1. A strain of seawater Psychrobacter (Psychrobacter aquimaris) A4N01, which is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 20 days in 2021, and has a biological preservation number of CCTCC NO: m2021120.

2. A microbial agent comprising a metabolite of psychrobacter maritima A4N01 and/or psychrobacter maritima A4N01 according to claim 1.

3. A halophilic nitrogen assimilating microbiome comprising a metabolite of the psychrobacter marinum A4N01 and/or the psychrobacter marinum A4N01 of claim 1;

preferably, the halophilic nitrogen assimilation microorganism group is obtained by inoculating the psychrobacter marinum A4N01 into high-salt sewage for culture;

preferably, the halophilic nitrogen assimilating microbiome is present in a form comprising high salinity sewage or activated sludge.

4. The method for constructing a saltcompatible nitrogen assimilating microorganism according to claim 3, characterized in that it comprises the following steps:

and (3) inoculating the psychrobacter marinum A4N01 into a bioreactor containing high-salinity sewage, and operating until the ammonia nitrogen removal efficiency and the sedimentation performance are stabilized to obtain the halophilic nitrogen assimilation microorganism group.

5. The method of claim 4, wherein the inoculated cells are controlled to be not less than 5 g/L;

the high-salinity sewage is simulated high-salinity sewage, the salinity of the simulated high-salinity sewage is not lower than 3% (w/w), and the high-salinity sewage is prepared by adopting old seawater;

preferably, the simulated high salinity wastewater component comprises: 0.8g/L of glucose, 0.5g/L of sodium acetate, 0.55g/L of ammonium chloride, 0.14g/L of dipotassium hydrogen phosphate and 0.25mg/L of peptone, and the salt content is 3.3 percent.

6. The construction method according to claim 4, characterized by comprising:

the bioreactor is a sequencing batch bioreactor;

adopting a continuous mode to operate, taking high-salinity sewage with the salt content of 3-7% as inlet water, controlling DO to be maintained at 2-3mg/L by aeration, and enabling the carbon-nitrogen ratio of the inlet water to be not less than 10;

preferably, the operation period is 8 hours, comprising 5min water inlet, 450min aeration, 15min sedimentation and 10min water outlet, without discharging sludge.

7. Use of a psychrophilum marigold (Psychrobacter aquimaris) A4N01 according to claim 1, a microbial inoculant according to claim 2 and/or a halophilic nitrogen assimilating microbiome according to claim 3 in any one or more of:

a) synthesizing single-cell protein;

b) recycling nitrogen and phosphorus nutrients;

c) treating wastewater;

d) preparing an organic fertilizer;

e) improving the soil fertility;

f) bioremediation of saline-alkali soil;

g) treating water eutrophication;

h) repairing water body pollution;

i) greenhouse gas emission reduction and carbon neutralization.

8. The use according to claim 7,

in said b), nitrogen is metabolized on assimilation;

in the step c), the wastewater comprises high-salinity wastewater, marine culture wastewater, industrial saline wastewater and seawater toilet flushing wastewater;

in said g), said body of water comprises fresh water and seawater, preferably seawater.

9. A method for the integrated resource conversion of nutrients in a high-salt environment is characterized by comprising the following steps: applying the Acidophilus marinus (Psychrobacter aquimaris) A4N01 of claim 1, the microbial agent of claim 2, and/or the saltphilic nitrogen assimilating microbiome of claim 3 to a high salt environment.

10. The method of claim 9, wherein the high salt environment is a high salt water environment;

preferably, the salinity of the high-salinity water environment is not lower than 3% (w/w), and is further preferably 3-7% (w/w).

