Salt-tolerant, dephosphorizing and denitrifying pha-producing bacterial strain and application thereof

Abstract

The application belongs to the technical field of microbial application, and relates to a salt-tolerant, dephosphorizing and denitrifying PHA strain and application thereof. Zobellella denitrificans ) ZD10, which has been preserved in the China Center for Type Culture Collection, located in Wuhan, Wuhan University, China, and has a preservation number of CCTCC NO: M 20242632 and a preservation date of November 25, 2024. The strain has the following applications: application in the remediation of eutrophic water bodies and the treatment of high-salt and high-ammonia-nitrogen wastewater; application in the treatment of ordinary ammonia-nitrogen wastewater; application in the treatment of industrial high-ammonia-nitrogen wastewater; application in the treatment of eutrophic seawater; application in the production of PHA by using various carbon sources; and application in marine circular economy. The strain has the functions of simultaneous dephosphorization and denitrification, can realize simultaneous nitrification and denitrification, dephosphorization and the production of raw material PHA with unique physical properties under high-salt (3%) and non-sterilized conditions, and can play a unique role in the construction of a marine circular economy system and the treatment of high-ammonia-nitrogen wastewater.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and relates to a salt-tolerant, phosphorus-removing, and nitrogen-removing PHA-producing strain and its application, specifically to a Zobellella denitrificans strain ZD10 and its application in the remediation of eutrophic water bodies and the treatment of high-salt and high-ammonia-nitrogen wastewater. Background Technology

[0002] High-ammonia nitrogen industrial wastewater (ammonia concentration of 500-2500 mg / L) and eutrophication in nearshore waters caused by domestic wastewater discharge, high-ammonia nitrogen wastewater discharge, and unregulated fish and shrimp farming are two major challenges hindering water purification. Currently, the main treatment methods for high-ammonia nitrogen industrial wastewater include sedimentation, stripping, adsorption, ion exchange, membrane filtration, and advanced oxidation. These methods often require large amounts of energy and chemicals, causing secondary environmental pollution and significant energy consumption, making them uneconomical and unsustainable. Furthermore, these methods cannot be implemented in large-scale water bodies for the treatment of algal blooms. Therefore, how to remove nitrogen and phosphorus in open waters to inhibit algal blooms is a major challenge.

[0003] Biological nitrogen removal (BNR) is an effective method for treating industrial wastewater by removing low concentrations of ammonia nitrogen. It is considered a sustainable and relatively economical treatment method (if implemented properly), and therefore theoretically applicable to the treatment of eutrophication in large bodies of water. However, due to high fermentation costs and uncontrollable factors such as water temperature and ocean currents, many problems remain to be solved in practical applications. Furthermore, most algal blooms are caused by excessive inorganic phosphorus content, so strains with only denitrification capabilities cannot meet the requirements for treating eutrophic water bodies. For wastewater with high ammonia nitrogen levels, current BNR methods are not economically effective at removing ammonia nitrogen because as ammonia concentration increases, the oxygenation required for nitrification using traditional autotrophic bacteria rises rapidly, increasing the cost of ammonia nitrogen removal to some extent. When using heterotrophic nitrifying and agrotrophic denitrifying bacteria for treatment, sufficient chemically pure carbon sources (such as sodium acetate, sodium citrate, and sodium succinate) must be added to effectively remove ammonia nitrogen, increasing the cost of treatment. Furthermore, there are few strains that can tolerate high ammonia nitrogen levels and organic solvents, thus presenting certain limitations. Therefore, it is essential to develop biosafe strains from marine environments that can adapt to indigenous environments and utilize the complex carbon sources of seawater for nitrogen and phosphorus removal in open environments. Simultaneously, if high-value-added products can be harvested from the obtained bacterial cells, it can, to some extent, offset the increased costs caused by seed culture fermentation, making this treatment method more sustainable.

