Low-temperature-resistant denitrification strain and application thereof in recirculating aquaculture system

Abstract

The invention relates to the technical field of microorganisms, in particular to a low-temperature-resistant denitrification strain and application thereof in a recirculating aquaculture system. The nitrogen removal bacterial strain is halomonas piezotolans YX98, and the preservation number of the halomonas piezotolans YX98 is CCTCC (China Center for Type Culture Collection) NO: M 20242820. The strain YX98 has an efficient denitrification function and also has the characteristics of low temperature resistance and halophile. The strain is inoculated into simulated wastewater with the temperature of 15 DEG C and the salinity of 20% o, and the degradation rates of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen are as high as 96.15%, 98.20% and 99.99% respectively; when the strain is applied to a salmon and trout circulating mariculture system as a low-temperature fungicide, the NH4 < + >-N and NO2 <->-N degradation function of a moving bed bio-membrane reactor can be enhanced, so that the duration of a function starting period is shortened by 43.75%, and the duration of a function stable period is shortened by 26.32%.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a low-temperature resistant denitrification strain and application thereof in a circulating water aquaculture system. Background Art

[0002] The rapid development of seawater recirculating aquaculture has brought about widespread nitrogen pollution, especially the toxic effects of ammonia nitrogen and nitrite nitrogen on aquatic organisms. In the recirculating aquaculture system (RAS), the enhanced moving bed biofilm reactor (MBBR) biofilm treatment of ammonia nitrogen and nitrite nitrogen has two ways: natural strain biofilm method and bacterial agent biofilm method. Compared with the former, the bacterial agent biofilm method promotes the formation of dominant strain biofilm by adding bacterial agents with nitrogen removal function to the MBBR. This type of biofilm has stronger tolerance in extreme environments such as high nitrogen and high salt; more obvious growth advantages; higher nitrogen removal efficiency; lower operating costs; higher potential economic value, and broader application prospects in the recirculating aquaculture market.

[0003] However, it is difficult to apply the biofilm method in low-temperature seawater RAS systems. Most of the highly efficient denitrification strains discovered so far are usually mesophilic or mesophilic bacteria, and their optimal growth temperature is usually in the range of 25℃-40℃. In water treatment processes, environmental conditions with water temperatures below 25℃ are usually defined as medium-low temperature conditions. Under extreme aquaculture conditions such as low temperature and high salt, the growth, metabolic functions and efficiency of the strains will be severely inhibited, resulting in poor treatment of recirculating aquaculture effluent. Due to the lack of strains with corresponding characteristics, salmon and trout recirculating aquaculture are at greater risk of nitrogen toxicity, and the initial start-up of the recirculating aquaculture system is longer and the energy consumption is higher.

[0004] Relevant studies have found that some natural denitrification strains are also resistant to low temperatures. Therefore, under the low-temperature circulating seawater aquaculture environment, it is urgent to explore low-temperature resistant and tolerant denitrification strains and apply them to MBBR biofilm to optimize the denitrification process of low-temperature seawater RAS. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a low-temperature resistant denitrification strain and its application in a circulating aquaculture system.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0007] In the first aspect, Halomonas piezotolerans YX98 is provided, which has been deposited in the China Center for Type Culture Collection (CCTCC) on December 16, 2024, with the deposit number CCTCC NO: M 20242820.

[0008] In a second aspect, a microbial agent is provided, which is prepared from Halomonas piezotolerans YX98.

[0009] In a third aspect, the application of Halomonas piezotolerans YX98 or a microbial agent comprising Halomonas piezotolerans YX98 in the biological treatment of nitrogen-containing tail water from low-temperature seawater circulating aquaculture.