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The nutrient transformation in the high-salinity environment is easily inhibited by salinity, and the problems of difficult high-salinity sewage treatment, difficult substance transformation of saline-alkali soil, easy nutrition loss and the like exist. However, since the high-salt sewage contains a large amount of nitrogen and phosphorus and is easy to cause eutrophication of water bodies when discharged into the environment, the sewage needs to be treated before being discharged. At present, the biological treatment method aiming at the high-salinity sewage is mainly a biological method, and the substance conversion in the high-salinity environment also mainly depends on the action of microorganisms. The conversion of nitrogen is mainly realized by the nitrification-denitrification of microorganisms, ammonia nitrogen in the sewage is oxidized into nitrite by ammonia oxidizing bacteria, then the nitrite oxidizing bacteria are converted into nitrate, finally the nitrate is reduced into nitrite by the denitrification of bacteria and then converted into nitrogen, and finally the conversion and removal of nitrogen are realized. However, in the process, the salinity can inhibit the activity of microorganisms, and particularly nitrite oxidizing bacteria are very sensitive to the salinity, so that the nitrification-denitrification path is inhibited, the accumulation of nitrite occurs, and the nitrogen removal efficiency is reduced. At the same time, nitrogen is converted to poorly utilized nitrogen gas, resulting in loss of nitrogen in the ecosystem with concomitant production of nitrous oxide as an isothermal gas. In addition, in a high-salt environment, the removal of organic matters and phosphorus is also inhibited, so that the synchronous removal of high-salt sewage pollutants is difficult.

The key of the biological method for realizing the conversion of nutrient substances is to find a salt-tolerant strain with the capability of utilizing organic matters, nitrogen, phosphorus and other pollutants and realize the stabilizing effect of the functional strain in an environmental microbiome. Chinese patent CN107739086A discloses a method for denitrifying high salinity wastewater using sludge developed from marine sediments or sludge. And (3) gradually reducing the carbon-nitrogen ratio and increasing the ammonia nitrogen concentration and the total nitrogen concentration, wherein after each process reaches stable operation for about 30 days, the next step is carried out, and after 59 days, the acclimation process of the sludge is realized. When the concentration of chloride ions in the wastewater treated by the sludge is 10-30g/L, the assimilation nitrogen removal proportion accounts for 95% of the total nitrogen removal rate, and the total nitrogen removal rate reaches 90%. However, the inventors have found that the sludge used in this method has disadvantages such as a large number of microbial communities, unclear metabolic characteristics, complicated regulation and control method, long sludge acclimation time, and slow system start-up.

Disclosure of Invention

Based on the defects of the prior art, the invention provides a marine self-flocculating bacterium and a saltiness nitrogen assimilation microorganism group driven by the marine self-flocculating bacterium, as well as a construction method and application of the marine self-flocculating bacterium and the saltiness nitrogen assimilation microorganism group. The invention screens and obtains a strain of seawater psychrophilic bacillus from marine sediments, assimilates ammonia nitrogen to synthesize organic nitrogen and synchronously converts organic matters and phosphorus, and researches unexpectedly find that the halophilic nitrogen assimilation microorganism group developed by the halophilic bacillus also has good nutrient substance conversion removal capacity and sedimentation performance, has wide tolerance to salinity, can be used in the directions of high-salinity sewage treatment, saline-alkali soil fertilization and the like, realizes the recovery of nutrient elements such as nitrogen, phosphorus and the like in a high-salinity environment, and has the advantages of simple operation, low price and environmental friendliness, thereby having good value of practical application.

In order to achieve the technical purpose, the invention relates to the following technical scheme:

in one aspect of the present invention, a Psychrobacter marinum (Psychrobacter aquimaris) A4N01 is provided, which has been preserved in China center for type culture Collection (address: Wuhan university of Lodoop Gashan mountain of Wuchang city, Wuhan, Hubei) at 1 month and 20 days in 2021, and has a biological preservation number of CCTCC NO: m2021120.

In a second aspect of the present invention, there is provided a microbial agent comprising the above-mentioned psychrobacter marinum A4N 01.

In a third aspect of the present invention, there is provided a halophilic nitrogen assimilating microorganism group comprising the aforementioned Haemophilus marinus A4N 01; more specifically, the halophilic nitrogen assimilation microorganism group is obtained by inoculating the psychrobacter marinum A4N01 into high-salt sewage for culture; specifically, the salt-nitrogen-assimilating microorganism group can be high-salt sewage obtained through the treatment or activated sludge obtained through the treatment.

