Nitrifying bacteria synthetic flora preparation for aquaculture pond mouth expanding culture and application of nitrifying bacteria synthetic flora preparation

Abstract

The invention discloses a nitrifying bacteria synthetic flora preparation for aquaculture pond mouth expanding culture and application of the nitrifying bacteria synthetic flora preparation. The nitrifying bacteria synthetic flora preparation comprises a nitrifying bacteria synthetic flora, a culture medium and an attachment carrier, the nitrifying bacteria synthetic flora is formed by collecting biofilm formation filter stones of a biofilter of a circulating water culture system and domesticating and enriching the biofilm formation filter stones through a single nitrogen source culture medium, and the highest degradation efficiency of the nitrifying bacteria synthetic flora to nitrite is 32.45 mg / (L.h). After the penaeus vannamei boone is subjected to field expanding culture at a pond opening of penaeus vannamei boone shed culture, the nitrite content in the shed water body can be effectively controlled to be lower than 2.0 mg / L in the whole process, the prawn sub-poisoning state caused by nitrite in the later stage of high-density culture is remarkably relieved, the culture period is prolonged, the peak shifting marketing opportunity is grasped, the transportation resistance of the prawns is improved, and the economic benefit is increased. And the problems of short shelf life and high use cost of the nitrobacteria are solved, and the application bottleneck of the nitrobacteria in aquaculture is successfully overcome.

Description

Technical Field

[0001] This invention relates to the field of aquaculture, specifically to a nitrifying bacteria synthesis microbial community preparation for the expansion of aquaculture ponds and its application. Background Technology

[0002] With the rapid development of intensive aquaculture, excessive nitrite levels in water have become a key factor restricting the healthy development of aquaculture. Especially in the later stages of aquaculture, as feeding increases and uneaten feed and feces accumulate, nitrite levels in the water can even exceed 20 mg / L. The accumulation of nitrite can cause serious harm to aquatic animals such as fish and shrimp, including chronic poisoning, respiratory distress, stunted growth, and even death.

[0003] Currently, nitrite is generally treated in aquaculture using physical, chemical, and biological methods. Physical methods primarily use adsorbents such as zeolite powder and activated carbon to adsorb nitrite; however, this method often results in rapid rebound and only addresses the symptoms, not the root cause. Chemical methods mainly utilize chlorine dioxide, potassium persulfate, potassium ferrate, and polyferric sulfate, but improper use often disrupts the microbial balance of the aquaculture system and directly harms the farmed animals. Biological methods primarily utilize microorganisms to degrade nitrite produced in aquaculture, representing a green and environmentally friendly treatment method.

[0004] However, most current microbial products primarily utilize heterotrophic bacteria. Aquaculture water bodies are typically low in carbon and nitrogen. Under these conditions, traditional probiotics such as Bacillus and Lactobacillus preferentially utilize organic nitrogen, exhibiting very low efficiency in utilizing inorganic nitrogen. Consequently, their effectiveness in degrading nitrite is poor, far inferior to nitrifying bacteria. Furthermore, the nitrifying bacteria currently used in aquaculture also present numerous problems. Because nitrifying bacteria do not produce spores or endospores, the bacterial cells in liquid or ordinary solid formulations die quickly, resulting in extremely poor storage stability, a very short shelf life, and often failing to meet expected performance. Additionally, the high dosage required leads to excessively high costs, making it unacceptable to users. Therefore, there is an urgent need for a stable and cost-effective nitrifying bacteria product. Summary of the Invention

[0005] To address and overcome the problems and shortcomings of existing technologies, this invention provides a nitrifying bacteria synthesis microbial community preparation for aquaculture pond expansion and its application. The nitrifying bacteria synthesis microbial community preparation of this invention can effectively reduce the nitrite content in aquaculture ponds during expansion, and its application cost is low, making it suitable for practical aquaculture needs.

[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: This invention provides a nitrifying bacteria synthesis microbial preparation for aquaculture pond expansion, comprising nitrifying bacteria synthesis microbial groups, culture medium, and attachment carrier.

[0007] Furthermore, the mass ratio of the nitrifying bacteria synthesizing microbial community, the culture medium, and the attachment carrier is 20~25:1:2~4.

