Carbon bacterium mixture for aquaculture as well as preparation method and application of carbon bacterium mixture

Abstract

The invention discloses a carbon and bacterium mixture for aquaculture and a preparation method and application thereof, and belongs to the technical field of aquaculture. The carbon and bacterium mixture for aquaculture comprises the following raw materials in parts by volume: 150-200 parts of a fermentation carbon source and 15-20 parts of a composite flora, the fermentation carbon source comprises an intermediate product of grain fermentation; the composite flora comprises at least two of bacillus tequilensis, bacillus coagulans and bacillus licheniformis. The carbon and bacterium mixture disclosed by the invention can effectively reduce the contents of ammonia nitrogen, nitrite, total nitrogen and chemical oxygen demand in an aquaculture water body, has no obvious adverse effects on the environmental basic indexes such as dissolved oxygen, pH value and total phosphorus in the water body, and can maintain the stability and safety of water body ecology while realizing efficient degradation of pollutants.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to a carbon-based bacterial mixture for aquaculture, its preparation method, and its application. Background Technology

[0002] Under the current high-density, intensive aquaculture model, the large amount of feed and high biological load per unit water body lead to the rapid accumulation of uneaten feed, excrement and metabolic products in the water, causing an imbalance in the carbon-nitrogen ratio, a significant increase in the concentration of ammonia nitrogen and nitrite, and a large amount of organic matter deposition. This, in turn, leads to a series of problems such as the excessive reproduction of harmful algae, the imbalance of the indigenous microbial community structure, the proliferation of pathogens and a sudden drop in dissolved oxygen, which seriously affect the healthy growth of farmed animals and the stable operation of the aquaculture system.

[0003] To control the aforementioned problems, traditional methods typically involve interventions such as "large-scale water exchange + chemical bottom improvement." However, these methods suffer from high water consumption, high energy consumption, and high operating costs. They also easily cause side effects such as exogenous pollution, drug residues, and disruption of the microecology, which are detrimental to the sustainable development of aquaculture systems. In recent years, biological regulation technology has gradually gained attention, mainly by applying probiotics (such as Bacillus and nitrifying bacteria) or carbon sources (such as brown sugar and honey) to improve water quality. However, its practical application still has many shortcomings, such as: the bacterial strain formula is too simple to adapt to complex and changing aquatic environments; and the carbon source addition method is too crude, making it difficult to achieve stable ecological regulation effects.

[0004] Therefore, accurately supplementing available carbon sources to promote the targeted proliferation of beneficial bacteria, build a stable micro-ecosystem, and promote the transformation of harmful substances has become a key technological requirement for achieving green and efficient aquaculture. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a carbon-based microbial mixture for aquaculture, its preparation method, and its application. The carbon-based microbial mixture of this invention can efficiently degrade various pollutants in aquaculture water, while also exhibiting environmental compatibility and safety.

[0006] The technical solution of the present invention is as follows: A carbon-based microbial mixture for aquaculture comprises the following raw materials in the following proportions: 150-200 parts by volume of fermentation carbon source and 15-20 parts by mass of compound microbial community. The fermentation carbon source includes intermediate products from grain fermentation; The complex microbial community includes at least two of Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis.

[0007] Preferably, the fermentation carbon source includes intermediate products from corn fermentation; The fermentation carbon source contains ≥120g / L of organic carbon and ≥40g / L of small molecule peptides.

[0008] Preferably, the method for preparing the fermentation carbon source includes the following steps: S1. Mix starch raw material powder with water to obtain a slurry, then add amylase to obtain an enzyme-containing slurry. Spray liquefy and flash evaporate the enzyme-containing slurry at temperature T1, cool it down to temperature T2, and keep it at the temperature until the iodine test is qualified to obtain a liquefied liquid. S2. Filter a portion of the liquefied liquid to remove the filter residue and obtain a clear sugar solution. Mix the remaining liquefied liquid and sugar solution in a certain proportion, dilute, add a nitrogen source to make a culture medium and sterilize it. S3. Inoculate the sterilized culture medium with Aspergillus niger spores to ferment, prepare fermentation broth, filter, concentrate, and obtain fermentation carbon source.

[0009] Preferably, the method for preparing the fermentation carbon source specifically includes the following steps: S1. After crushing the corn starch raw material, pass it through a 60-80 mesh sieve to obtain corn starch raw material powder. Mix the corn starch raw material powder with water to obtain a slurry. Control the slurry concentration to 25-35% (w / v), adjust the pH to 5.0-6.0, and then add acid-resistant α-high temperature amylase to obtain an enzyme-containing slurry. Spray liquefy and flash evaporate the enzyme-containing slurry at 95-100℃, cool it to 90-95℃, and keep it at this temperature for 2-4 hours until the iodine test is qualified to obtain a liquefied liquid. The amount of acid-resistant α-high temperature amylase added is 30-50 U / g corn starch raw material powder. S2. Filter 75-95% of the total volume of the liquefied liquid to remove the filter residue and obtain a clear sugar solution. Mix the remaining liquefied liquid with the sugar solution at a volume ratio of 1:(3-5). Dilute the total sugar concentration of the mixture with water to 10-16% (w / v) to obtain a diluted solution. Add a nitrogen source to make the total nitrogen volume concentration in the diluted solution reach 0.06-0.2% to prepare a culture medium. Sterilize the culture medium at 115-125℃ for 20-30 minutes and then cool it down to 30-40℃. S3. Inoculate the sterilized culture medium with Aspergillus niger spores to achieve a spore concentration of 250,000 to 550,000 spores / mL. Ferment the medium at 30 to 40°C to prepare a fermentation broth. Filter the fermentation broth through a plate and frame filter, then through a multi-layer membrane filter. Concentrate the filtrate 25 to 40 times at 70 to 90°C in an MVR concentration system to obtain the fermentation carbon source.

[0010] Preferably, the ratio of viable Bacillus tegmentatus, Bacillus coagulans, and Bacillus licheniformis in the complex microbial community is (0.5~1.5):(1~3):(6~8).

[0011] Preferably, the viable count of the *Bacillus tegmentatus* is 500-1.5 billion CFU / mL; the viable count of the *Bacillus coagulans* is 1-3 billion CFU / mL; and the viable count of the *Bacillus licheniformis* is 6-8 billion CFU / mL.

[0012] Preferably, the method for preparing the composite microbial community includes: mixing activated Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis according to the proportion of live bacteria to obtain the composite microbial community.

[0013] The second aspect of this invention protects a method for preparing the carbon-bacterial mixture for aquaculture as described in the first aspect, wherein the fermentation carbon source and the composite microbial community are mixed in a certain proportion to obtain the carbon-bacterial mixture for aquaculture.

