Application of rhodococcus coryneform CGMCC No.23799 in improvement of COD and ammonia nitrogen in water body

Abstract

The invention belongs to the technical field of aquatic micro-ecological preparations, and particularly relates to application of rhodococcus coryneform CGMCC No.23799 in improvement of COD (Chemical Oxygen Demand) of a water body and denitrification. The rhodococcus coryneform HQ40 disclosed by the invention can be used for efficiently degrading organic pollutants in a low-oxygen environment and synchronously realizing total nitrogen removal; the adaptability to 0%-3% salinity (NaCl) is high, and the growth and COD degradation performance is stable; the compound has the characteristics of oxidizing sulfides, relieving bottom mud acidification and not changing the pH value of the water body, is safe and non-toxic to cultured animals, and is suitable for repairing the cultured water body.

Description

Technical Field

[0001] This invention belongs to the field of aquatic microecological preparation technology, specifically involving the application of a strain of Corynebacterium-like Rhodococcus CGMCC No. 23799 in improving COD and ammonia nitrogen in water bodies. Background Technology

[0002] In high-density, intensive aquaculture, large amounts of organic matter, such as uneaten feed, feces, and animal and plant remains, accumulate. The accumulation and decomposition of this organic matter severely damages the aquatic environment, harms the health of aquatic animals, and hinders the sustainable development of the industry. Therefore, the rapid and effective degradation of organic matter in aquaculture ponds is a key research issue in the regulation of pond aquaculture water environments. Utilizing microorganisms to degrade organic matter is a recognized environmentally friendly, safe, and sustainable method. The key to this method is screening and applying probiotic strains that can efficiently decompose organic waste such as uneaten feed and feces, and can transform or remove toxic substances such as ammonia nitrogen and nitrite.

[0003] Currently, numerous studies have isolated microorganisms with degradation functions from aquaculture environments, confirming their ability to degrade organic matter and ammonia nitrogen under laboratory conditions. However, applying laboratory strains to actual aquaculture environments still faces many challenges: on the one hand, aquaculture environments are complex and variable, with factors such as temperature, salinity, pH, dissolved oxygen, and carbon-to-nitrogen ratio (C / N) significantly affecting the growth, colonization, and metabolic activity of strains; on the other hand, the degradation capacity of a single strain is very limited, making it difficult to cope with the diverse pollutants in aquaculture water. Therefore, the development of compound microecological preparations composed of non-single strains has become a trend, aiming to achieve more stable water purification effects through synergistic effects of microbial communities. Nevertheless, existing compound microbial agents still suffer from problems such as insufficient scientific compatibility of strains, weak tolerance to environmental mutations, and unstable efficacy in actual aquaculture water.

[0004] Therefore, the current technological need is to screen out native beneficial microorganisms with strong adaptability, clear functions and synergistic effects from specific aquaculture environments, and to develop microecological preparations that have stable preparation processes, can continuously and efficiently degrade organic pollutants in aquaculture water, improve water quality and ensure biosafety of aquaculture organisms. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of a strain of *Rhodococcoides corynebacterioides* HQ40 in improving COD and ammonia nitrogen levels in aquaculture water. *Rhodococcoides corynebacterioides* HQ40 was deposited on November 15, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and classified as *Rhodococcoides corynebacterioides*, with accession number CGMCC No. 23799.

[0006] Furthermore, the 16S rRNA nucleic acid sequence of the *Rhodococcus cordata* is shown in SEQ ID No. 1.

[0007] Furthermore, the application also includes the degradation of total nitrogen content in water bodies.

[0008] Furthermore, the aquaculture water body mentioned is a seawater aquaculture water body.

[0009] Furthermore, the salinity of the water body is 0%~3% by mass / volume ratio of NaCl.

[0010] Furthermore, the aforementioned corynebacterium-like Rhodococcus is used in combination with Bacillus belye.

[0011] Preferably, the Bacillus belyssus has the accession number CGMCC No. 27887.

[0012] Furthermore, the bacterial concentration of the compound strain of *Rhodococcus cordata* and *Bacillus belyssae* in aquaculture water is 5.0 × 10⁻⁶. 4 CFU / mL ~5.0×10 6 CFU / mL.