[0008] Furthermore, the components of the nitrifying bacteria synthetic flora include 2.6 × 10⁶ basophilic Pseudomonas. 8 CFU / g ~3.0×10 8 CFU / g, Teratobacillus 2.5×10 8 CFU / g ~3.0×10 8 CFU / g, Corynebacterium praeparatum 1.5×10 8 CFU / g ~2.0×10 8 CFU / g, Terbinafine 1.0×10 8 CFU / g ~1.5×10 8 CFU / g, Pseudomonas stearothermiae 0.4×10 8 CFU / g ~0.5×10 8 CFU / g, Pseudomonas taurine 2.0 × 10⁻⁶ 8 CFU / g ~2.5×10 8 CFU / g.

[0009] Furthermore, by weight, the components of the culture medium include: 1 to 1.5 parts magnesium sulfate, 0.6 to 1.0 parts manganese sulfate, 2.0 to 3.0 parts potassium dihydrogen phosphate, 2.0 to 3.0 parts dipotassium hydrogen phosphate, 0.4 to 0.6 parts sodium carbonate, and 90 to 95 parts sodium nitrite.

[0010] Furthermore, the attachment carrier is at least one of diatomaceous earth, montmorillonite powder, and zeolite powder; the particle size of the attachment carrier is greater than or equal to 300 mesh.

[0011] Preferably, the attachment carrier is 300-mesh diatomaceous earth.

[0012] This invention also provides the application of the aforementioned nitrifying bacteria synthetic microbial preparation in improving the water quality of aquaculture water bodies.

[0013] Furthermore, the nitrifying bacteria synthetic microbial preparation is expanded in aquaculture ponds to obtain expansion solution; the expansion solution is then sprayed into aquaculture water at a dosage of 2L / mu·m to 4L / mu·m, and used once every three days.

[0014] Furthermore, the steps for expanding the aquaculture ponds are as follows: (1) Add water, culture medium and attachment carrier to a sterilized plastic bucket to prepare culture medium. Use a heating rod to control the temperature at 25~30℃ and fully aerate to remove residual chlorine. (2) Add the domesticated nitrifying bacteria to the culture medium, cover the bucket, and culture in the dark with aeration throughout the process. Detect the nitrite concentration of the culture medium every day and calculate the degradation rate. (3) When the nitrite concentration of the culture medium drops to 0 mg / L~10 mg / L, 0.25 kg of culture medium is added to the culture medium until the nitrite degradation rate increases to more than 20 mg / L·h, and the expansion culture medium of the nitrifying bacteria synthetic microbial group preparation is obtained; (4) Take half of the total volume of the expansion solution and sprinkle it into the aquaculture water. Then add an equal amount of water to the remaining expansion solution. Add 0.25 kg of culture medium and 0.125 kg of attachment carrier to every 100 L of expansion solution. Then continue to culture until the nitrite concentration drops to 0 mg / L~10 mg / L. Take half of the total volume of the expansion solution again and sprinkle it into the aquaculture water. Add an equal amount of water, culture medium and attachment carrier to the remaining expansion solution. Continue to culture. Repeat this cycle.

[0015] Furthermore, the acclimatization steps for the nitrifying bacteria synthesizing microbial community are as follows: (1) Add water, culture medium and attachment carrier to the sterilized plastic bucket, use a heating rod to control the temperature at 25~30℃, and fully aerate to remove residual chlorine; (2) After aeration for 24 hours, add biofilm-coated filter stones from the biological filter of the recirculating aquaculture system to the container, cover the container, and then aerate and incubate in the dark throughout the process; test the nitrite concentration of the culture medium and calculate the degradation rate every day; (3) When the nitrite concentration in the culture medium is less than 10 mg / L, continue to add 0.25 kg of culture medium per 100 L of culture medium and acclimate until the nitrite degradation rate is higher than 30 mg / L·h to obtain the acclimatized nitrifying bacteria synthetic community.

[0016] Furthermore, the nitrifying bacteria synthesis culture medium can effectively reduce the nitrite content in aquaculture water, and its use throughout the aquaculture process can control the nitrite content to below 2.0 mg / L.

