Salt-tolerant, dephosphorizing and denitrifying PHA-producing strain and application thereof

Abstract

The invention belongs to the technical field of microorganism application, and relates to a salt-resistant, dephosphorizing and denitrifying PHA-producing strain and application thereof. The strain has a taxonomic name of Zobells denitrificans ZD10, is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20242632, and the preservation date is November 25, 2024. The strain can be applied to eutrophic water body remediation and high-salt and high-ammonia-nitrogen wastewater treatment; application in the aspect of common ammonia-nitrogen wastewater treatment; the composite material is applied to the aspect of industrial high-ammonia-nitrogen sewage treatment; the method is applied to the aspect of eutrophication seawater treatment; the application is used for producing PHA by adopting various different carbon sources; the method is applied to ocean circular economy. The strain has synchronous dephosphorization and denitrification functions, can realize synchronous nitrification, denitrification and dephosphorization under high-salt (3%) and non-sterilization conditions, can generate a degradable plastic raw material PHA with unique physical properties, and can play a unique role in construction of a marine circular economy system and high-ammonia-nitrogen sewage treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and relates to a salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain and an application thereof, and specifically relates to a denitrifying Zobellella denitrificans strain ZD10 and an application thereof in eutrophic water restoration and high-salt, high-ammonia nitrogen wastewater treatment. Background Art

[0002] High ammonia nitrogen industrial sewage (ammonia concentration is 500-2500mg / L) and the eutrophication problem of offshore waters caused by life and high ammonia nitrogen sewage discharge and irregular fish and shrimp farming are two major difficulties that plague water purification. At present, the main treatment methods of high ammonia nitrogen industrial sewage include precipitation, stripping, adsorption, ion exchange, membrane filtration and advanced oxidation. These methods need to invest a large amount of energy and chemicals, causing secondary environmental pollution and a large amount of energy consumption, so they are uneconomical and unsustainable methods, and these methods cannot be implemented in large-scale waters for the treatment of algal blooms. Therefore, how to remove nitrogen and phosphorus elements in open waters to inhibit algal blooms is a difficulty among difficulties.

[0003] The biological nitrogen removal (BNR) method can effectively remove low-concentration ammonia nitrogen to treat industrial wastewater. It is considered to be a sustainable and relatively economical treatment method (if implemented properly). Therefore, it can theoretically be used to treat eutrophication in large waters. However, due to the high fermentation cost and the influence of uncontrollable factors such as water temperature and ocean currents, there are still many problems to be solved in the actual application process. In addition, the cause of most algal blooms is the excessive inorganic phosphorus content, so strains with pure denitrification function cannot meet the requirements for treating eutrophic water bodies. For high-ammonia nitrogen wastewater, the currently used BNR cannot remove ammonia nitrogen economically and effectively. The reason is that with the increase of ammonia concentration, the amount of oxygen required for removal when using traditional autotrophic bacteria for nitrification reaction increases rapidly, which increases the cost of ammonia nitrogen removal to a certain extent. When using heterotrophic nitrifying and denitrifying bacteria for treatment, sufficient chemical pure carbon sources (such as sodium acetate, sodium citrate and sodium succinate, etc.) must be added to effectively remove ammonia nitrogen, which increases the cost of treatment. In addition, there are few strains that can tolerate high ammonia nitrogen and organic solvents, so there are certain shortcomings. Therefore, it is necessary to develop biosafety strains from the marine environment that can adapt to the indigenous environment and use the complex carbon source of seawater in the open environment for nitrogen and phosphorus removal. At the same time, if high value-added products can be harvested from the obtained bacteria, the cost increase caused by seed liquid fermentation can be compensated to a certain extent, making this treatment method more sustainable.

[0004] The emergence of heterotrophic nitrification-aerobic denitrification bacteria provides a new idea for solving the above problems. This type of strain can use the oxidation of organic matter to provide electrons for ammonia oxidation and bacterial respiration, overcoming the disadvantage of slow growth caused by insufficient electron supply of chemoautotrophic ammonia oxidizing bacteria. In theory, if the strain can use organic matter in eutrophic seawater and transfer electrons to the electron transport chain of ammonia oxidation, it can achieve the purpose of removing nitrogen while removing organic matter. However, in order to remove phosphorus from seawater or high-ammonia nitrogen anhydrous water to inhibit the excessive growth of toxic algae and other microorganisms, phosphorus needs to be removed. In theory, different microbial flora can be used to remove ammonia nitrogen and phosphorus respectively, but due to the interaction between microorganisms, this effect is not easy to control, and the process of removing ammonia nitrogen is not easy to design. It is easier to design a process for removing ammonia nitrogen and phosphorus using one microorganism (heterotrophic nitrification aerobic denitrification polyphosphate bacteria). So far, a few strains of heterotrophic nitrification aerobic denitrification polyphosphate bacteria have been reported, and these strains have potential application value in treating high-ammonia nitrogen wastewater and managing water. However, there are certain application limitations. For example, most of these strains cannot use cheap carbon sources to remove nitrogen and phosphorus, and have poor tolerance to environmental changes. Another more realistic problem is that the cost is too high when applied to large open waters, and the growth conditions of the strains cannot be controlled. Therefore, so far, there has been no successful case of using these strains to treat real high-ammonia nitrogen sewage or large-scale algal blooms. Summary of the invention