[0013] Preferably, the bacterial concentration of the compound strain in the aquaculture water is 5.0 × 10⁻⁶. 5 CFU / mL.

[0014] Furthermore, the application was carried out in a low-oxygen water environment, wherein the dissolved oxygen (DO) concentration in the water was 0.7~1.3 mg / L, and the 24-hour degradation rate of COD by the strain was 32.35%±0.5%, and the 48-hour degradation rate was 56.28%±0.5%.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] HQ40 *Rhodococcus cordata* can efficiently degrade organic pollutants (COD) in low-oxygen environments (58.27% degradation rate in 24 hours) and simultaneously remove total nitrogen (50.14% degradation rate). It exhibits strong adaptability to salinity (NaCl) of 0%-3%, and shows stable growth and COD degradation performance (68%-70% degradation rate in 72 hours). It also has the characteristics of oxidizing sulfides, alleviating sediment acidification, and not changing the pH of the water body. It is safe and non-toxic to farmed animals and is suitable for the remediation of seawater aquaculture water bodies.

[0017] This invention constructs a composite microecological preparation with synergistic degradation function by combining broad-spectrum, salt-tolerant corynebacterium-like Rhodococcus HQ40 with highly efficient nitrogen-reducing Bacillus belyssus H16. This preparation not only significantly enhances the simultaneous removal of organic matter and ammonia nitrogen by aquaculture systems but also exhibits good environmental adaptability, making it suitable for various aquaculture pond water quality conditions. Its preparation process is simple and low-cost; it can be applied directly by spraying or using existing aeration facilities without requiring modification to existing aquaculture infrastructure, thus combining economic efficiency with environmental friendliness.

[0018] This compound microbial preparation can achieve a COD degradation rate of 84.60%, a total nitrogen degradation rate of 58.55%, and an ammonia nitrogen degradation rate of 59.27% ​​in aquaculture water with a COD concentration of approximately 405 mg / L, a total nitrogen concentration of 345 mg / L, and an ammonia nitrogen concentration of 30 mg / L, demonstrating excellent water purification capabilities.

[0019] Instruction manual illustrations

[0020] Figure 1 The growth and organic matter degradation capacity of 42 bacterial strains in the performance testing medium.

[0021] Figure 2 : Figure 2 -A represents the growth and organic matter degradation capacity of strain HQ40 in the performance testing medium over 96 h; Figure 2 -B represents the total nitrogen and ammonia nitrogen degradation capacity of strain HQ40 within 96 h in the performance testing medium.

[0022] Figure 3 : Figure 3 -A represents the effect of different salt concentrations on the growth of strain HQ40; Figure 3 -B represents the effect of different salt concentrations on the organic matter degradation ability of strain HQ40.

[0023] Figure 4 : Figure 4 -A represents the effect of hypoxia on the growth of strain HQ40; Figure 4 -B represents the effect of hypoxia on the organic matter degradation ability of strain HQ40.

[0024] Figure 5 : Figure 5 -A represents the organic matter degradation capacity of the compound microecological preparation; Figure 5 -B represents the total nitrogen degradation capacity of the compound microecological preparation; Figure 5 -C represents the ammonia nitrogen degradation capacity of the compound microecological preparation.

[0025] Figure 6 : Figure 6 -A represents the effect of a compound microecological preparation on the degradation rate of organic matter in the water of Litopenaeus vannamei ponds. Figure 6-B represents the effect of a compound microecological preparation on the total nitrogen content in ponds used for Litopenaeus vannamei farming. Figure 6 -C represents the effect of a compound microecological preparation on the ammonia nitrogen content in ponds used for Litopenaeus vannamei farming. Detailed Implementation

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0027] Example 1: Study on the organic matter degradation of *Rhodococcus pyogenes* HQ40 (a type of corynebacterium)

[0028] Rhodococcoides corynebacterioides HQ40 was deposited on November 15, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, under the classification name: Rhodococcoides corynebacterioides, with accession number CGMCC No. 23799. See patent CN202610039095.0 for details.