[0017] Compared with the prior art, the advantages and technical effects of the present invention are: The nitrifying bacteria synthesized in this invention are synthesized by continuously acclimating biofilm filter stones from a recirculating aquaculture system in the presence of a suitable culture medium and attachment carrier. Their nitrite degradation efficiency is greater than 30 mg / (L·h), and after being expanded and cultivated in aquaculture ponds, they can effectively reduce the nitrite content in aquaculture water. This invention solves the problems of poor storage stability and inconsistent effectiveness of existing nitrifying bacteria. Furthermore, once successfully expanded, they can be used continuously for a long period, greatly reducing operating costs and showing broad application prospects in aquaculture. Attached Figure Description

[0018] Figure 1 The changes in nitrite degradation rate during the acclimatization of the nitrifying bacteria synthetic microbial community in Example 1; Figure 2 The effect of different dosages of nitrifying bacteria synthesizing microbial communities on nitrite removal in Example 3; Figure 3 The different containers used for on-site expansion of the nitrifying bacteria synthetic microbial community preparation in Example 4; Figure 4 The changes in nitrite levels during the on-site expansion of the nitrifying bacteria synthesis microbial community preparation in Example 4; Figure 5 The changes in nitrite levels in the aquaculture water after using the culture medium for the nitrifying bacteria synthesizing microbial community in Example 5; Figure 6 The average nitrite content in the pool water of the control group and the experimental group in Example 5; Figure 7 The average residual chlorine content in the pool water of the control group and the experimental group in Example 5. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail with reference to the following specific examples.

[0020] Example 1: Domestication of Nitrifying Bacteria Synthetic Microbiota Sterilize and clean the covered plastic bucket and aeration pump. Then, add water, culture medium, and attachment carrier to the sterilized plastic bucket at a mass ratio of 100:0.25:0.5 to prepare the culture solution. Use a heating rod to control the temperature at 25-30℃, and simultaneously use the aeration pump to fully aerate and remove residual chlorine. The culture medium components include 1.2 parts magnesium sulfate, 0.8 parts manganese sulfate, 2.5 parts potassium dihydrogen phosphate, 2.0 parts dipotassium hydrogen phosphate, 0.5 parts sodium carbonate, and 93 parts sodium nitrite; the attachment carrier is 300-mesh diatomaceous earth. All components of the culture medium and the 300-mesh diatomaceous earth are commercially available products.

[0021] After aeration of the culture medium for 24 hours, biofilm-coated filter stones from the recirculating aquaculture system were added to the tank, with 500g of biofilm-coated filter stones added per 100L of culture medium. The tank was then covered. The culture was carried out under aeration and in the dark throughout the process, with nitrite concentration monitored daily and degradation rate calculated. When the nitrite concentration was below 10mg / L, 0.25kg of culture medium was added per 100L of culture medium. To reduce the feedback inhibition of nitrate accumulation on the growth of nitrifying bacteria, half of the acclimatization solution was discarded and replaced with fresh culture medium on days 17, 22, and 26 during the acclimatization period. After 30 days of acclimatization, the final nitrite degradation rate increased to 32.45mg / (L·h). Figure 1 A nitrifying bacterial biosynthesis community meeting the requirements was obtained. 16S rDNA classification and sequencing were used to determine the bacterial composition, and the results showed that the nitrifying bacterial biosynthesis community consisted of 2.6 × 10⁻⁶ bacteria.8 CFU / g of alkalophilic Pseudomonas ( Pseudomonas alcaliphila ), 2.5×10 8 CFU / g of *Geobacterium tumefaciens*, 1.5 × 10 8 CFU / g of Corynebacterium praeparatum, 1.0×10 8 CFU / g of Terbinafine, 0.4×10 8 CFU / g of Pseudomonas stearothermia ( Stutzerimonas stutzeri ) and 2.0×10 8 CFU / g of Pseudomonas aeruginosa ( Pseudomonas toyotomiensis )composition.