[0010] The beneficial effects of the present invention are:

[0011] The present invention screened a new type of Halomonas piezotolerans YX98 from circulating aquaculture seawater. Compared with heterotrophic nitrification-aerobic denitrification strains of other genera, strain YX98 has both low temperature resistance, halophilic characteristics and efficient denitrification function. When inoculated into simulated wastewater with a temperature of 15°C and a salinity of 20%, the degradation rates of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen (initial concentrations of 100 mg / L) in 96 hours were as high as 96.15%, 98.20% and 99.99% respectively. The strain is applied to the circulating aquaculture system of salmon and trout, and the NH4 + -N and NO2 - -N degradation function, shortening the functional startup period by 43.75% and the functional stabilization period by 26.32‰. The denitrification strain of the present invention has strong environmental adaptability, simple domestication, and obvious denitrification function advantages, and is particularly suitable for biological treatment of nitrogen-containing tail water in low-temperature seawater circulating aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The salt and low temperature resistance characteristics of strain YX98 isolated in Example 1; (a) the growth characteristics of strain YX98 at different salinities; (b) the growth characteristics of strain YX98 at different temperatures;

[0013] Figure 2 The denitrification trend of strain YX98 isolated in Example 1 in a mixed nitrogen source;

[0014] Figure 3 The denitrification trend of strain YX98 isolated in Example 1 in a single nitrogen source; wherein (a) ammonia nitrogen is the only nitrogen source; (b) nitrite nitrogen is the only nitrogen source; (c) nitrate nitrogen is the only nitrogen source;

[0015] Figure 4 The dynamic change trend of water quality nitrogen concentration in each stage of the MBBR bioreactor in Example 2. DETAILED DESCRIPTION

[0016] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0017] Example

[0018] In this example, a novel Halomonas piezotolerans YX98 was screened out from circulating aquaculture seawater. The specific steps of the culture method and strain identification of Halomonas piezotolerans YX98 are as follows:

[0019] Step 1: Prepare 2216 medium by dissolving 5 g / L peptone, 1 g / L yeast powder, 0.1 g / L ferric citrate, and 30 g / L sodium chloride in deionized water, sterilize under high pressure at 121°C for 20 min, and then cool down;

[0020] Step 2: Inoculate the YX98 strain into 2216 seawater culture medium and place it in a shaker with the temperature set at 15°C and the speed set at 200 r / min for enrichment culture.

[0021] Step 3: Add 15 g / L agar powder to the components of the 2216 medium in step 2, sterilize under high pressure at 121°C for 20 min, cool to 60°C, shake well and pour onto a plate to make 2216 agar medium, spread the enriched bacterial suspension onto a 2216 solid plate for culture, pick a single clone colony and repeat the enrichment culture in step 2 twice, then perform streak separation and purification twice to obtain the purified strain YX98, and finally perform 16s rRNA sequencing on it.

[0022] Step 4: Results and Analysis

[0023] The 16s rRNA sequence of strain Halomonas piezotolerans YX98 is shown below. The sequence has been uploaded to the National Center for Biotechnology Information (NCBI) with the gene accession number PQ759011.1

[0024]

[0025] Test Example 1

[0026] Low temperature resistance, salt tolerance and denitrification characteristics of Halomonas piezotolerans YX98:

[0027] Step 1: Prepare simulated aquaculture wastewater, whose basic components (expressed in grams per liter of water) include 3.375 g of sodium succinate, 6.76 g of Na2HPO4, 3.0 g of KH2PO4, 0.49 g of MgSO4·7H2O, and 0.01 g of CaCl2.

[0028] Step 2: Salt tolerance. Add NaCl to the simulated aquaculture wastewater in step 1 to adjust the salinity to 10‰, 20‰, 30‰, 40‰ and 50‰, respectively, and add 0.383g of NH4Cl at the same time. Pick the monoclonal colony of strain YX98 and activate it in 2216 culture medium, remove the supernatant, resuspend it with physiological saline, and inoculate it into a conical flask containing 100ml of the above-mentioned simulated aquaculture wastewater with different salinities at 1% (volume ratio). Culture for 24 hours under shaking conditions at 25°C and 200r / min. After the end, take samples to detect OD600. Each group of experiments was repeated three times. The bacterial density OD600 is determined by measuring the absorbance value of the bacterial cells at 600nm. Use the initial detection value of salinity as the horizontal axis and OD600 as the vertical axis to draw a mixing curve. Results are shown in Figure 1 (a).