[0004] The emergence of heterotrophic nitrifying-aerobic denitrifying bacteria offers a new approach to solving the aforementioned problems. These strains can utilize the oxidation of organic matter to provide electrons for ammonia oxidation and bacterial respiration, overcoming the slow growth caused by insufficient electron supply in chemoautotrophic ammonia-oxidizing bacteria. Theoretically, if this strain can utilize organic matter in eutrophic seawater and transfer electrons to the electron transport chain of ammonia oxidation, it can achieve the goal of simultaneously removing nitrogen while removing organic matter. However, to simultaneously remove phosphorus from seawater or high-ammonia-nitrogen aqueous water to inhibit the excessive growth of toxic algae and other microorganisms, phosphorus removal is also necessary. Theoretically, different microbial communities can be used to remove ammonia nitrogen and phosphorus separately; however, due to the interactions between microorganisms, this interaction is difficult to control, making the ammonia nitrogen removal process difficult to design. A process using a single microorganism (heterotrophic nitrifying-aerobic denitrifying polyphosphate-accumulating bacteria) for ammonia nitrogen and phosphorus removal is easier to design. To date, a few heterotrophic nitrifying-aerobic denitrifying polyphosphate-accumulating bacteria strains have been reported, and these strains have potential application value in treating high-ammonia-nitrogen wastewater and water treatment. However, there are also certain limitations to their applications. For example, most of these strains cannot utilize inexpensive carbon sources for nitrogen and phosphorus removal, and they have poor tolerance to environmental changes. Another practical problem is that their application in large-scale open water bodies is too costly, and the growth conditions of the strains cannot be controlled. Therefore, to date, there are no successful cases of using these strains for the treatment of real high-ammonia nitrogen wastewater or large-scale algal blooms. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention aims to provide a salt-tolerant, heterotrophic nitrification-aerobic denitrification, polyphosphate-accumulating, and PHA-producing bacterium and its applications.

[0006] The present invention adopts the following technical solution:

[0007] A salt-tolerant, phosphorus- and nitrogen-removing PHA-producing strain, taxonomically named Zobellella denitrificans ZD10, has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242632 and deposit date November 25, 2024.

[0008] In the above technical solution, a salt-tolerant, phosphorus-removing, and nitrogen-removing PHA-producing strain is isolated from high-salt, eutrophic fish and shrimp aquaculture bottom sediment. It is salt-tolerant, can remove nitrogen through heterotrophic nitrification-aerobic denitrification, can remove phosphorus through the formation of polyphosphoric acid, and can utilize seawater and various carbon sources to produce biodegradable plastics.

[0009] The second objective of this invention is to provide an application of a salt-tolerant, phosphorus-removing, and nitrogen-removing PHA-producing strain in the remediation of eutrophic water bodies and the treatment of high-salt and high-ammonia-nitrogen wastewater.

[0010] The third objective of this invention is to provide an application of a salt-tolerant, phosphorus-removing, and nitrogen-removing PHA-producing strain in the treatment of ordinary ammonia nitrogen wastewater.

[0011] The fourth objective of this invention is to provide an application of a salt-tolerant, phosphorus-removing, and nitrogen-removing PHA-producing strain in the treatment of industrial high-ammonia-nitrogen wastewater.

[0012] The fifth objective of this invention is to provide an application of a salt-tolerant, phosphorus-removing, and nitrogen-removing PHA-producing strain in the treatment of eutrophic seawater.

[0013] The sixth objective of this invention is to provide an application of a salt-tolerant, dephosphorization- and denitrification-resistant PHA-producing strain in the production of PHA using various carbon sources.

[0014] The seventh objective of this invention is to provide an application of a salt-tolerant, dephosphorization, and nitrogen-removing PHA-producing strain in the marine circular economy.

[0015] The eighth objective of this invention is to provide a bacterial agent comprising the salt-tolerant, phosphorus-removing, and nitrogen-removing PHA-producing strains described above.

[0016] Combining all the above technical solutions, the advantages and positive effects of this invention are as follows:

[0017] 1. The (Zobellella denitrificans) ZD10 provided by this invention can grow using ammonia nitrogen, nitrite, and nitrate as the sole nitrogen source in a high-salt environment. It can also achieve the simultaneous removal of ammonia nitrogen, nitrite, nitrate, and inorganic phosphorus using multiple carbon sources under aerobic and microaerobic conditions. Furthermore, it can convert organic matter in wastewater into PHA, a raw material for biodegradable plastics, thus partially solving the problems of treating high-salt and high-ammonia nitrogen wastewater and removing nitrogen and phosphorus from large water bodies.