[0014] The third aspect of this invention protects the application of the carbon-bacterial mixture for aquaculture described in the first aspect in the field of aquaculture.

[0015] Preferably, the carbon-bacterial mixture is added to the aquaculture water, and the final concentration of the fermented carbon source in the aquaculture water is 1.5~2 mL / m³. 3 The final concentration of the complex microbial community in the aquaculture water is 0.15~0.20 g / m³. 3 The carbon-bacterial mixture is added every 8 to 13 days.

[0016] The beneficial technical effects of this invention are as follows: (1) The carbon-bacterial mixture of the present invention uses the intermediate product of grain fermentation as a carbon source and combines it with a compound microbial community constructed according to a specific ratio of microbial species. The two work together to improve the utilization efficiency of carbon source in aquaculture water and promote the proliferation and function of beneficial microbial community, thus avoiding the problems of insufficient carbon source utilization or limited microbial community activity.

[0017] (2) The carbon-bacterial mixture of the present invention can effectively reduce the content of ammonia nitrogen, nitrite, total nitrogen and chemical oxygen demand (COD) in aquaculture water. The peak degradation rate of ammonia nitrogen can exceed 40%, the peak degradation rate of nitrite can exceed 30%, and the average degradation rate of ammonia nitrogen, nitrite, total nitrogen and COD can reach more than 20%. The pollutant degradation process is rapid and long-lasting. The mixture has no significant adverse effects on the basic environmental indicators such as dissolved oxygen (DO), pH value and total phosphorus (TP) in the water. It can maintain the stability and safety of the aquatic ecosystem while achieving efficient degradation of pollutants. The biofloc based on bacteria can be used as a feed source to supplement the insufficient nutrition of compound feed, which is in line with the development direction of green aquaculture. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the embodiments.

[0019] A carbon-based microbial mixture for aquaculture, characterized in that it comprises the following proportions of raw materials: 150-200 parts by volume of fermentation carbon source and 15-20 parts by mass of compound microbial community. The fermentation carbon source includes intermediate products from grain fermentation; The complex microbial community includes Bacillus tekiria ( Bacillus tequilensis Bacillus coagulans ( Bacillus coagulans ), Bacillus licheniformis ( Bacillus licheniformis At least two of them.

[0020] In some embodiments, the volume parts: mass parts = mL:g.

[0021] The Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis used in the following embodiments and comparative examples of the present invention can all be purchased.

[0022] In some embodiments, the fermentation carbon source includes an intermediate product of corn fermentation; it is a dark brown viscous liquid. Its main components include fermentation organic acids, reducing sugars, small peptides, and easily degradable carbohydrates; the fermentation carbon source has an organic carbon content ≥120 g / L and a small molecule peptide content ≥40 g / L.

[0023] In some embodiments, the method for preparing the fermentation carbon source specifically includes the following steps: S1. After crushing the corn starch raw material, pass it through a 60-80 mesh sieve to obtain corn starch raw material powder. Mix the corn starch raw material powder with water to obtain a slurry. Control the slurry concentration to 25-35% (w / v), adjust the pH to 5.0-6.0, and then add acid-resistant α-high temperature amylase to obtain an enzyme-containing slurry. Spray liquefy and flash evaporate the enzyme-containing slurry at 95-100℃, cool it to 90-95℃, and keep it at this temperature for 2-4 hours until the iodine test is qualified to obtain a liquefied liquid. The amount of acid-resistant α-high temperature amylase added is 30-50 U / g corn starch raw material powder. S2. Filter 75-95% of the total volume of the liquefied liquid to remove the filter residue and obtain a clear sugar solution. Mix the remaining liquefied liquid with the sugar solution at a volume ratio of 1:(3-5). Dilute the total sugar concentration of the mixture with water to 10-16% (w / v) to obtain a diluted solution. Add a nitrogen source to make the total nitrogen volume concentration in the diluted solution reach 0.06-0.2% to prepare a culture medium. Sterilize the culture medium at 115-125℃ for 20-30 minutes and then cool it down to 30-40℃. S3. Inoculate the sterilized culture medium with Aspergillus niger spores to achieve a spore concentration of 250,000 to 550,000 spores / mL. Ferment the medium at 30 to 40°C to prepare a fermentation broth. Filter the fermentation broth through a plate and frame filter, then through a multi-layer membrane filter. Concentrate the filtrate 25 to 40 times at 70 to 90°C in an MVR concentration system to obtain the fermentation carbon source.

[0024] The nitrogen source includes at least one of ammonium sulfate, urea, corn steep liquor powder, soybean peptone, and soybean meal.

[0025] In some embodiments, the ratio of viable counts of Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis in the composite microbial community is (0.5~1.5):(1~3):(6~8); preferably, the ratio of viable counts of Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis in the composite microbial community is 1:2:7.

[0026] In some embodiments of the above-mentioned carbon-bacterial mixture for aquaculture, the mixture is added to the aquaculture water, and the final concentration of the fermented carbon source in the aquaculture water is 1.5~2 mL / m³. 3 The final concentration of the complex microbial community in the aquaculture water is 0.15~0.20 g / m³. 3 .

[0027] In some embodiments, the carbon-bacterial mixture is added every 8 to 13 days; preferably, the carbon-bacterial mixture is added every 10 days.

[0028] Example 1 A carbon-bacterial mixture for aquaculture comprises the following raw materials in the following proportions: 150 parts by volume of fermentation carbon source and 15 parts by mass of compound microbial community; the fermentation carbon source is an intermediate product of corn fermentation; the ratio of viable bacteria of Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis in the compound microbial community is 1:2:7; wherein the viable bacteria count of Bacillus tekirae is 1 billion CFU / mL, the viable bacteria count of Bacillus coagulans is 2 billion CFU / mL, and the viable bacteria count of Bacillus licheniformis is 7 billion CFU / mL.

[0029] A method for preparing a carbon-bacterial mixture for aquaculture includes the following steps: mixing a fermentation carbon source with a complex microbial community in a certain proportion to obtain a carbon-bacterial mixture for aquaculture.

[0030] The fermentation carbon source is prepared by the following method: S1. After crushing the corn starch raw material, pass it through a 60-mesh sieve to obtain corn starch raw material powder. Mix the corn starch raw material powder with water to obtain a slurry. Control the slurry concentration to 30% (w / v), adjust the pH to 5.5, and then add acid-resistant α-high temperature amylase to obtain an enzyme-containing slurry. Spray liquefy and flash evaporate the enzyme-containing slurry at 100℃, cool it to 95℃, and keep it at this temperature for 3 hours until the iodine test is qualified to obtain a liquefied liquid. The amount of acid-resistant α-high temperature amylase added is 40U / g corn starch raw material powder.