[0022] The formula for calculating the nitrite degradation rate (the rate of change of nitrite concentration over a specific time period) is as follows: V=ΔC / Δt, In the formula, ΔC represents the change in nitrite concentration (mg / L), Δt represents the change in time (h), and the unit of degradation rate V is mg / (L·h).

[0023] Example 2: Validation of the shelf life of nitrifying bacteria synthetic microbial communities Nitrifying bacteria with an initial nitrite degradation rate of 30.50 mg / (L·h) were stored at room temperature and 4℃ for different periods of time, and their nitrite reduction effect was tested again. The shelf life of the nitrifying bacteria was determined by whether they could be cultured again and whether the nitrite reduction rate could be gradually increased.

[0024] The results (Table 1) show that, regardless of whether stored at room temperature or 4°C, the longest shelf life of the nitrifying bacteria synthesizing flora is 21 days. Storage at 4°C is slightly better, allowing for rapid culturing after 21 days, while culturing is slower at room temperature. After 4 weeks of storage, neither of these conditions can effectively revive the flora. Therefore, the shelf life of the nitrifying bacteria synthesizing flora is 3 weeks.

[0025] Table 1. Results of reculturing nitrifying bacteria biosynthetic communities after different storage times.

[0026] Example 3: Validation of the effective dosage for nitrifying bacteria to synthesize microbial communities A test solution with a nitrite content of 53.48 mg / L was prepared using sodium nitrite reagent. 0.5% 300-mesh diatomaceous earth was added, and under sufficient aeration to ensure dissolved oxygen, 0.5%, 1.0%, 2.5%, and 5.0% of nitrifying bacteria (with an initial nitrite degradation rate of 30.50 mg / (L·h)) were added respectively. Each addition was repeated three times, and the nitrite content was monitored daily. The results showed ( Figure 2 As the amount of nitrifying bacteria synthesizing microbiota increases, the removal rate of nitrite in the test solution increases. At a concentration of 5.0%, nitrite levels drop to 0 on day 4; at a concentration of 2.5%, it takes 5 days to reach 0. Even at concentrations of 1.0% and 0.5%, there is still an effect on reducing nitrite, albeit more slowly. This is generally consistent with the lowest standard dosage of nitrifying bacteria on the market, which is 0.4%.

[0027] Example 4: Expansion cultivation of nitrifying bacteria-synthesized microbial communities in aquaculture ponds According to Examples 2 and 3, two problems were encountered when using nitrifying bacteria synthesizing microbial communities in aquaculture ponds: First, as shown in Example 2, the shelf life of nitrifying bacteria synthesizing microbial communities is 3 weeks, while the average circulation period of aquaculture products is 3-5 months; second, direct use results in excessively high dosages, leading to high costs and inconvenience in operation. To overcome these two contradictions, this invention adopts on-site propagation to solve the problems of short shelf life and high usage costs.

[0028] Therefore, this invention designs a nitrifying bacteria synthesis microbial community preparation, which consists of three parts: nitrifying bacteria synthesis microbial community, culture medium, and attachment carrier. The nitrifying bacteria synthesis microbial community obtained through domestication in Example 1 is used as the seed package for the synthesis microbial community. The components of the culture medium in Example 1 are compounded into a culture medium package, and the carrier package is 300-mesh diatomaceous earth.

[0029] When expanding the nitrifying bacteria synthetic microbial community preparation in aquaculture ponds, first, disinfect and clean the covered plastic bucket and aeration pump with potassium permanganate. Then, add water, culture medium, and attachment carrier to the disinfected plastic bucket at a mass ratio of 100:0.25:0.5 to prepare the culture solution. Use a heating rod to control the temperature at 25-30℃, and simultaneously use the aeration pump to fully aerate and remove residual chlorine. After aeration for 24 hours, add 5L of synthetic microbial community seed per 100L of culture solution and cover the bucket. Continue aeration throughout the culture process, monitoring nitrite concentration and calculating the degradation rate daily. When the nitrite concentration is 0-10mg / L, add 0.25kg of culture medium per 100L of expansion solution until the nitrite degradation rate reaches 20mg / (L·h) or higher, and the nitrite concentration is 0-10mg / L. The desired synthetic microbial community expansion solution is then obtained. The expansion container can be a round or square large bucket (white or other colors are acceptable), a cement fermentation tank, a simple fermentation tank, etc. Figure 3 As shown. Note that direct sunlight should be avoided during the cultivation process. The culture container should be placed indoors. If it is outdoors, it is advisable to cover it with a black film to block light.