In a fourth aspect of the present invention, there is provided a method for constructing the above halophilic nitrogen assimilating microorganism group, the method comprising:

inoculating the psychrobacter marinum A4N01 into a bioreactor containing high-salinity sewage, and operating until the ammonia nitrogen removal efficiency and the sedimentation performance are stabilized to obtain the halophilic nitrogen assimilation microorganism group.

In a fifth aspect of the present invention, the use of the aforementioned psychrophilic bacillus marinus (Psychrobacter aquimaris) A4N01, microbial agents and/or halophilic nitrogen assimilating microbiome in any one or more of:

a) synthesizing single-cell protein;

b) recycling nitrogen and phosphorus nutrients;

c) treating wastewater;

d) preparing an organic fertilizer;

e) improving the soil fertility;

f) bioremediation of saline-alkali soil;

g) treating water eutrophication;

h) repairing water body pollution;

i) greenhouse gas emission reduction and carbon neutralization;

wherein, in b), nitrogen is metabolized based on assimilation;

in the step c), the wastewater comprises high-salinity wastewater, marine culture wastewater, industrial saline wastewater and seawater toilet flushing wastewater;

in said g), said body of water comprises fresh water and seawater, preferably seawater.

In a sixth aspect of the present invention, a method for the integrated resource transformation of nutrients in a high-salt environment is provided, which comprises: the aforementioned Haemophilus marinus (Psychrobacter aquimaris) A4N01, microbial agents, and/or salttolerant nitrogen assimilating microbiome are applied to a high salt environment.

More specifically, the high salinity environment is a high salinity environment, and the salinity of the high salinity environment is not lower than 3% (w/w), and is more preferably 3% -7% (w/w).

The beneficial technical effects of one or more technical schemes are as follows:

1. according to the technical scheme, a strain of Psychrobacter marinum (Pseudomonas aquimaris) A4N01 with flocculation and nitrogen assimilation functions is obtained through screening and separation, and researches show that the strain can metabolize ammonia nitrogen in an assimilation mode, does not produce nitrate and nitrite, does not produce nitrogen loss, has a wide tolerance range on salinity, and can realize the integrated removal of carbon, nitrogen and phosphorus nutrient substances. The strain can drive the formation of a saltiness nitrogen assimilation microorganism group, and the metabolic direction of the microorganism group developed by the strain is regulated and controlled by managing the structure of the microorganism group;

2. according to the method for developing the salt-nitrogen-assimilating microorganism group by using the nitrogen-assimilating marine self-flocculating bacteria, the microorganism group is started and cultured in the simulated wastewater with the salinity of 3%, salinity gradient domestication is not performed, the starting is quick, the time consumption is low, the operation is simple, and the cost is low;

3. the halophilic nitrogen assimilation microorganism group in the technical scheme removes ammonia nitrogen in an assimilation mode, nitrous acid and nitrate are not generated when high-salt wastewater is treated, the ammonia nitrogen and total nitrogen removal efficiency is high, no nitrogen is lost, the method has the advantages of being green, efficient and the like, and a new way is provided for removing nitrogen from biological high-salt wastewater;

4. the salt-nitrogen-assimilating microorganism group in the technical scheme has good organic matter and phosphorus removal capacity and good settling property, and can be used for recycling nutrient substances in high-salinity wastewater; the halophilic nitrogen assimilation microorganism group contains a large amount of organic matters and organic nitrogen, can be used for preparing organic fertilizers, and has wide application prospects in the fields of soil biological improvement, saline-alkali soil fertility immobilization and the like;

5. the salt nitrogen assimilation microorganism group in the technical scheme has certain removal efficiency on ammonia nitrogen in high-salinity sewage with the salinity of 3% -7% (w/w), has strong impact tolerance on different salinity, can be used for high-salinity sewage treatment such as marine culture wastewater, industrial salt-containing sewage, seawater toilet flushing wastewater and the like, and has wide industrial application prospect in a sewage treatment system.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows a phylogenetic tree of A4N01 of Achilles crassoensis (Psychrobacter aquimaris) according to the present invention.