[0031] S2. Filter 80% of the total volume of liquefied liquid to remove the filter residue and obtain a clear sugar solution. Mix the remaining liquefied liquid with the sugar solution at a volume ratio of 1:4. Dilute the total sugar concentration of the mixture with water to 12% (w / v) to obtain a diluted solution. Add ammonium sulfate to make the total nitrogen volume concentration in the diluted solution reach 0.2% to prepare a culture medium. Sterilize the culture medium at 121℃ for 25 min and then cool it to 40℃ for later use.

[0032] S3. Inoculate the sterilized culture medium with Aspergillus niger ( Aspergillus niger Spores were collected to achieve a spore concentration of 500,000 spores / mL and fermented at 35°C to prepare a fermentation broth. The fermentation broth was then filtered through a plate and frame filter, followed by multi-layer membrane filtration. The filtrate was concentrated 30 times at 80°C in an MVR concentration system to obtain the fermentation carbon source.

[0033] The composite microbial community is prepared by the following method: activated Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis are mixed according to the proportion of live bacteria to obtain the composite microbial community.

[0034] Example 2 A carbon-bacterial mixture for aquaculture is basically the same as that in Example 1, except that it includes the following proportions of raw materials: 200 parts by volume of fermentation carbon source and 20 parts by mass of compound microbial community.

[0035] A method for preparing a carbon-based bacterial mixture for aquaculture is the same as in Example 1.

[0036] The method for preparing the fermentation carbon source is the same as in Example 1.

[0037] The method for preparing the complex microbial community is the same as in Example 1.

[0038] Comparative Example 1 A fermented carbon source for aquaculture is basically the same as that in Example 1, except that it includes the following proportions of raw materials, uses only 200 parts by volume of fermented carbon source, and does not use compound microbial communities.

[0039] The method for preparing the fermentation carbon source is the same as in Example 1.

[0040] Comparative Example 2 A compound microbial community for aquaculture is basically the same as that in Example 1, except that it includes the following proportions of raw materials, uses only 20 parts by weight of the compound microbial community, and does not use a fermentation carbon source.

[0041] The method for preparing the complex microbial community is the same as in Example 1.

[0042] Comparative Example 3 A carbon-bacterial mixture for aquaculture is basically the same as that in Example 1, except that it includes the following proportions of raw materials: 50 parts by volume of fermentation carbon source and 15 parts by mass of compound microbial community.

[0043] A method for preparing a carbon-based bacterial mixture for aquaculture is the same as in Example 1.

[0044] The method for preparing the fermentation carbon source is the same as in Example 1.

[0045] The method for preparing the complex microbial community is the same as in Example 1.

[0046] Comparative Example 4 A carbon-bacterial mixture for aquaculture is basically the same as that in Example 1, except that it includes the following proportions of raw materials: 150 parts by volume of fermentation carbon source and 10 parts by mass of compound microbial community.

[0047] A method for preparing a carbon-based bacterial mixture for aquaculture is the same as in Example 1.

[0048] The method for preparing the fermentation carbon source is the same as in Example 1.

[0049] The method for preparing the complex microbial community is the same as in Example 1.

[0050] Comparative Example 5 A carbon-bacterial mixture for aquaculture is basically the same as that in Example 1, except that it includes the following proportions of raw materials: 150 parts by volume of fermentation carbon source and 30 parts by mass of compound microbial community.

[0051] A method for preparing a carbon-based bacterial mixture for aquaculture is the same as in Example 1.

[0052] The method for preparing the fermentation carbon source is the same as in Example 1.

[0053] The method for preparing the complex microbial community is the same as in Example 1.

[0054] Comparative Example 6 A carbon-based bacterial mixture for aquaculture is basically the same as in Example 1, except that the ratio of viable bacteria of Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis in the complex is 2:7:1; wherein the viable bacteria count of Bacillus tekirae is 2 billion CFU / mL, the viable bacteria count of Bacillus coagulans is 7 billion CFU / mL, and the viable bacteria count of Bacillus licheniformis is 1 billion CFU / mL.

[0055] A method for preparing a carbon-based bacterial mixture for aquaculture is the same as in Example 1.

[0056] The method for preparing the fermentation carbon source is the same as in Example 1.

[0057] The method for preparing the complex microbial community is the same as in Example 1.

[0058] Comparative Example 7 A carbon-based bacterial mixture for aquaculture is basically the same as in Example 1, except that the ratio of viable bacteria of Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis in the complex bacterial group is 1:1:1; wherein the viable bacteria count of Bacillus tekirae is 3.3 billion CFU / mL, the viable bacteria count of Bacillus coagulans is 3.3 billion CFU / mL, and the viable bacteria count of Bacillus licheniformis is 3.3 billion CFU / mL.

[0059] A method for preparing a carbon-based bacterial mixture for aquaculture is the same as in Example 1.

[0060] The method for preparing the fermentation carbon source is the same as in Example 1.

[0061] The method for preparing the complex microbial community is the same as in Example 1.

[0062] Test example: The carbon-based bacterial mixtures for aquaculture prepared in Examples 1-2 and Comparative Examples 1-7 of the present invention were tested as follows.

[0063] Water quality analysis was conducted in accordance with the Technical Specifications for Clinical Trials of Aquatic Drugs, relevant national standard analytical methods, and analytical methods in the Monitoring and Analysis Methods for Water and Wastewater.

[0064] The water quality determination method of this invention is shown in the table below.

[0065] Table 1: Water Quality Testing Methods

[0066] Test Example 1 Test method: Six Litopenaeus vannamei shrimp farming ponds with similar conditions were selected (each pond has an area of ​​5 mu and a water depth of 1.5 meters). Three ponds were selected as experimental ponds and three as control ponds. The stocking density was basically the same and the same amount of feed was fed.

[0067] Experimental pond: During the mid-stage of aquaculture (when feeding rates increase and water quality load intensifies), the carbon-based bacterial mixture from Example 1 was added. The dosage was as follows: based on water volume, the final concentration of the fermented carbon source in the aquaculture water was 1.5 mL / m³. 3 The final concentration of the compound microbial community in the aquaculture water was 0.15 g / m³.

[0068] Control pond: Standard aquaculture management was adopted, and no carbon-based bacteria mixture for aquaculture was added.