[0029] The 16S rRNA nucleic acid sequence of *Rhodococcus pyogenes* HQ40 is shown in the sequence listing as SEQ ID No. 1. Studies have shown that this *Rhodococcus pyogenes* strain can oxidize sulfides under low dissolved oxygen conditions without lowering the pH of the water, thus reducing sulfide pollution in aquaculture water, alleviating sediment acidification, and having no toxic effects on farmed animals.

[0030] This strain originated from 18 aquaculture water and sediment samples collected from marine fish farming ponds in Fujian Province. A total of 192 single-strain strains were isolated from these 18 samples, and 39 non-hemolytic strains were obtained after inoculation with hemolytic plates. Additionally, analysis of commercially available aquaculture water purification products (fecal sulfur flocs) yielded three strains with COD degradation capabilities: strain A2-1 (Rhodopseudomonas palustris), strain A2-4 (Bacillus subtilis), and strain A2-9 (intestinal symbiotic bacteria).

[0031] To investigate their organic matter degradation performance, 39 non-hemolytic bacterial strains, including HQ40, and 3 commercially available bacterial strains isolated from culture products were selected and inoculated into performance testing medium. After incubation at 30°C and 180 rpm for 24 h, the growth and proliferation of 42 bacterial strains in the performance testing medium were measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600 The COD content and COD degradation rate were determined using the alkaline potassium permanganate oxidation method (GB11914-89) according to national environmental protection standards.

[0032] Performance testing culture medium: Weigh 5g of shrimp feed, grind it thoroughly into powder, dissolve it in 1L of deionized water, mix well, seal the bottle mouth with sealing film, and soak for 48h to ensure that the nutrients in the feed are fully dissolved. Filter the supernatant through a 200-mesh sieve, repeat the filtration 3-5 times, centrifuge at 8000rpm for 15min, adjust the pH to 7.0, sterilize at 121℃ for 15min, and set aside.

[0033] Depend on Figure 1 It can be seen that strain HQ40 achieved a COD degradation rate of 58.27% after 24 h, exhibiting the best degradation ability, with a corresponding bacterial biomass OD. 600 The value was 0.745. The COD degradation rates of the isolated strains A2-1, A2-4, and A2-9 reached 26.63%, 27.01%, and 35.71%, respectively, with corresponding bacterial biomass OD values ​​of 0.745. 600 The values ​​were 0.673, 0.677 and 0.326, respectively.

[0034] Example 2: Determination of organic matter degradation capacity and denitrification effect of strain HQ40

[0035] Corynebacterium-like Rhodococcus HQ40 was inoculated into performance testing medium (COD: 405 mg / L, TN: 345 mg / L and NH4+). + The bacterial strain was cultured in a solution of -N: 30 mg / L at 30 °C with shaking on a shaker at 180 rpm. Samples were taken at 24 h, 48 h, 72 h, and 96 h to determine the bacterial biomass (OD). 600 Chemical oxygen demand (COD) degradation and nitrogen removal (TN and NH4+) + -N) effect. By Figure 2 -A indicates that *Rhodococcus pyogenes* HQ40 grows rapidly within the first 48 hours of culture, with OD... 600 After reaching a concentration of 1.059, the growth concentration decreased slightly, and then remained relatively stable after 72 hours. The COD content of the culture medium water decreased rapidly within 0-48 hours, from 405 mg / L to 82.23 mg / L, then rebounded to 135.81 mg / L at 72 hours, and decreased again to 107.48 mg / L at 96 hours, indicating that the degradation effect was optimal at 48 hours. Figure 2 -B indicates that the total nitrogen content of *Rhodococcus pyogenes* HQ40 decreased to 172 mg / L within 0-96 h, with a total nitrogen degradation rate of 50.14%; NH4 + The -N content increased to 33.21 mg / L at 48 h, began to decrease at 72 h, and decreased to 21.23 mg / L at 96 h. NH4+ + The degradation rate of -N was 29.23%.

[0036] Total nitrogen (TN) content was determined using a rapid water quality analyzer, based on the alkaline potassium persulfate method (National Standard: HJ 636-2012).