[0030] Half the total volume of the expansion culture medium was used in aquaculture. Half the volume of water was then added to the expansion culture medium. For every 100L of expansion culture medium, 0.25kg of culture medium and 0.125kg of attachment carrier were added. The culture was continued until the nitrite concentration dropped to 0-10mg / L. Half the total volume of the expansion culture medium was then used in aquaculture. The process of adding water, culture medium, and attachment carrier was repeated as described above, and the culture was continued in this cycle. The changes in nitrite concentration during the expansion process are shown below. Figure 4 As shown, the changes in nitrite levels during continuous expansion can be seen to indicate that the initial expansion phase takes 3-5 days, while the expansion rate stabilizes at 1-2 days in the later stages.

[0031] Example 5: Application of nitrifying microbial culture preparation in small-scale culture of Litopenaeus vannamei Experiment 1: Short-term effect of reducing nitrite In November 2023, an experiment was conducted in a small-scale shrimp farming area in Dongtai City, Yancheng City, Jiangsu Province. Five sheds were selected, each with a water volume of 0.6 mu·m. After 90 days of farming, the shrimp reached a size of approximately 30 individuals, with a stocking density of 85,000 shrimp per shed. The salinity was 15‰, ammonia nitrogen content was 0%, and nitrite content was 2-20 mg / L. The nitrifying bacteria synthesis culture medium from Example 4 was used at a dosage of 2L / shed, applied every three days. The results are as follows: Figure 5 As shown, the nitrite content in the water body decreased significantly on the 3rd to 4th day after use. The nitrite removal rates of the three sheds were 84%, 90% and 95% on the 5th day, respectively. The initial nitrite content of the other two sheds was higher (15 mg / L and 20 mg / L, respectively), and the nitrite removal rates were 80% and 50% on the 9th day.

[0032] Experiment 2: Long-term use and its effect on reducing nitrite In March 2024, at the Tongzhou Bay Shrimp Farming Park in Rudong County, Nantong City, Jiangsu Province, 12 days after the start of shrimp farming, four zones were selected, with 10 sheds in each zone, for the experiment. Zones A and C were the experimental groups, and Zones B and E were the control groups. Each shed had 0.6 mu·m of water volume, a stocking density of 45,000 shrimp per shed, and a water salinity of 20‰. The experimental groups used nitrifying bacteria synthesis culture medium at a dosage of 2L / shed, applied every three days, while the control groups did not use the product. In early June 2024, after 100 days of farming, the shrimp reached a size of 30 shrimp per jin (approximately 150g). Samples were taken and the nitrite and residual chlorine content in the water of each shed in both the experimental and control groups was measured.

[0033] The results showed that ( Figure 6The nitrite content in each experimental group was generally lower than that in the control group. The highest nitrite content was found in control group E (3-day average > 7 mg / L), followed by control group B (3-day average 5.5–6.4 mg / L). Experimental groups A and C maintained low nitrite levels (3-day average < 2 mg / L), with group C having the lowest, ranging from 0.61 to 0.69 mg / L. Furthermore, residual chlorine in the pond water was analyzed, and the results showed… Figure 7 The nitrite content and residual chlorine content in the pond water showed a strong correlation, meaning that ponds with high residual chlorine levels also tended to have high nitrite content. It is speculated that residual chlorine is the reason why the nitrite content in experimental group A was higher than that in experimental group C.

[0034] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A synthetic flora preparation of nitrifying bacteria for expansion and cultivation in aquaculture ponds, characterized in that: The nitrifying bacteria synthetic flora preparation comprises a nitrifying bacteria synthetic flora, a culture medium and an attachment carrier.

2. The nitrifying bacteria synthetic flora preparation according to claim 1, characterized in that: The mass ratio of the nitrifying bacteria synthetic flora, culture medium and attachment carrier is 20-25:1:2-4.