[0018] 2. This strain has the function of simultaneous phosphorus and nitrogen removal. It can achieve simultaneous nitrification and denitrification, phosphorus removal and produce PHA, a raw material for biodegradable plastics with unique physical properties, under high salt (3%) and non-sterilization conditions. It can play a unique role in the construction of marine circular economy system and the treatment of high ammonia nitrogen wastewater. Attached Figure Description

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, it should be understood that these drawings are designed for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless specifically indicated, these drawings are intended only to conceptually illustrate the structural construction described herein and are not necessarily drawn to scale.

[0020] Figure 1 This is a diagram showing the results of the systematic classification of the strains using NCBI blast.

[0021] Figure 2 These are diagrams showing the characteristics of single colonies and bacterial growth of the strain.

[0022] Figure 3 This is a morphological image of a single bacterial cell under a scanning electron microscope.

[0023] Figure 4 This is a graph showing the effect of the strain on ammonia nitrogen removal.

[0024] Figure 5 This is a graph showing the effect of the strain on the removal of total phosphorus.

[0025] Figure 6 These are images showing the polyphosphate and PHA produced by the strain. In the images, A and B show the production of polyphosphate particles observed using two different dyes, and C and D show the production of PHA observed using two different dyes.

[0026] Figure 7 This is a diagram showing the results of the strain's denitrification and phosphorus removal in high-salinity (approximately 3%) seawater.

[0027] Figure 8 shows the PHA produced by the strain using seawater and some of its physical characteristics, where: ABCD are the Fourier transform infrared (FTIR), proton nuclear magnetic resonance (1HNMR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) parameters of the strain's PHA production, respectively. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific implementation methods. These embodiments are only for illustrative purposes, and the scope of the invention is not limited to the specific implementation methods, but is defined by the scope of the claims. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the percentage contents refer to the mass or volume content per liter of culture medium.

[0029] Example 1: The culture medium formulation used in this example is as follows:

[0030] (1) Polyphosphate enrichment medium: CH3COONa 3.6g, MgSO4·7H2O 0.132g, Na2HPO4·12H2O 0.029g, K2SO4 0.027g, NH4Cl 0.057g, CaCl2·2H2O 0.017g, HEPES buffer 12mL, trace elements 2mL, pH 7.2.

[0031] (2) Nitrification culture medium: (NH4)2SO4 0.71 g, sodium citrate 3.94 g, K2HPO4·3H2O 6.5 g, MgSO4·7H2O 2.5 g, NaCl 2.5 g, FeSO4·7H2O 0.05 g, MnSO4·H2O 0.04 g, NaCl content 0-15%.

[0032] (3) LB medium: peptone 10.00 g / L, yeast extract 5.00 g / L, pH adjusted to 7.0, NaCl 0-15%;

[0033] (4) Denitrification medium: NaNO3 0.84 g / NaNO2 0.25 g, FeSO4·7H2O 0.5 g, MnSO4·H2O 0.4 g, K2HPO4·3H2O 6.5 g, MgSO4·7H2O 2.5 g, sodium citrate 5.1 g, trace element solution 2 mL / L, NaCl 0-15%, pH 7.0-7.5;

[0034] (5) Simulated seawater culture medium: obtained from the fish and shrimp breeding pond of Tianjin Changlu Haijing Group Co., Ltd., without any sterilization treatment, and directly used for the culture of the strain at room temperature and 100 rpm.

[0035] Example 2: Isolation, Identification and Preservation of Strains

[0036] (1) Isolation of bacterial strains: Seawater sediment was serially diluted and spread on polyphosphate agar plates. After incubation at 28°C for 48 hours, approximately 80 single colonies were obtained, including bacteria, fungi, and actinomycetes. These colonies were labeled, and nitrogen- and phosphorus-removing bacteria were preliminarily screened. Colonies with fast growth rates, numbered 2, 6, 7, 9, 10, 11, 12, 13, 14, and 16, were selected for streak purification and inoculated into phosphorus-rich medium. After shake-flask incubation, the changes in nitrogen and phosphorus content in the medium were detected. Based on the nitrogen and phosphorus removal rates, strain number 10 was selected as a candidate strain.