[0069] During the experiment, water samples were collected at a fixed point 0.5m from the pond on the same day as the addition of the carbon-bacterial mixture and on days 1, 3, 5, 7, 10 and 14 after the addition.

[0070] Water quality analysis was conducted in accordance with the "Technical Specifications for Clinical Trials of Aquatic Drugs," relevant national standard analytical methods, and the analytical methods in "Methods for Monitoring and Analyzing Water and Wastewater." The water quality determination methods are shown in Table 1.

[0071] The final experimental data were the average of the results measured in the three experimental ponds and the average of the results measured in the three control ponds. The experimental results are shown below.

[0072] (1) Ammonia nitrogen (NH4) in shrimp ponds after the use of carbon-bacterial mixture + Changes in -N) Table 2: Changes in ammonia nitrogen in shrimp ponds after the use of carbon-based bacteria mixture

[0073] As shown in Table 2, the improvement in water quality in the experimental pond exhibited a clear regularity during the 14-day monitoring period, with significant ammonia nitrogen degradation. The effect was initially apparent on day 1, significantly improved on day 3, and reached its peak between days 5 and 7, with a peak degradation rate of 46.6% on day 7. While the effect slightly decreased between days 10 and 14, it still maintained a high level of degradation efficiency. This indicates that the complex microbial community in the carbon-bacterial mixture of this invention underwent a rapid colonization, reproduction, and effectiveness process in the water, and its action cycle perfectly matched the frequency of carbon-bacterial mixture addition.

[0074] Table 2 also shows that during the entire monitoring period, the average degradation rate of ammonia nitrogen in the experimental pond was 29.7%, while the average change rate of ammonia nitrogen in the control pond increased by 6.7%. This indicates that the carbon-bacterial mixture of the present invention has a significant and stable degradation effect on ammonia nitrogen in aquaculture ponds, takes effect rapidly, has a long effective duration, and can effectively solve the problem of ammonia nitrogen accumulation in aquaculture water.

[0075] (2) Changes in nitrite (NO2-N) in shrimp ponds after the use of carbon-bacterial mixture Table 3: Changes in nitrite levels in shrimp ponds after the use of carbon-based bacteria mixtures

[0076] As shown in Table 3, the nitrite concentration in the control pond increased throughout the entire 14-day monitoring period, with an average increase of 6.8%. This is because, under the conventional aquaculture model, as feeding continues, uneaten feed and excrement accumulate, leading to obstruction of the nitrogen cycle and the natural accumulation of nitrite as an intermediate product.

[0077] In contrast, the average nitrite degradation rate in the experimental pond reached 21.7% during the complete 14-day monitoring period, with the effect peaking on the 7th day at a degradation rate of 36.5%. This indicates that the carbon-bacterial mixture of the present invention exhibits a significant and stable degradation effect on nitrite in water, which can meet the industry's needs for nitrite control.

[0078] This further illustrates that adding the carbon-bacterial complex of the present invention to aquaculture ponds can promote nitrification while enhancing the denitrification process, thus fundamentally solving the problem of nitrite accumulation in water, rather than temporarily adsorbing or masking nitrite.

[0079] (3) Changes in total nitrogen (TN) in shrimp ponds after the use of carbon-based bacteria mixture Table 4: Changes in total nitrogen (TN) in shrimp ponds after the use of carbon-based bacterial mixtures

[0080] As shown in Table 4, the total nitrogen degradation in the experimental pond peaked on day 5 (-35.3%). Although it rebounded slightly afterward, it remained significantly lower than the initial value throughout the 14-day monitoring period, with an average degradation rate of 23.12%, consistent with the degradation patterns of ammonia nitrogen and nitrite. In contrast, the total nitrogen in the control pond increased throughout the monitoring period, with an average increase of 5.98%.

[0081] This demonstrates that the carbon-bacterial mixture of the present invention exhibits a significant and sustained degradation effect on total nitrogen (TN) in water, effectively reducing the nitrogen load within the aquaculture system and also reducing the risk of environmental pollution from wastewater.

[0082] (4) Changes in chemical oxygen demand (COD) in shrimp ponds after the use of carbon-bacterial mixture. Table 5: Changes in Chemical Oxygen Demand (COD) in Shrimp Ponds After the Application of Carbon-Based Microbial Mixture

[0083] As shown in Table 5, during the complete 14-day monitoring period, the COD concentration in the experimental pond showed a stable downward trend, with the degradation effect rapidly manifesting after application and reaching its peak on day 5, with a peak degradation rate as high as 34.5%. Throughout the monitoring period, the average COD degradation rate in the experimental pond reached 21.6%, ultimately stabilizing the COD concentration at 65.8 mg / L, significantly lower than the initial value. In contrast, the COD in the control pond increased by an average of 2.67% during the entire monitoring period. This demonstrates that the carbon-bacterial mixture of the present invention has a significant and lasting effect on COD degradation in water, exhibiting a continuous removal capacity for organic pollutants in water.

[0084] (5) Changes in total phosphorus (TP) in shrimp ponds after the use of carbon-based bacteria mixture Table 6: Changes in total phosphorus (TP) in shrimp ponds after the use of carbon-based bacterial mixtures

[0085] As shown in Table 6, during the complete 14-day monitoring period, the total phosphorus (TP) concentration in the experimental sugars fluctuated very little (-4.4% ~ 2.2%), with an average change rate of -1.8%. This indicates that the carbon-bacterial mixture of the present invention, while efficiently degrading nitrogenous pollutants (ammonia nitrogen, nitrite, total nitrogen) and organic pollutants (COD), did not significantly affect the total phosphorus content in the aquaculture ponds, maintaining the natural balance of the phosphorus cycle in the water body. It has high environmental safety and compatibility, avoiding the risk of abnormal algal proliferation that may be caused by drastic fluctuations in phosphorus content. This further demonstrates that the solution of the present invention has good targeting and ecological safety in the management of complex aquatic environments.

[0086] (6) Changes in pH of shrimp ponds after the use of carbon-based bacteria mixture Table 7: Changes in pH of shrimp ponds after the application of carbon-based bacteria mixture

[0087] As shown in Table 7, during the complete monitoring period of 1 to 14 days, the pH of the experimental pond remained within the range of 7.70 to 7.75, with minimal fluctuations and an average pH change rate of -1.30%, while the average pH change rate of the control pond was -1.87%. This indicates that there was no significant difference in pH variation between the experimental and control ponds, demonstrating that the carbon-based bacterial mixture of this invention did not significantly affect the pH of the aquaculture ponds and effectively maintained the stability of the pH in the aquaculture water.