[0037] Ammonia nitrogen was determined using the Nessler's reagent method (National Standard: HJ535-2009) as per the national environmental protection standard.

[0038] Example 3: Effects of different salt concentrations on the growth and organic matter degradation capacity of strain HQ40

[0039] The frozen strain HQ40 was activated and inoculated into three groups of performance testing media with initial NaCl concentrations of 0.0%, 1.5%, and 3.0%. The initial COD and culture conditions were the same as in Example 2, and the timed sampling and detection methods were the same as above. Sampling was performed at 24 h, 48 h, 72 h, and 96 h to determine the bacterial biomass (OD). 600 The degradation effects of chemical oxygen demand (COD) and chemical oxygen demand (COD) are determined by... Figure 3 -A indicates that the growth curves of strain HQ40 almost overlapped in 1.5% and 3.0% NaCl concentration media, but the growth concentration was higher at 0.0% NaCl. Figure 3 As shown in section B, the COD degradation rate curves of strain HQ40 in media with NaCl concentrations of 0.0%, 1.5%, and 3.0% exhibit similar trends. The COD degradation rate increases most rapidly within 0-48 h, increases slowly within 48-72 h, and then slowly decreases within 72-96 h, reaching 68.64%, 69.14%, and 70.12%, respectively. The optimal degradation effect is observed at 72 h. In summary, NaCl concentrations of 0%-3% have little impact on the growth and organic matter degradation ability of strain HQ40.

[0040] Example 4: Growth and organic matter degradation capacity of strain HQ40 under hypoxic conditions

[0041] The frozen strain HQ40 was activated and inoculated into the performance testing medium. Dissolved oxygen (DO) was controlled at 1.0 ± 0.3 mg / L (static culture) by replacing oxygen with a nitrogen / carbon dioxide mixture to simulate the hypoxic state at the bottom of an aquaculture pond. HQ40 cultured under normoxic conditions (normal shake-flask culture) served as a control. Initial COD was the same as in Example 2. Sampling was taken at 24 h, 48 h, 72 h, and 96 h to determine the bacterial biomass (OD). 600 The degradation effects of chemical oxygen demand (COD) and chemical oxygen demand (COD) are determined by... Figure 4 -A indicates that the growth curves of strain HQ40 show a high degree of overlap under hypoxic conditions and normal shaking culture, but the growth concentration is higher under normal shaking conditions. From Figure 4As shown in section -B, the COD degradation rate curves of strain HQ40 under hypoxic and normal shaking culture exhibit similar trends. Under hypoxic conditions, the degradation rates at 24 h, 48 h, 72 h, and 96 h were 32.35%, 56.28%, 62.47%, and 63.21%, respectively. Under normal culture conditions, the degradation rates at 24 h, 48 h, 72 h, and 96 h were 44.94%, 67.16%, 71.85%, and 68.64%, respectively. In conclusion, hypoxic conditions have no significant effect on the growth and organic matter degradation ability of strain HQ40.

[0042] Example 5 Preparation of compound microbial preparation

[0043] Single colonies of *Rhodococcus pyogenes* HQ40 were inoculated into YPD medium and cultured at 180 rpm and 30°C for 12 h until the OD600 reached 0.6. The prepared primary seed culture was then inoculated into YPD medium at an inoculation rate of 1.0% and cultured at 180 rpm and 30°C for 16 h until the OD600 reached 0.7, thus obtaining the secondary seed culture of strain HQ40.

[0044] Single colonies of Bacillus belye H16 were inoculated into MNB medium and cultured at 180 rpm and 33°C for 10 h until the OD600 reached 0.6. The prepared primary seed culture was then inoculated into MNB medium at an inoculation rate of 1.0% and cultured at 180 rpm and 33°C for 12 h until the OD600 reached 0.7, thus obtaining the secondary seed culture of strain H16.

[0045] Bacillus velezensis H16 is a strain developed in this study and was deposited on July 14, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNo. 27887. See patent CN202311454341.1 for details.