FIG. 2 shows the effect of Bacillus psychrophilus (Pseudomonas aquimaris) A4N01 on removing ammonia nitrogen, nitrite nitrogen and urea.

FIG. 3 shows the total nitrogen balance of Bacillus marinus (Psychromobacter aquimaris) A4N01 using ammonia nitrogen in the present invention.

FIG. 4 shows the removal of ammonia nitrogen from Haemophilus marinus (Psychrobacter aquimaris) A4N01 under different salinity conditions.

FIG. 5 shows the removal of ammonia nitrogen, total nitrogen, COD and total phosphorus in a Sequencing Batch Reactor (SBR) according to the present invention.

FIG. 6 is a nitrogen balance batch experiment of the moderately saline nitrogen assimilating microbiome of the present invention.

FIG. 7 shows the operation of the moderately saline nitrogen assimilating microbiome of the present invention at different salinity levels.

FIG. 8 shows the removal efficiency of ammonia nitrogen, total nitrogen and total phosphorus of moderately saline nitrogen assimilation microorganism group in the invention at different salinity.

FIG. 9 shows the effect of the saltiness nitrogen assimilation microorganism group on ammonia nitrogen removal in the invention.

FIG. 10 shows the structural composition of the consortium of moderately saline nitrogen-assimilating microorganisms according to the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As mentioned above, the nutrient conversion in the high-salinity environment is easily inhibited by salinity, and the problems of difficult high-salinity sewage treatment, difficult substance conversion in saline-alkali soil, easy nutrition loss and the like exist. The key of the biological method for realizing the conversion of nutrient substances is to find a salt-tolerant strain with the capability of utilizing organic matters, nitrogen, phosphorus and other pollutants and realize the stabilizing effect of the functional strain in an environmental microbiome. Therefore, there is a need to develop a method for developing halophilic microorganisms and microorganism groups, which have a definite metabolic pathway, can rapidly enrich and start, and have a good comprehensive conversion capability to nutrients in a salt environment.

In view of the above, in one embodiment of the present invention, a Psychrobacter marinum (Psychrobacter aquimaris) A4N01 is provided, which has been deposited in the chinese typical culture collection center at 20/1/2021 (address: university of marthang, lord, mountain lod, marthang, north Hubei), and has a biological preservation number of CCTCC NO: m2021120. The strain can efficiently metabolize ammonia nitrogen by utilizing a nitrogen assimilation way, has self-flocculation capacity, does not produce nitrate and nitrite in a metabolic process, does not produce nitrogen loss, has wide tolerance range on salinity, and can realize integrated removal of carbon, nitrogen and phosphorus nutrients.

The metabolite of the psychrobacter marinum A4N01 also belongs to the protection scope of the invention.

In another embodiment of the present invention, the metabolite of the psychrobacter marinum A4N01 can be obtained from fermentation broth of the psychrobacter marinum A4N 01. The metabolite of the psychrobacter marinum A4N01 can be prepared by the following method: inoculating the psychrobacter marinum A4N01 into a liquid fermentation culture medium for fermentation culture, and removing the psychrobacter marinum A4N01 in the liquid culture (fermentation liquid) to obtain the metabolite of the psychrobacter marinum A4N 01.

Wherein the liquid fermentation medium is preferably a seawater LB medium.

The fermentation culture conditions are specifically as follows: culturing at 20-30 ℃ (preferably 25 ℃) for 20-30 h (preferably 24h), rotating speed: 180-250r/min (preferably 200 r/min).

The seawater LB culture medium comprises the following components:

10g/L of peptone, 3g/L of yeast extract and aged seawater, wherein the salinity of the seawater is 3.3 percent.

In another embodiment of the present invention, there is provided a microbial agent comprising a metabolite of the above-mentioned psychrobacter marinum A4N01 and/or psychrobacter marinum A4N 01.