[0088] (7) Changes in dissolved oxygen (DO) in shrimp ponds after the use of carbon-based bacteria mixture Table 8: Changes in dissolved oxygen (DO) in shrimp ponds after the use of carbon-based bacterial mixtures

[0089] As shown in Table 8, during the complete 14-day monitoring period, the dissolved oxygen (DO) concentration in the experimental pond remained within the safe range of 5.9–6.3 mg / L, with minimal fluctuations and an average change rate of only -1.6%. Furthermore, around day 5, when organic matter degradation was at its most intense, the DO level in the experimental pond began to recover, reaching +1.6% on day 7. This indicates that the carbon-bacterial mixture of the present invention was efficiently utilized for microbial metabolism without triggering a drastic oxygen-consuming process. This further demonstrates that the carbon-bacterial mixture of the present invention achieves a highly efficient synergy between water quality improvement and dissolved oxygen stability, without sacrificing dissolved oxygen for water cleanliness, but rather forming a highly efficient, low-consumption, and stable ecosystem.

[0090] In summary, after the carbon-bacterial mixture of Example 1 of this invention was added to the aquaculture water, during a 14-day monitoring period, the peak degradation rates of ammonia nitrogen exceeded 40%, nitrite exceeded 30%, chemical oxygen demand (COD) exceeded 30%, and the average removal rate of total nitrogen (TN) reached 23.12%, with an effective duration of 7-10 days. Furthermore, it had no negative impact on the basic environment of the aquaculture water; dissolved oxygen (DO) levels remained stably maintained within the safe range of 5.9-6.3 mg / L, pH fluctuations did not exceed 1.30%, and total phosphorus (TP) content remained stable (average change rate of only -1.8%). All indicators remained within the optimal range for aquaculture organisms, fully demonstrating its extremely high environmental safety and ecological compatibility.

[0091] Test Example 2 Test method: One largemouth bass farming pond was selected (each pond has an area of ​​4 mu, a water depth of 2 meters, and a stocking density of 3600 fish / mu). During the mid-stage of the farming process (when the feeding amount increases and the water quality load becomes heavier), the carbon-bacterial mixture used in aquaculture as described in Example 2 was added. The dosage was as follows: calculated by water volume, the final concentration of the fermented carbon source in the farming water was 2 mL / m³, and the final concentration of the compound bacterial community in the farming water was 0.2 g / m³.

[0092] During the experiment, water samples were collected at a fixed point 0.5m from the pond on the day of use of the carbon-bacterial mixture and on days 1, 2, 3, 5, 7, 9 and 12 after use.

[0093] Water quality analysis was conducted in accordance with the "Technical Specifications for Clinical Trials of Aquatic Drugs," relevant national standard analytical methods, and the analytical methods in "Methods for Monitoring and Analyzing Water and Wastewater." The water quality determination methods are shown in Table 1.

[0094] The experimental results are shown in the table below.

[0095] Table 9: Changes in pond water quality in Test Example 2

[0096] As shown in Table 9, after adding the carbon-bacterial mixture from Example 2: the peak degradation rate of ammonia nitrogen reached 46.43% on day 5, with an average degradation rate of 27.55%; the peak degradation rate of nitrite reached 42.11% on day 5, with an average degradation rate of 22.56%; the peak degradation rate of chemical oxygen demand (COD) reached 31.51% on day 5, with an average degradation rate of 18.01%; and the peak degradation rate of total nitrogen (TN) reached 41.54% on day 5, with an average degradation rate of 22.64%. For ammonia nitrogen, nitrite, COD, and TN, the effective action time of the carbon-bacterial mixture from Example 2 can last for 7-9 days, effectively degrading ammonia nitrogen, nitrite, COD, and TN in water.

[0097] In addition, regarding basic water quality indicators, the addition of the carbon-bacterial mixture from Example 2 had no negative impact on the aquatic environment. The dissolved oxygen (DO) level remained stable within the safe range of 5.8~6.2 mg / L, the pH value fluctuated within a range of less than 2.0%, and the total phosphorus (TP) content remained stable (with an average change rate of only -2.1%). All indicators were within the optimal range for aquaculture organisms, demonstrating good environmental safety and ecological compatibility.

[0098] Test Example 3 Test method: Select one largemouth bass farming pond (4 mu in area, 2 meters in depth, and a stocking density of 3600 fish / mu). During the mid-stage of the farming process (when feeding increases and water quality becomes more demanding), add the fermented carbon source used in Comparative Example 1 for aquaculture. The dosage is as follows: calculated by water volume, the final concentration of the fermented carbon source in the farming water is 2 mL / m³.

[0099] During the experiment, water samples were collected at a fixed point 0.5m in the pond on the day the fermentation carbon source was used and on days 1, 2, 3, 5, 7, 9 and 12 after use.

[0100] Water quality analysis was conducted in accordance with the "Technical Specifications for Clinical Trials of Aquatic Drugs," relevant national standard analytical methods, and the analytical methods in "Methods for Monitoring and Analyzing Water and Wastewater." The water quality determination methods are shown in Table 1.

[0101] The experimental results are shown in the table below.

[0102] Table 10: Changes in pond water quality in Test Example 3

[0103] As can be seen from Table 10, after adding the fermentation carbon source of Comparative Example 1, the peak degradation rate of ammonia nitrogen reached 25.00% on the 7th day, which is lower than that of the embodiment of the present invention, and the effect could not be maintained. By the 9th day, it had declined to 17.86%, and the average degradation rate was only 14.80%, which is also lower than that of the embodiment of the present invention.

[0104] As can be seen from Table 10, after adding the fermentation carbon source of Comparative Example 1, the peak degradation rate of nitrite was 26.32% on the 5th day, and the degradation effect declined after the 5th day, with an average degradation rate of only 14.29%, which is lower than that of the embodiment of the present invention.

[0105] As shown in Table 10, after adding the fermented carbon source of Comparative Example 1, the peak COD degradation rate on day 7 was 10.96%. This is because: residual feed and feces in the aquaculture water will lead to excessive nitrogen (N) content and relatively insufficient carbon (C). This C / N ratio imbalance will limit the growth of beneficial microorganisms. After supplementing the carbon source, the beneficial bacteria will proliferate and the water quality will improve relatively. However, the average degradation rate during the usage period is only 5.75%, which is far lower than that of the embodiment of the present invention.