[0046] The prepared seed cultures of strains HQ40 and H16 were mixed evenly at a 1:1 ratio and then inoculated into a seed tank for co-fermentation. The fermentation temperature was 30 ℃, the rotation speed was 180 rpm, the liquid volume was 40%, the tank pressure was 0.5 MPa, the aeration ratio was 1:0.5, the pH value was 7.0, and the fermentation time was 12 h.

[0047] After the seed tank fermentation was completed, all the contents were transferred to the fermenter for fermentation. The fermentation temperature was 30 ℃, the rotation speed was 180 rpm, the liquid volume was 50%, the tank pressure was 0.5 MPa, the aeration ratio was 1:0.5, the pH value was 7.0, and the fermentation time was 24 h. The viable count of *Rhodococcus* var. *corynebacterium* in the prepared fermentation broth was 8.0 × 10⁻⁶.9 CFU / mL, Bacillus viable count was 5.0 × 10⁻⁶. 8 CFU / mL.

[0048] The culture medium used for seed tank fermentation and fermenter fermentation consisted of: 16 g / L sucrose, 2 g / L peptone, 8 g / L yeast extract, 2 g / L KH2PO4, 1-2 g / L Na2HPO4, 0.5-1 g / L MgSO4·7H2O, 0.1-0.2 g / L CaCl2, and pH 7.0-7.2.

[0049] Example 6: Determination of the degradation performance and denitrification effect of the compound microecological preparation on organic matter.

[0050] The prepared compound microecological preparation was inoculated into the performance testing medium, with three replicates per group, and the culture conditions were the same as above. Chemical oxygen demand (COD) and ammonia nitrogen (NH4) in the culture medium were sampled and tested periodically. + -N) and total nitrogen content (TN). (The text abruptly ends here, so the translation stops as well.) Figure 5 -A indicates that before 48 hours, the COD degradation rates of the compound microbial community and HQ40 were similar, significantly higher than that of strain H16. After 48 hours, the COD degradation rate of strain HQ40 decreased significantly to 66.47%, while the COD degradation rate of the compound microecological preparation increased to 84.60%. At 96 hours, the COD degradation rates of the compound microbial community, strains HQ40, and H16 were 84.60%, 73.46%, and 63.46%, respectively. Figure 5 -B indicates that the degradation effect of TN by the complex bacterial community was better than that by the single strain. After 96 h, TN decreased from 345 mg / L to 143 mg / L, with a degradation rate of 58.55%. Figure 5 -C indicates that the ammonia nitrogen level in the HQ40 treatment group first increased and then decreased, reaching 24.23 mg / L after 96 h. The combined bacterial community effectively controlled NH4+. + The degradation effect of -N was significantly better than that of strain HQ40, with ammonia nitrogen levels decreasing throughout the process. After 96 h, the ammonia nitrogen levels of the composite microbial community, strains HQ40, and H16 were reduced to 12.13, 24.23, and 14.23 mg / L, respectively, with degradation rates of 59.57%, 19.23%, and 52.56%. In summary, the co-fermentation of strains H16 and HQ40 significantly improved COD degradation and denitrification.

[0051] Example 7: Effects of compound microecological preparations on organic matter and nitrogen content in pond water for Litopenaeus vannamei farming.

[0052] The culture experiment was conducted in an indoor recirculating aquaculture system. Pacific white shrimp were temporarily reared for one week under the experimental conditions (salinity 28-35‰, water temperature 30℃) with a feed intake of 5% of their body weight. After temporary rearing, 600 healthy Pacific white shrimp weighing approximately 1.4 g were randomly divided into 5 treatment groups (G0-G4), with 3 replicates per treatment and 40 shrimp per replicate. These shrimp were randomly assigned to 15 500 L culture tanks. The control group (G0) received no bacterial solution; groups G1, G2, and G3 each received a compound bacterial solution in each culture tank, resulting in a bacterial concentration of 5.0 × 10⁻⁶ in the culture water. 4 CFU / mL, 5.0×10 5 CFU / mL and 5.0×10 6 CFU / mL, supplemented every 5 days; Group G4 was supplemented with a commercially available compound probiotic water purifier with good feedback, at the recommended dosage, supplemented every 5 days. During the experiment, normal commercial feed was provided three times a day (08:00, 13:00, and 18:00), and the ammonia nitrogen level in the water was regulated by controlling the circulating water system. When the ammonia nitrogen concentration exceeded 6 mg / L, the circulating water system was turned on to replace 80.0% of the water, and turned off at other times. The circulating water system was turned on at a fixed time each day to replace 10.0% of the water, and turned off at other times. The experiment was observed for 10 days, and samples were taken at the same time every afternoon before feeding to measure the COD, total nitrogen, and ammonia nitrogen content in the aquaculture water.