[0037] (2) Identification of the strain: Fresh single colonies were picked and placed in 50 μL of Takara microbial lysis buffer, treated at 85°C for 15 minutes, and after slight centrifugation, no more than 1.5 μL of supernatant was used as a template. After PCR amplification, a 16S rDNA fragment of approximately 1435 bp was obtained. The product was sent to BGI Genomics for sequencing using primers 27F and 1492R. By comparing the obtained sequence with the NCBI Genebank database, a phylogenetic tree was constructed in MEGA5.0 software using the neighbor-joining method. Based on the phylogenetic relationship, the strain was identified as *Zobellella denitri ficans*, and further named *Zobellella denitrificans* ZD10. The identification results are shown in the appendix to the instruction manual. Figure 1 As shown.

[0038] Colony morphology: Fresh single colonies were streaked onto LB agar plates and incubated at 30°C for 48 hours. After single colonies grew on the plates, they were observed. The colonies of this strain were light yellow with a moist and smooth surface. The results are as shown in the attached instructions. Figure 2 As shown.

[0039] Scanning electron microscopy (SEM) morphology of the bacterial strain: The bacteria are rod-shaped, approximately 2.5 micrometers in length and 1 micrometer in width. Results are shown in the attached instruction manual. Figure 3 As shown.

[0040] (3) Preservation of the strain: The Zobellella denitrificans in this invention was obtained from the fish and shrimp farming pond of Tianjin Changlu Haijing Group Co., Ltd. Based on the above identification and analysis results and the 16S rDNA sequence homology analysis results, it was identified as a Zobellella genus bacterium and named Zobellella denitrificans ZD10. It has been deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO: M 20242632 and deposit date of November 25, 2024.

[0041] Example 3: Determination of ammonia nitrogen removal capacity of the strain. The specific steps are as follows:

[0042] Fresh ZD10 single colonies were inoculated into 100 mL of heterotrophic nitrification culture medium and cultured on a shaker at 30℃ and 150 rpm. Samples were taken every 24 hours, and the bacteria were removed by centrifugation at 12000 rpm for 5 min. The concentration of residual ammonia nitrogen was then measured using a Lianhua Technology multi-parameter water quality analyzer (5B-6C) to determine the ammonia nitrogen removal capacity. The results are shown in the attached instructions. Figure 4 As shown in the results, strain 10 achieved an ammonia nitrogen removal rate of 86% within 48 hours. This indicates that the strain does indeed possess a certain denitrification ability, and the denitrification effect is quite good.

[0043] Example 4: Total phosphorus removal effect of the strain

[0044] Similar to ammonia nitrogen removal, fresh single colonies of the strain were inoculated into polyphosphate liquid medium and cultured at 30°C with shaking at 150 rpm. Samples were taken every 24 hours and centrifuged at 12,000 rpm to collect the supernatant. Changes in total phosphorus in the supernatant were detected using a Lianhua Technology multi-parameter water quality analyzer. Results are shown in the attached instruction manual. Figure 5 As shown in the results, the strain achieved a total phosphorus removal rate of 61.5% within 48 hours, demonstrating a high phosphorus removal capacity while also removing ammonia nitrogen.

[0045] Example 5, Polyphosphates from Strains and the PHAs They Produce

[0046] To observe the polyphosphate accumulation and PHA production in the bacterial cells, Sudan Black and Nile Red staining were used to preliminarily determine whether the strain had the ability to produce PHA. The results are shown in the appendix to the instruction manual. Figure 6 As shown in A and 6B. In the Sudan Black staining method, under an optical microscope, the bacterial cells appear red, and the lipid substance PHA appears black. In the Nile Red staining method, red fluorescence (polyphosphate particles) was observed under a fluorescence microscope, indicating that this strain can produce polyphosphate and PHA. The results are shown in the attached instructions. Figure 6 As shown in C and 6D.

[0047] Example 6: Results of nitrogen and phosphorus removal by the strain in high-salinity (approximately 3%) seawater.

[0048] To verify the strain's ability to remove nitrogen and phosphorus and produce PHA in real seawater, the strain was first cultured in LB liquid medium to the logarithmic growth phase. The bacterial cells were collected by high-speed centrifugation at 12,000 rpm, and washed and resuspended with sterile water to remove nitrogen and phosphorus from the medium. The resuspended bacterial cells were then inoculated into 500 mL of aquaculture seawater and cultured with microaerobic shaking (50 rpm). During this period, the concentrations of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, and total phosphorus were continuously sampled and monitored. The results are shown in the appendix to the instruction manual. Figure 7 As shown.