3. The nitrifying bacteria synthetic flora preparation according to claim 1, characterized in that: The components of the nitrifying bacteria synthetic flora include 2.6×10 8 CFU / g ~3.0×10 8 CFU / g, Land Bacteroids 2.5×10 8 CFU / g ~3.0×10 8 CFU / g, Indian Ocean microcoryneformis 1.5×10 8 CFU / g ~2.0×10 8 CFU / g, Land Bacillus 1.0×10 8 CFU / g ~1.5×10 8 CFU / g, Pseudomonas stutzeri 0.4×10 8 CFU / g ~0.5×10 8 CFU / g, Pseudomonas tolerans 2.0×10 8 CFU / g ~2.5×10 8 CFU / g.

4. The nitrifying bacteria synthetic flora preparation according to claim 1, characterized in that: In parts by weight, the components of the culture medium include: 1 to 1.5 parts of magnesium sulfate, 0.6 to 1.0 parts of manganese sulfate, 2.0 to 3.0 parts of potassium dihydrogen phosphate, 2.0 to 3.0 parts of dipotassium hydrogen phosphate, 0.4 to 0.6 parts of sodium carbonate, and 90 to 95 parts of sodium nitrite.

5. The nitrifying bacteria synthetic flora preparation according to claim 1, characterized in that: The attachment carrier is at least one of diatomaceous earth, montmorillonite powder and zeolite powder; the particle size of the attachment carrier is greater than or equal to 300 meshes.

6. Use of the nitrifying bacteria synthetic flora preparation according to any one of claims 1 to 5 in improving the water quality of aquaculture water.

7. The use according to claim 6, characterized in that: The nitrifying bacteria synthetic flora preparation is expanded in a breeding pond to obtain an expansion liquid; the expansion liquid is sprayed into an aquaculture water body at a dosage of 2L / mu·m2 to 4L / mu·m2, and is used once every three days.

8. The use according to claim 7, characterized in that: The steps of expanding the breeding pond are as follows: (1) Add water, culture medium and attachment carrier into a sterilized container to prepare culture solution, control the temperature at 25°C to 30°C, and provide sufficient aeration; (2) adding the domesticated nitrifying bacteria synthetic flora to the culture solution, sealing the culture solution, aerating and culturing in the dark, detecting the nitrite concentration of the culture solution every day and calculating the degradation rate; (3) When the nitrite concentration of the culture solution drops to 0 mg / L-10 mg / L, adding culture medium to the culture solution until the nitrite degradation rate increases to above 20 mg / L·h, thereby obtaining a culture solution for the synthetic flora preparation of nitrifying bacteria; (4) Sprinkling 1 / 2 of the total volume of the expansion liquid into the aquaculture water body, then adding an equal amount of water, culture medium and attachment carrier to the remaining expansion liquid, and then continuing to culture until the nitrite concentration drops to 0 mg / L~10 mg / L, and then taking 1 / 2 of the total volume of the expansion liquid again and splashing it into the aquaculture water body, and adding an equal amount of water, culture medium and attachment carrier to the remaining expansion liquid, and continuing to culture, and repeating the cycle.

9. The use according to claim 8, characterized in that: In the step (3), 0.25 kg to 0.3 kg of culture medium is added to every 100 L of culture medium; in the step (4), 0.25 kg to 0.3 kg of culture medium and 0.125 kg to 0.2 kg of attachment carrier are added to every 100 L of expansion culture medium.

10. The use according to claim 7, characterized in that: The domestication steps of the synthetic flora of nitrifying bacteria are as follows: (1) Add water, culture medium and attachment carrier into a sterilized container to prepare culture solution, control the temperature at 25°C to 30°C, and provide sufficient aeration; (2) After aeration for 24 hours, add the biofilter membrane filter stone of the circulating aquaculture system to the container, seal it and culture it in the dark during the whole process; test the nitrite concentration of the culture medium every day and calculate the degradation rate; (3) When the nitrite concentration of the culture medium is lower than 10 mg / L, culture medium is added to the culture medium and the culture medium is acclimated until the nitrite degradation rate is higher than 30 mg / L·h, thereby obtaining an acclimated nitrifying bacteria synthetic flora.

Images