and aerobic environments can be switched by fixing and removing the upper cover module. The inoculated strain is located at the interface between the solid supplemented medium and the solid phosphorus-containing medium; the height of the solid phosphorus-containing medium in the well of the deep well plate is 1 / 3 to 1 / 2 of the depth of the well.

2. The device for screening PAOs by alternating anoxic / aerobic culture according to claim 1, wherein, A gap is left between adjacent cover holes for the insertion of the hole sidewall of the deep hole plate. When the upper cover module and the deep hole plate are sealed together, the cover hole sidewall is inserted into the inner sidewall of the hole of the deep hole plate.

3. The device for screening PAOs by alternating anoxic / aerobic culture according to claim 1, wherein, The solid phosphorus-containing culture medium comprises: 3.20~3.30 g / L CH3COONa·3H2O, 0.03~0.04 g / L KH2PO4, 0.30~0.32 g / L NH4Cl, 0.09~0.10 g / L MgSO4·7H2O, 0.01~0.03 g / L K2SO4, 0.02~0.03 g / L CaCl2·2H2O, 0.8~1.2 mL / L trace element concentrate I, and 15~20 g / L agar powder; The trace element concentrate I has a pH of 6.0-6.5 and includes the following components: 0.4~0.6 g / L FeCl2·4H2O, 0.010~0.015 g / L MnCl2·4H2O, 0.06~0.08 g / L ZnCl2, 0.005~0.007 g / L H3BO3, 0.035~0.040 g / L Na2MoO4·2H2O, 0.010~0.015 g / L NiCl2·6H2O, and 0.001~0.003 g / L CuCl2·2H2O.

4. The device for screening phosphorus accumulating organisms by alternating anoxic / aerobic culture according to claim 1, wherein, The composition of the solid supplemental culture medium includes: 3.20~3.30 g / L CH3COONa·3H2O, 0.01~0.02 g / L KH2PO4, 0.30~0.32 g / L NH4Cl, 0.09~0.10 g / L MgSO4·7H2O, 0.01~0.03 g / L K2SO4, 0.02~0.03 g / L CaCl2·2H2O, 0.8~1.2 mL / L trace element concentrate II, and 6~15 g / L agar powder; The trace element concentrate II has a pH of 6.0-6.5 and includes the following components: 0.4~0.6 g / L FeCl2·4H2O, 0.010~0.015 g / L MnCl2·4H2O, 0.06~0.08 g / L ZnCl2, 0.005~0.007 g / L H3BO3, 0.035~0.040 g / L Na2MoO4·2H2O, 0.010~0.015 g / L NiCl2·6H2O, and 0.001~0.003 g / L CuCl2·2H2O.

5. A method for screening polyphosphate-accumulating bacteria through alternating anaerobic / aerobic culture, characterized in that, The apparatus described in any one of claims 1 to 4 is suitable for screening polyphosphate-accumulating bacteria through alternating anaerobic / aerobic culture. The method includes the following steps: S1. Enrichment and coating stage: Inoculate environmental samples into LB liquid medium for enrichment culture, dilute the enriched culture solution under aseptic conditions, and then coat it on LB solid medium. S2, Screening stage of alternating anaerobic / aerobic culture of polyphosphate-accumulating bacteria: S21. Select different single bacterial colony sites from LB solid medium and inoculate them onto the surface of solid phosphorus-containing medium in the wells of a deep-well plate. S22. First anaerobic / aerobic alternating culture screening: The cover module is sealed with the deep well plate to form a closed anaerobic environment by contacting the solid supplemented culture medium with the solid phosphorus-containing culture medium. After incubation until signs of colony growth are observed on the solid phosphorus-containing culture medium, the cover module is removed, and the deep well plate is sealed to form a closed aerobic environment. Incubation continues until colony diameter increases or colony color darkens on the solid phosphorus-containing culture medium. Then, a second anaerobic / aerobic alternating culture screening is performed. S23. Second Anaerobic / Aerobic Alternating Culture Screening: Remove the solid supplementary medium used in the previous anaerobic culture and wash the overlying module. After sterilization, add new solid supplementary medium. The second anaerobic / aerobic alternating culture screening process is the same as the first anaerobic / aerobic alternating culture screening process, except that the culture time is adjusted according to the colony growth. Once the colonies on the solid phosphorus-containing medium have grown into single colonies of moderate size and obvious morphology, proceed to the BCIP colorimetric observation stage. If the colonies on the solid phosphorus-containing medium are still growing, repeat the anaerobic / aerobic alternating culture screening process as described in the second anaerobic / aerobic alternating culture screening until the colonies on the solid phosphorus-containing medium have grown into single colonies of moderate size and obvious morphology. S3, BCIP colorimetric observation stage: Using sterile coating beads, the BCIP colorimetric agent is evenly coated onto the surface of the solid phosphorus-containing medium to obtain the initial screening polyphosphate strains that grow well on the solid phosphorus-containing medium and show obvious blue colony turning reaction.

6. The method according to claim 5, characterized in that, In step S3, the BCIP colorimetric agent is an N,N-dimethylformamide solution with a BCIP mass concentration of 0.1% to 2%, and the coating amount of the BCIP colorimetric agent is 2 to 5 μL / well.

7. The method of claim 5, wherein, In step S3, 5 to 8 coating beads with a diameter of 0.4 mm to 0.8 mm are placed in each well, and the BCIP colorimetric agent is evenly coated on the surface of the solid phosphorus-containing culture medium by shaking in a figure-eight manner.

8. The method of claim 5, wherein, The method further includes the following steps: S4. Colony transfer: The pre-screened polyphosphate strains are transferred to a deep-well plate containing liquid phosphorus-containing medium, sealed, and cultured on a shaker at 28-37°C. S5. Determination of total phosphorus degradation capacity: Centrifuge the deep-well plate from step S4 to remove the bacterial cells, take the supernatant and add colorimetric reagent, screen out the strain with the lowest absorbance, which is the strain with the strongest ability to degrade total phosphorus, and identify the strain.

9. The method of claim 8, wherein, In step S4, the liquid phosphorus-containing culture medium comprises: 3.2~3.3 g / L CH3COONa·3H2O, 0.03~0.04 g / L KH2PO4, 0.30~0.32 g / L NH4Cl, 0.09~0.10 g / L MgSO4·7H2O, 0.01~0.03 g / L K2SO4, 0.02~0.03 g / L CaCl2·2H2O, and 0.8~1.2 mL / L trace element concentrate IIII; The trace element concentrate IIII has a pH of 6.0-6.5 and includes the following components: 0.4~0.6 g / L FeCl2·4H2O, 0.010~0.015 g / L MnCl2·4H2O, 0.06~0.08 g / L ZnCl2, 0.005~0.007 g / L H3BO3, 0.035~0.040 g / L Na2MoO4·2H2O, 0.010~0.015 g / L NiCl2·6H2O, and 0.001~0.003 g / L CuCl2·2H2O.

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