[0106] Table 10 also shows that after adding the fermentation carbon source of Comparative Example 1, the peak total nitrogen degradation rate on day 3 was 23.08%, and the average degradation rate was only 13.19%, indicating a low average removal rate. This is because the removal of total nitrogen depends on the final conversion of various forms of nitrogen (ammonia nitrogen, nitrite, nitrate) into nitrogen gas that escapes from the system. This requires the driving force of specific microorganisms, especially denitrification, which is a complex process dominated by microorganisms. However, Comparative Example 1 only had a fermentation carbon source, resulting in a short-lived effect of the carbon-bacterial mixture.

[0107] In summary, ammonia nitrogen, nitrite, COD, and TN all peaked between days 5 and 7 and then began to decline significantly, reaching near their initial levels by day 12. This indicates low average efficiency and rapid decline. This demonstrates that when the microbial content is too low, the degradation of carbon sources through fermentation cannot be sustained, the action cycle is shortened, and it is impossible to maintain stable, high-quality water throughout the entire usage period.

[0108] Test Example 4 Experimental Method: One largemouth bass farming pond was selected (4 mu in area, 2 meters in depth, and a stocking density of 3600 fish / mu). During the mid-stage of the farming process (when feeding increased and water quality became more demanding), the carbon-based bacterial mixture used in Comparative Example 2 was added. The dosage was as follows: calculated by water volume, the final concentration of the compound bacterial community in the farming water was 0.2 g / m³.

[0109] During the experiment, water samples were collected at a fixed point 0.5m in the pond on the day of use of the compound microbial community and on days 1, 2, 3, 5, 7, 9 and 12 after use.

[0110] Water quality analysis was conducted in accordance with the "Technical Specifications for Clinical Trials of Aquatic Drugs," relevant national standard analytical methods, and the analytical methods in "Methods for Monitoring and Analyzing Water and Wastewater." The water quality determination methods are shown in Table 1.

[0111] The experimental results are shown in the table below.

[0112] Table 11: Changes in pond water quality in Test Example 4

[0113] As shown in Table 11, after adding the compound microbial community, the ammonia nitrogen degradation rate reached a peak of -32.14% on day 5, which was significantly lower than that in the example. The effective action time was about 5-7 days, with a short action cycle and a significant rebound after day 7. The nitrite degradation rate reached a peak of -26.32% on day 7, which was lower than that in the example. The degradation process fluctuated, and the effective action time was maintained for about 7 days. The chemical oxygen demand (COD) degradation rate reached a peak of -25.33% on day 7, with an average degradation rate of only -14.51%. Both the peak and average degradation rates were significantly lower than those in the example. The total nitrogen (TN) degradation rate reached a peak of -33.85% on day 5, with an average degradation rate as low as -13.85%. The effective action time was about 5-7 days, which were also lower than those in the example.

[0114] The above data indicate that in the absence of exogenous fermentation carbon sources, the metabolic activity and systemic degradation capacity of the complex microbial community are fundamentally limited. Despite the addition of the complex microbial community, the carbon source shortage and the resulting imbalance in the water's carbon-to-nitrogen ratio (C / N) limit microbial proliferation and prevent the complete incomplete denitrification process. Consequently, the overall metabolic capacity of the microbial community is insufficient and unsustainable. This leads to a comprehensive and significant decrease in the peak degradation rate, average removal rate, and effective duration of various pollutants, failing to achieve the highly efficient and sustained purification effect of a "carbon-microbe mixture."

[0115] In terms of environmental safety, the addition of this single bacterial agent has no negative impact on the basic aquatic environment. The dissolved oxygen (DO) level remains stable, the pH value fluctuates within a small range, and the total phosphorus (TP) content remains stable, meeting the requirements for aquaculture safety.

[0116] Test Example 5 Test method: Select one largemouth bass farming pond (4 mu in area, 2 meters in depth, and a stocking density of 3600 fish / mu). During the mid-stage of the farming process (when feeding increases and water quality becomes more demanding), add the carbon-based microbial mixture used in Comparative Example 3. The dosage is as follows: calculated by water volume, the final concentration of the fermented carbon source in the farming water is 0.5 mL / m³, and the final concentration of the compound microbial community in the farming water is 0.15 g / m³.

[0117] During the experiment, water samples were collected at a fixed point 0.5m from the pond on the day of use of the carbon-bacterial mixture and on days 1, 2, 3, 5, 7, 9 and 12 after use.

[0118] Water quality analysis was conducted in accordance with the "Technical Specifications for Clinical Trials of Aquatic Drugs," relevant national standard analytical methods, and the analytical methods in "Methods for Monitoring and Analyzing Water and Wastewater." The water quality determination methods are shown in Table 1.

[0119] The experimental results are shown in the table below.

[0120] Table 12: Changes in pond water quality in Test Example 5

[0121] As can be seen from Table 12, after adding the carbon-bacterial mixture of Comparative Example 3, the peak degradation rate of ammonia nitrogen reached 35.71% on the 7th day, which was lower than that of the embodiment of the present invention, and the effect could not be maintained. By the 9th day, it had declined to 17.86%, and the average degradation rate was only 19.39%, which was also lower than that of the embodiment of the present invention.

[0122] As can be seen from Table 12, after adding the carbon bacteria mixture of Comparative Example 3, the peak COD degradation rate was 23.84% on day 5, but its average degradation rate was only 12.99%, which is much lower than that of the embodiment of the present invention.

[0123] As shown in Table 12, after adding the carbon-bacterial mixture of Comparative Example 3, the peak degradation rate of nitrite was 31.58% on day 5. The reason for the high degradation rate at this time is that the carbon source was insufficient to support the complete denitrification process. As a product of nitrification and a substrate for denitrification, the conversion process of nitrite was hindered in the intermediate stage, resulting in the temporary accumulation of nitrite being suppressed, but it could not be completely converted into nitrogen gas for removal. However, the degradation effect declined rapidly after day 5, with an average degradation rate of only 16.54%, indicating that the effect of the carbon-bacterial mixture was short-lived and had a rebound phenomenon. This further illustrates that the low carbon source content in the carbon-bacterial mixture of Comparative Example 3 led to a carbon source shortage, which severely restricted the activity of denitrifying bacteria and could not continuously and thoroughly eliminate nitrite, a key toxic factor.

[0124] Table 12 also shows that after adding the carbon-bacterial mixture of Comparative Example 3, the peak total nitrogen degradation rate on day 5 was 35.38%, and the average degradation rate was only 18.68%, indicating a low average removal rate. This is because the removal of total nitrogen depends on the final conversion of various forms of nitrogen (ammonia nitrogen, nitrite, nitrate) into nitrogen gas that escapes from the system. This process is highly dependent on organic carbon sources (especially denitrification). However, the carbon source content in the carbon-bacterial mixture of Comparative Example 3 was too low, resulting in a short-lived effect of the mixture.