In another embodiment of the present invention, the microbial agent further contains a carrier in addition to the active ingredient. The carrier may be one that is commonly used in the field of microbial agents and is biologically inert.

The carrier can be a solid carrier or a liquid carrier;

the solid carrier can be a mineral material, a plant material or a high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, bean flour and starch; the high molecular compound can be polyvinyl alcohol or/and polyglycol;

the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane or/and dodecane.

The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

In still another embodiment of the present invention, there is provided a halophilic nitrogen assimilating microorganism group comprising the above-mentioned psychrobacter marinum A4N01 and/or metabolites of psychrobacter marinum A4N 01; more specifically, the halophilic nitrogen assimilation microorganism group is obtained by inoculating the psychrobacter marinum A4N01 into high-salt sewage and culturing.

In another embodiment of the present invention, the halophilic nitrogen assimilating microorganism group may be high-salinity sewage obtained by the above treatment or activated sludge obtained by the above treatment.

In another embodiment of the present invention, there is provided a method for constructing the above halophilic nitrogen assimilating microorganism group, the method comprising:

inoculating the psychrobacter marinum A4N01 into a bioreactor containing high-salinity sewage, and operating until the ammonia nitrogen removal efficiency and the sedimentation performance are stabilized to obtain the halophilic nitrogen assimilation microorganism group. The research of the invention unexpectedly discovers that the psychrobacter marinum A4N01 can drive the formation of the halophilic nitrogen assimilation microorganism group, regulate and control the metabolic direction of the microorganism group developed by the psychrobacter marinum through managing the structure of the microorganism group, and has the advantages of no need of salinity gradient acclimation, quick start, less time consumption, simple operation and low cost. The salt and nitrogen assimilation microorganism group has good organic matter and phosphorus removal capacity and good settling property, and can recover nutrient substances in high-salinity wastewater; meanwhile, the salt nitrogen assimilation microorganism group contains a large amount of organic matters and organic nitrogen, can be used for preparing organic fertilizers, and has good practical application value.

Wherein the inoculated thallus is controlled to be not less than 5 g/L.

The high-salinity wastewater can be actual high-salinity wastewater or simulated high-salinity wastewater; wherein, the salinity of the simulated high-salinity sewage is not lower than 3 percent (w/w), and the seawater is prepared.

More specifically, the simulated high salinity wastewater components comprise: 0.8g/L of glucose, 0.5g/L of sodium acetate, 0.55g/L of ammonium chloride, 0.14g/L of dipotassium hydrogen phosphate and 0.25mg/L of peptone, and the salt content is 3.3 percent.

The bioreactor is a Sequencing Batch Reactor (SBR), the sequencing batch reactor is an intermittent activated sludge system adopting a pool body, and the pool body is used as the bioreactor and a sedimentation tank. When treating a continuous flow of sewage, at least two or more tanks are required.

The construction method comprises the following steps:

the continuous operation is adopted, high-salinity wastewater with the salt content of 3-7% is taken as inlet water, DO is controlled to be 2-3mg/L by aeration, and the carbon-nitrogen ratio of the inlet water is not less than 10; the operation period is 8h, including 5min water inlet, 450min aeration, 15min sedimentation and 10min water outlet, without discharging sludge.

The volume exchange rate was 62.5% and the Hydraulic Retention Time (HRT) was 12.8 h.

In another embodiment of the present invention, the use of the aforementioned psychrophilic bacillus marinus (Psychrobacter aquimaris) A4N01, microbial agents and/or halophilic nitrogen assimilating microbiome in any one or more of the following:

a) synthesizing single-cell protein;

b) recycling nitrogen and phosphorus nutrients;

c) treating wastewater;

d) preparing an organic fertilizer;

e) improving the soil fertility;

f) bioremediation of saline-alkali soil;

g) treating water eutrophication;

h) repairing water body pollution;

i) greenhouse gas emission reduction and carbon neutralization;

wherein, in b), nitrogen is metabolized based on assimilation;

in the step c), the wastewater comprises high-salinity wastewater, marine culture wastewater, industrial saline wastewater and seawater toilet flushing wastewater;

in said g), said body of water comprises fresh water and seawater, preferably seawater.