[0029] Low temperature resistance. Add NaCl to adjust the salinity to 20‰, set the shaking table temperature to 5℃, 10℃, 15℃, 20℃ and 25℃ respectively, and the other parameters are the same as the salt resistance. Figure 1 (b)

[0030] Step 3: Add 0.128g NH4Cl, 0.167g NaNO2, and 0.224g NaNO3 to the simulated aquaculture wastewater in step 1, pick a monoclonal colony of the strain Halomonas piezotolerans YX98 and activate it in 2216 medium, remove the supernatant, resuspend it with physiological saline, and inoculate it into a conical flask containing 100ml simulated aquaculture wastewater at 1% (volume ratio). Cultivate it in a shaking table at 15℃ and 200r / min for 240h, and take samples every 24h to detect NH4 + -N, NO2 - -N, NO3 - -N, COD concentration and OD 600 All experiments were repeated 3 times. + -N concentration was determined by Nessler's reagent spectrophotometry (HJ 535-2 009); NO3 --N concentration was determined by zinc-cadmium reduction method (GB / 17 378.4-2 007); NO2 - -N concentration was determined by naphthylethylenediamine spectrophotometry (GB / 17 378.4-2 007); COD concentration was determined by HACH kit (2 415 815-CN); bacterial density OD 600 The determination is made by measuring the absorbance of bacterial cells at 600nm. The initial detection values ​​of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, DOC, and OD600 are used as the ordinate, and the time is used as the abscissa to draw the parameter-time mixing curve. Figure 2 .

[0031] Step 4: Add a single nitrogen source to the simulated aquaculture wastewater in step 1. Add 0.383g of NH4Cl to the wastewater group with ammonia nitrogen as the single nitrogen source, add 0.479g of NaNO2 to the wastewater group with nitrite nitrogen as the single nitrogen source, and add 0.607g of NaNO3 to the wastewater group with nitrate nitrogen as the single nitrogen source. The other operating principles are the same as step 3. Results are shown in Figure 3 .

[0032] Step 5: Results and Analysis

[0033] Depend on Figure 1 (a) The OD of strain YX98 in the salinity range of 10‰-40‰ 600 The value gradually increased from 0.406 to a peak value of 0.801, and the OD600 value under the high salinity condition of 50‰ still reached 0.746, indicating that it has a wide range of salt tolerance. Figure 1 (b) It can be seen that strain YX98 grows slowly at 5℃-15℃, but grows vigorously at 15℃-25℃. 600 The value increased sharply from 0.263 to a peak value of 0.781, and only slightly decreased at 20°C-25°C, indicating that it can tolerate low temperature environments of 5°C to 25°C. This step confirmed the cold and salt tolerance of strain YX98.

[0034] Depend on Figure 2 It can be seen that Halomonas piezotolerans YX98 has a strong degradation effect on ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in simulated aquaculture wastewater with a low temperature of 15°C and a salinity of 20‰. The degradation rates in 96 hours are as high as 96.15%, 98.20% and 99.99% respectively. After 168 hours, the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen are all degraded to below 0.01 mg / L; the COD concentration is close to 0 mg / L, and the wastewater is efficiently purified; after the bacterial liquid concentration reaches the peak, the metabolism is slow and the concentration is stable. This step confirms the efficient denitrification ability of strain YX98.

[0035] Depend on Figure 3 It can be seen that in the wastewater with ammonia nitrogen, nitrite nitrogen and nitrate nitrogen as the single nitrogen source, the degradation rate of ammonia nitrogen and nitrate nitrogen by strain YX98 reached the highest 99.99%, but its adaptation period to ammonia nitrogen source wastewater was the shortest, the strain growth level was the highest, the COD utilization rate was the highest, the sewage purification capacity was the most significant, and the peak degradation rate of nitrate nitrogen was the highest. It is worth noting that the strain YX98 had a longer adaptation period to nitrite nitrogen source wastewater, but the nitrite nitrogen degradation rate of 240h was still as high as 84.37, and the strain grew vigorously, with a high COD utilization rate, showing good decontamination ability. This step confirms the efficient degradation ability and degradation characteristics of strain YX98 for different nitrogen sources.

[0036] Test Example 2

[0037] The application of Halomonas piezotolerans YX98 in promoting the start-up of MBBR function to treat RAS nitrogen-containing tail water is as follows:

[0038] Step 1: Prepare low-temperature bacterial agent. Dissolve 5 g / L peptone, 1 g / L yeast powder, and 20 g / L sea crystal nutrient source in deionized water, sterilize under high pressure at 121°C for 20 min, and obtain the bacterial agent culture medium after cooling; inoculate strain YX98 into the bacterial agent culture medium at 1% (volume ratio), place in a shaker at a temperature of 15°C and a speed of 200 r / min for 48 hours to obtain the domesticated low-temperature YX98 bacterial agent.