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

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

[0007] A salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain, whose taxonomic name is Zobellella denitrificans ZD10, has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit number is CCTCC NO: M 20242632, and the deposit date is November 25, 2024.

[0008] In the above technical scheme, a salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain is a salt-tolerant bacteria isolated from high-salt, eutrophic fish and shrimp aquaculture mud, which can remove nitrogen through heterotrophic nitrification-aerobic denitrification, remove phosphorus by forming polyphosphate, and use seawater and a variety of carbon sources to produce degradable plastics.

[0009] The second invention object of the present invention is to provide a salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain for use in eutrophic water restoration and high-salt, high-ammonia nitrogen wastewater treatment.

[0010] The third invention objective of the present invention is to provide a salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain for use in the treatment of common ammonia nitrogen wastewater.

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

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

[0013] The sixth object of the present invention is to provide a salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain for use in producing PHA using various carbon sources.

[0014] The seventh invention objective of the present invention is to provide a salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain for use in marine circular economy.

[0015] The eighth invention objective of the present invention is to provide a bacterial agent, which comprises the above-mentioned salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain.

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

[0017] 1. The (Zobellella denitrificans) ZD10 provided by the present invention can grow in a high-salinity environment using ammonia nitrogen, nitrite and nitrate as the only nitrogen source, and can use multiple carbon sources under aerobic and microaerobic conditions to achieve the simultaneous removal of ammonia nitrogen, nitrite, nitrate and inorganic phosphorus, and convert organic matter in sewage into PHA, a raw material for degradable plastics, partially solving the problems of high-salinity and high-ammonia nitrogen wastewater treatment and nitrogen and phosphorus removal in vast waters.

[0018] 2. This strain has the function of simultaneous dephosphorization and denitrification. It can achieve simultaneous nitrification and denitrification, remove phosphorus and produce PHA, a raw material for degradable plastics with unique physical properties, under high salt (3%) and non-sterile conditions. It can play a unique role in the construction of marine circular economy system and the treatment of high ammonia nitrogen wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments, but it should be understood that these drawings are designed only for explanation purposes and are not intended to limit the scope of the present invention. In addition, unless otherwise specified, these drawings are intended only to conceptually illustrate the structural configurations described herein and are not necessarily drawn to scale.

[0020] Figure 1 This is the result of systematic classification of strains through NCBI blast.

[0021] Figure 2 It is the single colony and bacterial lawn characteristic diagram of the strain.

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

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

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

[0025] Figure 6 These are diagrams showing the effects of polyphosphate in the strain and the production of PHA, where: A and B use two different dyes to observe the production of polyphosphate particles; C and D also use two different dyes to observe the production of PHA.

[0026] Figure 7 This is a graph showing the results of the strain removing nitrogen and phosphorus in high-salinity (about 3%) seawater.

[0027] FIG8 is a diagram of PHA produced by the strain using seawater and some of its physical properties, wherein: ABCD are the Fourier transform infrared spectrum (FTIR), proton nuclear magnetic resonance spectrum (1HNMR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) parameters of PHA produced by the strain, respectively. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with specific implementation methods. The embodiments are only used to explain the present invention. The scope of the present invention is not limited to the specific implementation methods, but is limited by the scope of the claims. The experimental methods used in the following examples are conventional methods unless otherwise specified; the percentage contents are the mass or volume content per liter of culture medium unless otherwise specified.

[0029] Example 1, the culture medium formula used in the example is as follows:

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

[0031] (2) Nitration 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 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 culture 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., directly used for strain cultivation without any sterilization treatment, the temperature was room temperature, and the rotation speed was 100 rpm.

[0035] Example 2, Isolation, identification and preservation of strains

[0036] (1) Isolation of strains: The seawater sediment was diluted in a gradient manner and spread on a polyphosphate medium plate. After culturing at 28 degrees for 48 hours, about 80 single colonies were obtained, including bacteria, fungi, and actinomycetes. They were labeled and preliminarily screened for denitrification and phosphorus removal bacteria. The fast-growing colonies No. 2, 6, 7, 9, 10, 11, 12, 13, 14, and 16 were selected for streaking purification and inoculated into a phosphorus-rich medium for shake flask culture. The changes in the nitrogen and phosphorus contents in the medium were detected, and strain No. 10 was selected as a candidate strain based on the nitrogen and phosphorus removal rates.