In another embodiment of the present invention, a method for the integrated resource transformation of nutrients in a high salinity environment is provided, which comprises: the aforementioned Haemophilus marinus (Psychrobacter aquimaris) A4N01, microbial agents, and/or salttolerant nitrogen assimilating microbiome are applied to a high salt environment.

In yet another embodiment of the present invention, the high salinity environment is a high salinity water environment having a salinity of not less than 3% (w/w), and more preferably 3% to 7% (w/w).

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

A method for screening and culturing a Psychrobacter marinum (Psychrobacter aquimaris) A4N01 comprises the following steps:

the marine sediment from yellow sea (122 degrees 48 'E, 35 degrees 59' N) is inoculated in a sea water LB culture medium, the shaking culture is carried out for 24 hours in a shaking table under the condition of 25 ℃ and 200r/min, and the supernatant is taken out and streaked on a sea water LB solid plate.

Inoculating single colonies formed on the plate into seawater LB culture medium respectively, shaking and culturing in a shaking table at 25 deg.C and 200r/min for 24h, observing and screening microorganisms with flocculation capability.

Inoculating the strain with flocculation capability obtained by screening in an ammonia nitrogen culture medium, observing the ammonia nitrogen and total nitrogen removal capability of the strain, and screening the strain with high ammonia nitrogen removal efficiency, no generation of nitrite and nitrate and no loss of system total nitrogen, namely the psychrobacterium marianum (Psychrobacter aquimaris) A4N 01.

Inputting the tested 16s rDNA sequence into a GenBank database website of an NCBI website for similarity comparison, and drawing a phylogenetic tree. The phylogenetic tree results are shown in FIG. 1. The results show that the psychrophilic bacillus (Psychrobacter aquimaris) A4N01 has the highest similarity with the Psychrobacter aquimaris, and the strain is identified and named as Psychrobacter aquimaris A4N01 by combining physiological and biochemical properties, colony morphology and indexes.

Example 2

The method for determining the nitrogen metabolism physicochemical properties of the A4N01 strain of Haemophilus marinus (Psychrobacter aquimaris) is as follows

(1) The activated bacterium liquid is prepared by inoculating Bacillus marinus (Psychrobacter aquimaris) A4N01 into seawater LB culture medium, and shake culturing in shaker at 25 deg.C and 200r/min for 24 h. Firstly, centrifuging the prepared activated bacterium liquid of the Psychrobacter marinum (Pseudomonas aquimaris) A4N01 at 4000rpm for 10 min; ② after removing the supernatant, using sterilized normal saline (0.85% NaCl) to resuspend and wash the thalli; centrifuging at 4000rpm for 10 min. Thirdly, after the thalli are re-suspended by using sterilized normal saline, the thalli are respectively inoculated in culture media containing ammonia nitrogen, nitrite nitrogen and urea as nitrogen sources according to the inoculation amount of 10 percent, and the utilization and the transformation of the strains to various nitrogen sources are measured by sampling according to time points.

The ammonia nitrogen, nitrite nitrogen and urea culture medium in the method is prepared by old seawater, the salinity of the seawater is 3.3%, and other components are shown in the table 1:

TABLE 1

As can be seen from FIG. 2, the strain can utilize ammonia nitrogen, nitrite nitrogen and urea as nitrogen sources, and no nitrate or nitrite is produced in the ammonia nitrogen conversion process. As can be seen from figure 3, in the process of assimilating and utilizing ammonia nitrogen, total nitrogen in the system is converted from a water phase into biomass, and no nitrogen is lost.