[0053] The test results are attached. Figure 6 .like Figure 6 As shown in Figure A, the COD concentration in the experimental groups continued to rise over 10 days. The cumulative COD concentration in each treatment group (G1-G4) was significantly lower than that in the control group (G0), indicating that all treatments effectively slowed down the accumulation of organic matter. The COD concentration in group G2 (5.0 × 10⁻⁶) was the lowest among all treatments. 5 CFU / mL was most effective, along with G3 (5.0 × 10⁻⁶ CFU / mL). 6 The effects were similar in the CFU / mL groups, and the effects of groups G1-G3 were all better than those of group G4 (commercially available product group). Figure 6 As shown in Figure -B, the total nitrogen accumulation curves of all treatment groups (G1-G4) were significantly lower than those of the control group (G0), and the effects of the treatment groups differed significantly. The degradation rates of groups G1-G4 relative to group G0 were 26.1%, 44.4%, 47.0%, and 19.9%, respectively. Group G3 had the highest total nitrogen degradation, similar to that of group G2. Figure 6As shown in Figure -C, there were significant differences in the ammonia nitrogen accumulation rate among the groups, indicating that different treatments had different effects on the conversion or removal of ammonia nitrogen. At 10 days, the ammonia nitrogen concentrations in groups G0-G4 reached 2.52 mg / L, 1.85 mg / L, 1.61 mg / L, 1.65 mg / L, and 2.01 mg / L, respectively. The degradation rates of groups G1-G4 relative to group G0 were 26.6%, 36.1%, 34.5%, and 20.2%, respectively. Group G2 showed the best effect, which was basically consistent with the control effect on COD and total nitrogen, demonstrating the synergistic effect of the treatment. The above experimental results indicate that the application of this compound microecological preparation in aquaculture significantly slows down the accumulation of organic matter and the upward trend of ammonia nitrogen in the aquaculture water, reduces the frequency of water changes, improves water quality stability, and, considering both cost and effectiveness, the optimal concentration is 5.0 × 10⁻⁶. 5 The effect is best at CFU / mL.

[0054] sequence list

[0055] SEQ ID No. 1:

[0056]

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Rhodococcus pyogenes CGMCC No. 23799 in improving COD and ammonia nitrogen levels in aquaculture water.

2. The application according to claim 1, characterized in that, The 16S rRNA nucleic acid sequence of the *Rhodococcus cordata* is shown in SEQ ID No.

1.

3. The application according to claim 1, characterized in that, The application also includes the degradation of total nitrogen content in water bodies.

4. The application according to claims 1-3, characterized in that, The aquaculture water body mentioned is a seawater aquaculture water body.

5. The application according to claim 1, characterized in that, The salinity of the water body is 0%~3% by mass / volume ratio of NaCl.

6. The application according to claim 1, characterized in that, The aforementioned club-like Rhodococcus and Bacillus belye were used in combination.

7. The application according to claim 6, characterized in that, The preservation number of the Bacillus belyssus is CGMCC No. 27887.

8. The application according to claim 6, characterized in that, The bacterial concentration of the compound strain of *Rhodococcus cordata* and *Bacillus belyssae* in aquaculture water was 5.0 × 10⁻⁶. 4 CFU / mL ~5.0×10 6 CFU / mL.

9. The application according to claim 8, characterized in that, The concentration of the compound bacterial strain in aquaculture water was 5.0 × 10⁻⁶. 5 CFU / mL.

10. The application according to any one of claims 1-3, characterized in that: The application is carried out in a low-oxygen water environment, wherein the dissolved oxygen concentration in the water is 0.7~1.3 mg / L.

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