[0039] Step 2: Build a circulating water aquaculture system for steelhead trout. This embodiment builds two sets of low-temperature circulating water aquaculture systems (RAS) that simulate the tail water conditions of steelhead trout aquaculture, named as the enhanced system (microbial biofilm) and the control system (natural biofilm). RAS consists of a circulating water pump, aquaculture barrels, MBBR, a microfilter, a protein separator, an aerator, an air pump, an ultraviolet lamp, a central control power supply and a circulating pipeline, among which a circulating water pump of 35Hz, aquaculture barrels of 800L*10 barrels, and MBBR of 3.6m 3 (Water body 1.8m 3 , K5 filler filling rate 40%, hydraulic retention time 0.4h), turn off the microfilter, protein separator and UV lamp, the aerator and air pump are always on, the system water volume is about 10t, and it is circulated 24 times a day.

[0040] Step 3: RAS simulates low-temperature circulating aquaculture tail water configuration. Add NH4Cl and NaNO2 to the system to adjust the NH4 + -N and NO2 -The initial concentrations of -N were 10 mg / L and 5 mg / L, respectively, the salinity was set at about 20‰, the water temperature was set at about 15°C, and the pH was maintained between 6.5 and 8.5. To simulate the metabolic rate of steelhead trout feed residues, 2.0 g of Hanye salmon feed was added to each culture barrel every day, and 2 groups of air stones were added to each culture barrel for aeration.

[0041] Step 4: Adding microbial agents to the MBBR. Within 60 days after the start-up of the MBBR, add the low-temperature YX98 microbial agent acclimated in step 1 to the MBBR of the enhanced system at an inoculation rate of 1‰ (v / v) every 24 hours to enhance the function of the MBBR; add the same amount of microbial agent culture medium prepared in step 1 to the control system.

[0042] Step 5: Take water samples from the MBBR outlets of the two systems every 2 days to test for NH4 + -N and NO2 - -N concentration and analyzed.

[0043] Step 6: Results and Analysis

[0044] Results Figure 4 The addition of low-temperature YX98 bacterial agent significantly shortened the functional startup time and stabilization period of MBBR, and strengthened the system's MBBR startup to treat NH4 + -N and NO2 - -N time point is 21 days earlier than that of the control system, and the duration is shortened by 43.75%; NH4 + -N and NO2 - The time point when the -N concentration stabilized below 1 mg / L was 20 days earlier than that of the control system, and the duration was shortened by 26.32%. During the stable period, the addition of YX98 bacteria made the fluctuation of ammonia nitrogen and nitrite nitrogen concentrations in the enhanced system smaller than that of the control system, and stabilized at a low concentration level more quickly. The results show that the addition of low-temperature YX98 bacteria to MBBR can enhance the degradation of NH4 + -N and NO2 - -N is a feasible strategy, which can not only shorten the start-up time of MBBR, but also improve the treatment effect of RAS on nitrogen compounds in low-temperature circulating aquaculture effluent and enhance the stability of the treatment process.

[0045] The present invention screened a new type of Halomonas piezotolerans YX98 from circulating aquaculture seawater, domesticated and expanded it into a low-temperature bacterial agent by utilizing its low-temperature resistance, salt resistance and denitrification characteristics, innovatively invented a low-temperature bacterial agent biofilm method, optimized the MBBR biological biofilm process, and enhanced the degradation effect of ammonia nitrogen and nitrite nitrogen in low-temperature seawater RAS tail water, providing a theoretical basis and guiding experience for the actual production application of the strain and the low-temperature seawater RAS nitrogen-containing tail water treatment technology.

[0046] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0047] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Halomonas piezotolerans YX98, characterized in that Its deposit number is CCTCC NO: M20242820.

2. A microbial agent, characterized in that: It comprises the Halomonas piezotolerans YX98 as described in claim 1.

3. Use of the Halomonas piezotolerans YX98 according to claim 1; or the microbial agent according to claim 2 in the biological treatment of nitrogen-containing tail water from low-temperature seawater circulating aquaculture.

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