Example 3

The method for determining the nitrogen metabolism physicochemical properties of the A4N01 strain of Haemophilus marinus (Psychrobacter aquimaris) is as follows

The activated bacterium liquid is prepared by inoculating Bacillus marinus (Psychrobacter aquimaris) A4N01 into seawater LB culture medium, and shake culturing in shaker at 25 deg.C and 200r/min for 24 h. Firstly, centrifuging the prepared activated bacterium liquid of the Psychrobacter marinum (Pseudomonas aquimaris) A4N01 at 4000rpm for 10 min; ② after removing the supernatant, using sterilized normal saline (0.85% NaCl) to resuspend and wash the thalli; centrifuging at 4000rpm for 10 min; thirdly, after the thalli are re-suspended by using sterilized normal saline, the thalli are respectively inoculated into simulated wastewater with different salinity according to the inoculation amount of 10 percent, and the removal condition of the bacterial strain to ammonia nitrogen is measured by sampling according to time points.

The simulated wastewater with different salinity in the method is prepared by 1%, 2%, 3%, 4% and 5% (w/w) of sodium chloride, and the other components are shown in the following table 2:

TABLE 2

As can be seen from figure 4, the strain has wide tolerance range on salinity, and has higher removal effect on ammonia nitrogen under the salinity condition of 1-5%. In the salinity range of 1-3%, the ammonia nitrogen removal rate exceeds 74%, and in the salinity range of 4-5%, the ammonia nitrogen removal rate also exceeds 60%.

Example 4

A method for developing a halophilic nitrogen assimilating microbiome from Acidobacterium marinum (Psychrobacter aquimaris) A4N01, comprising the steps of:

(1) inoculating Bacillus marinus (Psychrobacter aquimaris) A4N01 in seawater LB culture medium, and shake culturing in shaker at 25 deg.C and 200r/min for 24 hr to obtain activated bacteria solution;

(2) inoculating the A4N01 strain of the Psychrobacter marinum (Pseudomonas aquimaris) prepared in the step (1) into a Sequencing Batch Reactor (SBR), and controlling the concentration of activated strains in the SBR to be about 5 g/L. The sequencing batch bioreactor (SBR) has effective volume of 3.2L, and operates in a continuous mode, and an air diffusion device is arranged at the bottom of the SBR to play roles in aeration and stirring. Each cycle of the reactor comprises four steps of water inlet, aeration, sedimentation and water discharge, including 5min of water inlet, 450min of aeration, 15min of sedimentation and 10min of water outlet, each period is 8 hours, the volume exchange rate is 62.5%, and the Hydraulic Retention Time (HRT) is 12.8 hours. Sludge is not discharged during the operation, and the salt nitrogen assimilation microorganism group with stable ammonia nitrogen removal efficiency and good sedimentation performance is obtained. Sequencing batch bioreactor operating parameters are shown in table 3.

The seawater LB culture medium in the step (1) comprises the following components:

10g/L of peptone, 3g/L of yeast extract and 3.3 percent of seawater salinity.

TABLE 3

When the Sequencing Batch Reactor (SBR) is operated, the components of the synthetic wastewater are adopted to simulate the sewage, the synthetic wastewater is prepared by old seawater, and the components are shown in the table 4:

TABLE 4

Example 5

The halophilic nitrogen assimilating microbiome of example 4 was treated in Sequencing Batch Reactor (SBR) in the same manner as described in example 4 for the simulated high salinity wastewater of example 4, and the COD, ammonia nitrogen, total nitrogen, and total phosphorus concentrations were measured by sampling periodically as described in example 4. The operation results are shown in fig. 3, and it can be seen from the results that, in the whole operation process, the treatment effect of the salt nitrogen assimilation microorganism group developed by the psychrobacterium marianum (pseudomonas aquimaris) A4N01 is good and stable, the final removal rate of ammonia nitrogen and total nitrogen in the simulated high-salinity wastewater can reach 84% and 75%, and the removal of nitrogen in the wastewater is mainly assimilation without accumulation of nitrite and nitrate. Meanwhile, the method has good removal capability on organic matters and phosphorus in the simulated high-salinity wastewater, and the final removal rates of COD and total phosphorus respectively reach more than 98% and 72%.