[0049] As shown in the figure, the strain achieved a total phosphorus removal rate of 68.41% and an ammonia nitrogen removal rate of 91.76% within 24 hours. It produced very small amounts of nitrate nitrogen, not exceeding 1 mg / L, and almost no nitrite nitrogen. These results indicate that the strain has the potential for nitrogen and phosphorus removal in seawater, has practical application value, and exhibits good nitrogen removal efficiency. This strain has the function of simultaneously removing ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen, as well as phosphorus, from high-salt, non-sterilized seawater.

[0050] Example 7: PHA produced by the strain using seawater and some of its physical characteristics

[0051] The above-mentioned seawater was cultured to OD 600When no further additions were observed, the bacterial cells were collected by centrifugation at 12,000 rpm, dried at 70°C, and the intracellular PHA produced was extracted using chloroform extraction. The PHA was then identified and characterized by infrared spectroscopy, nuclear magnetic resonance, and thermogravimetric analysis, with results shown in the appendix to the instruction manual. Figure 8A As shown in BCD.

[0052] In the infrared spectrum, a sharp absorption peak is observed at 1721 cm⁻¹, corresponding to the carbonyl (C=O) stretching of the ester, and another absorption peak is observed at 1277 cm⁻¹, corresponding to the -CH group. The presence of these absorption peaks has been reported and labeled as PHA markers. In the NMR spectrum, three different signals at 1.29, 2.5, and 5.27 ppm (ppm refers to chemical shift) represent methyl (CH₃), methylene (CH₂), and methanethiol (CH₂), respectively, all characteristic structures of PHB. Therefore, both infrared and NMR results confirm that the substance produced by the strain is PHB, and also demonstrate the strain's potential to produce PHA using multiple carbon sources. This indicates that the strain has the function of converting organic matter in wastewater into PHA, a raw material for biodegradable plastics.

[0053] Based on the above embodiments, it is evident that the strain in this invention can simultaneously remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen, as well as dephosphorize, from high-salt, non-sterilized seawater. More importantly, this strain can utilize nutrients in seawater to produce PHA, which has significant application value, and the enriched phosphorus can be used for phosphate fertilizer application. The above embodiments demonstrate that this strain can be applied in the remediation of eutrophic water bodies and the treatment of high-salt, high-ammonia nitrogen wastewater; in the treatment of ordinary ammonia nitrogen wastewater; in the treatment of industrial high-ammonia nitrogen wastewater; in the treatment of eutrophic seawater; and in the production of PHA using various carbon sources.

[0054] If photovoltaic power generation is used to construct floating fermentation devices in wide waters to provide a certain amount of electricity, it will also have a certain tourism effect and reduce the cost of fermentation. The tourism revenue and the generated PHA can also significantly reduce the costs of fermentation and production processes, creating a more sustainable way to treat wastewater and algal blooms, and playing a unique role in the construction of a marine circular economy system and the treatment of high ammonia nitrogen wastewater.

[0055] In summary, this strain possesses simultaneous phosphorus and nitrogen removal capabilities, enabling simultaneous nitrification and denitrification under high-salt (3%) and non-sterile conditions. It also removes phosphorus and produces PHA, a biodegradable plastic feedstock with unique physical properties. This strain can play a unique role in the construction of marine circular economy systems and the treatment of high-ammonia-nitrogen wastewater. This invention has broad application prospects and the potential to generate significant social, economic, and environmental benefits.

[0056] The above embodiments have provided a detailed description of the present invention, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain, characterized in that: Its taxonomic name is Denitrifying Zobel ( Zobellella denitrificans ZD10 has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242632 and deposit date November 25, 2024.

2. The application of the salt-tolerant, phosphorus-removing, and nitrogen-removing PHA-producing strain as described in claim 1 in the treatment of high-salt, ammonia-nitrogen wastewater with a salinity of 3%.

3. The application of the salt-tolerant, phosphorus-removing, and nitrogen-removing PHA-producing strain as described in claim 1 in denitrification and phosphorus removal in seawater with a salinity of 3%.

4. The application of a salt-tolerant, dephosphorization, and nitrogen-removing PHA-producing strain as described in claim 1 in the production of PHB.

5. A microbial agent, characterized in that: It contains the salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain with accession number CCTCC NO: M20242632 as described in claim 1.

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