[0125] In summary, ammonia nitrogen, nitrite, COD, and TN all peaked on day 5 and then began to decline significantly, reaching near their initial levels by day 12, indicating low average efficiency and rapid decline. This demonstrates that when the carbon source content is too low, bacterial activity cannot be sustained, leading to a shortened action cycle and an inability to stably maintain excellent water quality throughout the entire usage cycle of the carbon-bacterial mixture.

[0126] Test Example 6 Experimental Method: One largemouth bass farming pond was selected (4 mu in area, 2 meters in depth, and a stocking density of 3600 fish / mu). During the mid-stage of the farming process (when feeding increased and water quality became more demanding), the carbon-based microbial mixture used in Comparative Example 4 was added. The dosages were as follows: based on water volume, the final concentration of the fermented carbon source in the farming water was 1.5 mL / m³, and the final concentration of the compound microbial community in the farming water was 0.1 g / m³.

[0127] During the experiment, water samples were collected at a fixed point 0.5m from the pond on the day of use of the carbon-bacterial mixture and on days 1, 2, 3, 5, 7, 9 and 12 after use.

[0128] Water quality analysis was conducted in accordance with the "Technical Specifications for Clinical Trials of Aquatic Drugs," relevant national standard analytical methods, and the analytical methods in "Methods for Monitoring and Analyzing Water and Wastewater." The water quality determination methods are shown in Table 1.

[0129] The experimental results are shown in the table below.

[0130] Table 13: Changes in pond water quality in Test Example 6

[0131] As shown in Table 13, after adding the carbon-based bacteria mixture of Comparative Example 4: the ammonia nitrogen degradation rate reached a peak of 42.86% on day 5, which was lower than that of the example, with an effective action time of about 5-7 days, a short action period, and a rapid rebound in the later stage; the nitrite degradation rate reached a peak of 36.84% on day 5, with an effective action time of about 7 days; the chemical oxygen demand degradation rate reached a peak of 28.22% on day 5, with an average degradation rate of only 15.15%, both the peak and average degradation rates were lower than those of the example; the total nitrogen degradation rate reached a peak of 38.46% on day 5, with an effective action time of about 7 days, which was also lower than that of the example.

[0132] The above data indicate that, under sufficient carbon source conditions, the concentration of the compound microbial community is the key to determining the degradation efficiency and duration of action. In the carbon-microbial mixture of Comparative Example 4, the content of the compound microbial community was too low. Although there was sufficient carbon source as substrate, the overall metabolic capacity of the microbial community was insufficient, resulting in a significant decrease in the peak degradation rate, average removal rate and effective action time of various pollutants.

[0133] In terms of environmental safety, the addition of the carbon-based bacteria mixture in Comparative Example 4 had no negative impact on the basic aquatic environment. The dissolved oxygen (DO) level remained stable at 5.8~6.2 mg / L, the pH value fluctuated within a range of less than 1.8%, and the total phosphorus (TP) content remained stable, meeting the requirements for aquaculture safety.

[0134] Test Example 7 Experimental Method: One largemouth bass farming pond was selected (4 mu in area, 2 meters in depth, and a stocking density of 3600 fish / mu). During the mid-stage of the farming process (when feeding increased and water quality became more demanding), the carbon-based bacterial mixture used in Comparative Example 5 was added. The dosages were as follows: based on water volume, the final concentration of the fermented carbon source in the farming water was 1.5 mL / m³, and the final concentration of the compound bacterial community in the farming water was 0.3 g / m³.

[0135] During the experiment, water samples were collected at a fixed point 0.5m from the pond on the day of use of the carbon-bacterial mixture and on days 1, 2, 3, 5, 7, 9 and 12 after use.

[0136] Water quality analysis was conducted in accordance with the "Technical Specifications for Clinical Trials of Aquatic Drugs," relevant national standard analytical methods, and the analytical methods in "Methods for Monitoring and Analyzing Water and Wastewater." The water quality determination methods are shown in Table 1.

[0137] The experimental results are shown in the table below.

[0138] Table 14: Changes in pond water quality in Test Example 7

[0139] As shown in Table 14, after adding the carbon-based bacteria mixture of Comparative Example 5: the ammonia nitrogen degradation rate reached its peak of 46.43% on the 3rd day, and then the degradation rate dropped rapidly, indicating that its efficient action period was short and the system stability was poor; the nitrite degradation rate reached its peak of 36.84% on the 3rd day, and its effective action time was significantly shortened, basically rebounding to the initial level on the 9th day; the chemical oxygen demand (COD) degradation rate reached its peak of 27.40% on the 3rd day, and its average degradation rate during the monitoring period was only 14.09%, both the peak and average degradation rates were lower than those of the example, indicating that its overall removal capacity for organic matter was insufficient; the total nitrogen degradation rate reached its peak of 36.92% on the 3rd day, and the rebound trend was obvious in the later stage, and the effective action time was shortened.

[0140] Based on the data above, it can be seen that the carbon-bacterial mixture in Comparative Example 5 exhibits the following characteristics: rapid degradation in the early stage, but significant and rapid rebound in the later stage, resulting in insufficient overall degradation effect and limited comprehensive treatment efficiency.

[0141] Test Example 8 Experimental Method: One largemouth bass farming pond was selected (4 mu in area, 2 meters in depth, and a stocking density of 3600 fish / mu). During the mid-stage of the farming process (when feeding increases and water quality becomes more demanding), the carbon-based microbial mixture used in Comparative Example 6 was added. The dosage was as follows: based on water volume, the final concentration of the fermented carbon source in the farming water was 1.5 mL / m³. 3 The final concentration of the compound microbial community in the aquaculture water was 0.15 g / m³.

[0142] During the experiment, water samples were collected at a fixed point 0.5m from the pond on the day of use of the carbon-bacterial mixture and on days 1, 2, 3, 5, 7, 9 and 12 after use.

[0143] Water quality analysis was conducted in accordance with the "Technical Specifications for Clinical Trials of Aquatic Drugs," relevant national standard analytical methods, and the analytical methods in "Methods for Monitoring and Analyzing Water and Wastewater." The water quality determination methods are shown in Table 1.

[0144] The experimental results are shown in the table below.

[0145] Table 15: Changes in pond water quality in Test Example 8

[0146] As shown in Table 15, after adding the carbon-based bacteria mixture of Comparative Example 6, the COD degradation rate reached its peak of 24.66% on the 7th day. The peak COD degradation was delayed and decreased. The average degradation rate during the monitoring period was 13.39%, which failed to reach a stable and efficient level. The average degradation rate and the duration of action were insufficient.