Example 6

The halophilic nitrogen assimilation microorganism group in example 4 was transferred into a batch culture medium at an inoculum size of 5g/L, the dissolved oxygen concentration was controlled at 2-3mg/L, sampling was performed at time points, and the ammonia nitrogen removal conversion and total nitrogen balance in an 8-hour operating period were determined.

The batch culture medium was prepared with aged seawater, and the composition is shown in table 5:

TABLE 5

As can be seen from fig. 6, the microbiome developed by Psychrobacter marinum (Psychrobacter aquimaris) A4N01 showed nitrogen utilization characteristics similar to that of the strain, ammonia nitrogen was still removed in a nitrogen assimilation manner, no nitrite and nitrate were produced during the process, and the removed ammonia nitrogen was accumulated into the biomass without nitrogen loss.

Example 7

The steps (1) to (3) are the same as the embodiment 4, then the saltiness nitrogen assimilation microorganism group is used for treating the synthetic simulated high-salinity wastewater with the salinity of 3 percent, 4 percent, 5 percent, 6 percent and 7 percent (w/w) in a Sequencing Batch Reactor (SBR), the operation mode and the parameters are the same as the embodiment 4, the carbon-nitrogen ratio of the simulated high-salinity wastewater is not lower than 10, the ammonia nitrogen is 100mg/L, and the total phosphorus is 20 mg/L. The ammonia nitrogen removal effect in the operation process of nearly 60 days is shown in figure 7, and the results show that the saltiness nitrogen assimilation microorganism group treatment system has shorter start-up time and stable operation effect under different salinity conditions. The average removal efficiency of ammonia nitrogen, total nitrogen and total phosphorus is shown in fig. 8, and it can be seen from the results that the halophilic nitrogen assimilation microorganism group obtained in example 4 has better salinity impact ability, the halophilic nitrogen assimilation microorganism group developed under 3% salinity (w/w) can directly realize the treatment of wastewater with higher salinity without salinity acclimation, and the halophilic nitrogen assimilation microorganism group has strong adaptability; the saltadapting nitrogen assimilation microorganism group has certain removal capacity for ammonia nitrogen in simulated high-salinity wastewater with salinity of 3% -7% (w/w), and when the salinity is not higher than 6%, the removal efficiency for ammonia nitrogen in the simulated high-salinity wastewater is not lower than 50%. And the removal of nitrogen in the wastewater is mainly assimilation, no nitrite and nitrate are accumulated, no nitrogen loss is caused, and meanwhile, the suitable salt-nitrogen assimilation microorganism group also has good removal capability for simulating phosphorus in the high-salinity wastewater.

Example 8

Steps (1) to (2) were carried out in the same manner as in example 4, while the Sequencing Batch Reactor (SBR) was operated, according to the parameters in Table 3 in example 4, and the ammonia nitrogen concentration was measured by sampling at regular intervals.

In this example, the composition of the synthetic wastewater, which is prepared from old seawater and has the composition shown in table 6, was simulated as sewage:

TABLE 6

As shown in FIG. 9, it can be seen that the halophilic nitrogen assimilation microorganism group treatment effect was good and stable during 170 days of operation, and the removal rate of ammonia nitrogen in the simulated high-salinity wastewater was maintained at 85% or more. FIG. 10 shows the structural composition changes of the community of salttolerant nitrogen assimilating microbiome.

In conclusion, the Psychrobacter marinum (Psychrobacter aquimaris) A4N01 has good salinity tolerance capability, can efficiently recover nitrogen, phosphorus and organic matters from high-salinity wastewater, can efficiently recover and convert nutrient substances in high-salinity wastewater due to no loss of nitrate, nitrite and nitrogen in the nitrogen metabolic process, and can be applied to the fields of high-salinity wastewater treatment, saline-alkali soil bioremediation and the like in future. The halophilic nitrogen assimilation microorganism group developed by the Psychrobacter marinum (Psychrobacter aquimaris) A4N01 is a good source of soil fertilizer due to its high content of nitrogen, phosphorus and organic matter. Can be made into fertilizer in the future and applied to the fields of soil improvement, saline-alkali soil bioremediation and the like.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.