[0037] (2) Identification of strains: Pick a fresh single colony into 50 μl Takara microbial lysis buffer, treat at 85 degrees for 15 minutes, centrifuge slightly, take no more than 1.5 μl of supernatant as a template, and amplify by PCR to obtain a 16SrDNA fragment of about 1435 bp in length. The product was sent to BGI for sequencing using 27F and 1492R primers. The obtained sequence was compared with the NCBI Genebank database, and a phylogenetic tree was constructed in the MEGA5.0 software program using the neighbor-joining method. The strain was identified as Zobellella denitri ficans based on the relationship, and further named as Zobellella denitrificans ZD10 strain. The identification results are shown in the attached manual. Figure 1 shown.

[0038] Colony morphology of strains: Take a fresh single colony and streak it on the LB plate, and culture it in a 30℃ incubator for 48 hours. After a single colony grows on the plate, observe it. The colony of the strain is light yellow with a moist and smooth surface. The results are as shown in the instructions. Figure 2 shown.

[0039] Microscopic morphology of the strain under scanning electron microscope: The bacteria are long rod-shaped, about 2.5 microns in length and about 1 micron in width. Figure 3 shown.

[0040] (3) Preservation of strains: The denitrifying Zobellella in the present invention was obtained from a fish and shrimp breeding pond of Tianjin Changlu Haijing Group Co., Ltd. According to the above identification and analysis results and the 16SrDNA sequence homology analysis results, it was determined to be a bacterium of the genus Zobellella and named as denitrifying Zobellella (Zobellella denitrificans) ZD10. It has been deposited in the China Center for Type Culture Collection, with a preservation address of Wuhan University, Wuhan, China, with a preservation number of CCTCCNO: M 20242632, and a preservation date of November 25, 2024.

[0041] Example 3, determination of ammonia nitrogen removal ability of strains, the specific steps are as follows:

[0042] Pick a fresh single colony of ZD10 and inoculate it into 100mL heterotrophic nitrification culture medium. Cultivate it in a shaking table at 30℃ and 150rpm. Take samples every 24 hours. Centrifuge at 12000rpm for 5min to remove the bacteria. Use Lianhua Technology Multi-parameter Water Quality Analyzer (5B-6C) to measure the concentration of residual ammonia nitrogen, so as to determine the ability to remove ammonia nitrogen. The results are shown in the attached manual. Figure 4 As shown in the results, it can be seen that the ammonia nitrogen removal rate of strain No. 10 within 48 hours is 86%, which shows that the strain does have a certain denitrification ability and the denitrification effect is good.

[0043] Example 4, the effect of strains on the removal of total phosphorus

[0044] Similar to the removal of ammonia nitrogen, a fresh single colony of the strain was inoculated into a polyphosphate liquid culture medium, cultured at 30 degrees and 150rpm, and sampled every 24 hours and centrifuged at 12000rpm to obtain the supernatant. The Lianhua Technology multi-parameter water quality meter was used to detect the changes in total phosphorus in the supernatant. The results are shown in the attached manual. Figure 5 As shown, it can be seen from the results that the total phosphorus removal rate of the strain within 48 hours is 61.5%, and it also has a high phosphorus removal ability while removing ammonia nitrogen.

[0045] Example 5, Phosphorus Polymerization by Strain and PHA Produced by Strain

[0046] In order to observe the polyphosphate accumulated in the bacteria and the PHA produced, Sudan black staining and Nile red staining were used to preliminarily determine whether the strain has the ability to produce PHA. The results are shown in the attached manual. Figure 6 In the Sudan black staining method, under an optical microscope, the bacteria appear red, and the lipid substance PHA appears black; in the Nile red staining method, red fluorescence (polyphosphate particles) is observed under a fluorescence microscope, indicating that the modified strain can produce polyphosphate and PHA. The results are shown in the attached manual. Figure 6 C and 6D.

[0047] Example 6, Results of denitrification and phosphorus removal by strains in high salinity (about 3%) seawater

[0048] In order to verify the ability of the strain to remove nitrogen and phosphorus and produce PHA in real seawater, the strain was first cultured in LB liquid medium to the logarithmic phase, the bacteria were collected by high-speed centrifugation at 12000rpm, washed and resuspended with sterile water to remove nitrogen and phosphorus elements in the culture medium, and the resuspended bacteria were inoculated into 500mL of aquaculture seawater for microaerobic shaking (50rpm) culture. During this period, samples were continuously taken to monitor the concentration changes of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and total phosphorus. The results are shown in the attached manual. Figure 7 shown.

[0049] As can be seen from the figure, the total phosphorus removal rate of the strain within 24 hours is 68.41%, and the ammonia nitrogen removal rate is 91.76%. A very small amount of nitrate nitrogen is produced, not exceeding 1 mg / L, and almost no nitrite nitrogen is produced. These results show that the strain has the potential to remove nitrogen and phosphorus in seawater, has practical application value, and has a good denitrification effect. The strain has the function of simultaneously removing ammonia nitrogen, nitrate nitrogen and nitrite nitrogen and removing phosphorus from high-salinity, non-sterile seawater.

[0050] Example 7. PHA produced by strains using seawater and some of its physical properties

[0051] The above seawater was cultured to OD 600When the cell count stopped increasing, the cells were collected by centrifugation at 12000 rpm, dried at 70°C, and the PHA produced in the cells was extracted by chloroform extraction. The PHA was identified and characterized by infrared, nuclear magnetic resonance and thermogravimetric analysis. The results are shown in the attached manual. Fig. 8A As shown in BCD.

[0052] In the infrared spectrum, there is a sharp absorption peak at 1721cm-1, corresponding to the carbonyl (C=O) stretching of the ester, and another absorption peak at 1277cm-1, corresponding to the -CH group. The existence of these absorption peaks has been reported and marked as PHA markers. In the nuclear magnetic resonance spectrum, the 1HNMR spectra of three different signals at 1.29, 2.5 and 5.27ppm (ppm refers to chemical shift) represent methyl (CH3), methylene (CH2) and methane (CH), which are all characteristic structures of PHB. Therefore, both infrared and nuclear magnetic resonance results confirm that the substance produced by the strain is PHB, and also illustrate that the strain has the potential to produce PHA using a variety of carbon sources. It can be seen that the strain has the function of converting organic matter in sewage into PHA, the raw material for degradable plastics.

[0053] Based on the above embodiments, it can be known that the strain of the present invention can simultaneously remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen and dephosphorize from high-salt, non-sterile seawater. More importantly, the strain can use the nutrients in seawater to produce PHA with important application value, and the enriched phosphorus element can be used for phosphate fertilizer application. Based on the above embodiments, it can be proved that this strain can be used in the restoration 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; in the production of PHA using various carbon sources.

[0054] If photovoltaic power generation is used to build a floating fermentation device in wide waters to provide a certain amount of electricity, there will be a certain sightseeing effect and reduce the cost of fermentation. The income from sightseeing and the PHA generated can also significantly reduce the cost of fermentation and production processes, build a more sustainable sewage and algal bloom management method, and play a unique role in the construction of the marine circular economy system and the treatment of high ammonia nitrogen sewage.

[0055] In summary, the strain has the functions of simultaneous dephosphorization and denitrification, can achieve simultaneous nitrification and denitrification, dephosphorization and produce PHA, a raw material for degradable plastics with unique physical properties, under high salt (3%) and non-sterile conditions, and can play a unique role in the construction of marine circular economy system and high ammonia nitrogen sewage treatment. The invention has broad application prospects and has the potential to generate high social, economic and environmental benefits.

[0056] The above embodiments describe the present invention in detail, but the contents are only preferred embodiments of the present invention and cannot be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

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

2. The salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain according to claim 1, characterized in that: It is a salt-tolerant bacterium isolated from high-salt, eutrophic fish and shrimp aquaculture mud, which can remove nitrogen through heterotrophic nitrification-aerobic denitrification, remove phosphorus by forming polyphosphate, and use seawater and a variety of carbon sources to produce degradable plastics.

3. An application of the salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain as claimed in claim 1 in the restoration of eutrophic water bodies and the treatment of high-salt, high-ammonia nitrogen wastewater.

4. Use of the salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain as claimed in claim 1 in the treatment of common ammonia nitrogen wastewater.

5. Use of the salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain as claimed in claim 1 in the treatment of industrial high-ammonia nitrogen wastewater.

6. Use of the salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain as claimed in claim 1 in the treatment of eutrophic seawater.

7. Use of the salt-tolerant, phosphorus-removing and nitrogen-removing PHA-producing strain as claimed in claim 1 in producing PHA using various carbon sources.

8. Use of the salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain as claimed in claim 1 in marine circular economy.

9. A bacterial agent, characterized in that: The invention comprises the salt-tolerant, phosphorus-removing, nitrogen-removing PHA-producing strain with the deposit number of CCTCC NO: M20242632 as claimed in claim 1.

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