[0147] As can be seen from Table 15, after adding the carbon-bacterial mixture of Comparative Example 6, the ammonia nitrogen degradation rate reached a peak of 35.71% on the 7th day, which was lower than that of the Example. Its effective action time was shortened to 5-7 days, and the degradation intensity was insufficient, indicating that the conversion efficiency of the carbon-bacterial mixture for ammonia nitrogen was greatly reduced.

[0148] As can be seen from Table 15, after adding the carbon-bacterial mixture of Comparative Example 6, the nitrite degradation rate reached a peak of 31.58% on the 7th day, which was lower than that of the Example. The effective action time was maintained for about 7 days, and the period of degradation rate being lower than 20% increased. The effect on nitrite was not good, and a significant decrease in nitrite concentration could not be achieved.

[0149] Table 15 also shows that after adding the carbon-bacterial mixture of Comparative Example 6, the total nitrogen degradation rate reached a peak of 32.31% on day 7, which was lower than that of the Example. The average degradation rate during the monitoring period was less than 20%, and the effective action time was also shortened to about 7 days, indicating that the denitrification efficiency was significantly reduced.

[0150] The proportions of each component in the complex microbial community in Comparative Example 6 carbon-bacterial mixture are not within the range limited by the present invention. Based on the above data, it can be seen that the purification efficiency of the carbon-bacterial mixture in Comparative Example 6 on aquaculture water is significantly reduced, and is worse than that of the above embodiments.

[0151] Test Example 9 Experimental Method: One largemouth bass farming pond was selected (4 mu in area, 2 meters in depth, and a stocking density of 3600 fish / mu). During the mid-stage of the farming process (when feeding increases and water quality becomes more demanding), the carbon-based microbial mixture used in Comparative Example 7 was added. The dosage was as follows: based on water volume, the final concentration of the fermented carbon source in the farming water was 1.5 mL / m³. 3 The final concentration of the compound microbial community in the aquaculture water was 0.15 g / m³.

[0152] During the experiment, water samples were collected at a fixed point 0.5m from the pond on the day of use of the carbon-bacterial mixture and on days 1, 2, 3, 5, 7, 9 and 12 after use.

[0153] Water quality analysis was conducted in accordance with the "Technical Specifications for Clinical Trials of Aquatic Drugs," relevant national standard analytical methods, and the analytical methods in "Methods for Monitoring and Analyzing Water and Wastewater." The water quality determination methods are shown in Table 1.

[0154] The experimental results are shown in the table below.

[0155] Table 16: Changes in pond water quality in Test Example 9

[0156] As can be seen from Table 16, after adding the carbon-bacterial mixture of Comparative Example 7, the COD degradation rate reached a peak of 22.74% on the 7th day, which was far lower than the peak value of the Example. The average degradation rate during the monitoring period was only 11.55%, which failed to reach a high level of efficient degradation, and the average efficiency and durability were insufficient.

[0157] As can be seen from Table 16, after adding the carbon-bacterial mixture of Comparative Example 7, the ammonia nitrogen degradation rate reached a peak of 32.14% on the 7th day, which was much lower than the peak value of the Example. The effective action time was shortened to 5-7 days, and the degradation intensity was weak throughout the entire cycle.

[0158] As shown in Table 16, after adding the carbon-bacterial mixture of Comparative Example 7, the peak nitrite degradation rate was 31.58%, which was lower than that of the Example. Its effective action time was maintained for about 7 days, and the overall degradation rate was less than 20%. The removal effect on nitrite was reduced, and the goal of significantly reducing the nitrite concentration could not be achieved.

[0159] Table 16 also shows that after adding the carbon-bacterial mixture of Comparative Example 7, the peak total nitrogen degradation rate was 29.23%, which was much lower than the peak of the example. The average degradation rate was only 13.85%, indicating that the denitrification capacity was seriously insufficient and the overall purification efficiency for total nitrogen (TN) was poor.

[0160] The proportions of each component in the complex microbial community in Comparative Example 7 carbon-bacterial mixture are not within the range specified in the present invention. Based on the above data, it can be seen that the carbon-bacterial mixture in Comparative Example 7 exhibits characteristics of slow onset of action, insufficient peak degradation capacity, and short effective period in purifying aquaculture water, and cannot form an efficient synergistic purification system.

[0161] In summary, the carbon-bacterial mixture of the present invention exhibits a systematic and efficient degradation capability for a variety of key pollutants after use, while having no significant impact on basic water quality indicators such as dissolved oxygen, pH, and total phosphorus (TP) in aquaculture ponds, demonstrating environmental compatibility and safety.

[0162] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A carbon-based bacterial mixture for aquaculture, characterized in that, It includes the following proportions of raw materials: 150-200 parts by volume of fermentation carbon source and 15-20 parts by mass of compound microbial community; The fermentation carbon source includes intermediate products from grain fermentation; The complex microbial community includes at least two of Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis.

2. The carbon-based bacterial mixture for aquaculture according to claim 1, characterized in that, The fermentation carbon source includes intermediate products from corn fermentation; The fermentation carbon source contains ≥120g / L of organic carbon and ≥40g / L of small molecule peptides.

3. The carbon-based bacterial mixture for aquaculture according to claim 1, characterized in that, The method for preparing the fermentation carbon source includes the following steps: S1. Mix starch raw material powder with water to obtain a slurry, then add amylase to obtain an enzyme-containing slurry. Spray liquefy and flash evaporate the enzyme-containing slurry at temperature T1, cool it down to temperature T2, and keep it at the temperature until the iodine test is qualified to obtain a liquefied liquid. S2. Filter a portion of the liquefied liquid to remove the filter residue and obtain a clear sugar solution. Mix the remaining liquefied liquid and sugar solution in a certain proportion, dilute, add a nitrogen source to prepare a culture medium and sterilize it. S3. Inoculate the sterilized culture medium with Aspergillus niger spores to ferment, prepare fermentation broth, filter, concentrate, and obtain fermentation carbon source.

4. The carbon-based bacterial mixture for aquaculture according to claim 3, characterized in that, The method for preparing the fermentation carbon source specifically includes the following steps: S1. After crushing the corn starch raw material, pass it through a 60-80 mesh sieve to obtain corn starch raw material powder. Mix the corn starch raw material powder with water to obtain a slurry. Control the slurry concentration to 25-35% (w / v), adjust the pH to 5.0-6.0, and then add acid-resistant α-high temperature amylase to obtain an enzyme-containing slurry. Spray liquefy and flash evaporate the enzyme-containing slurry at 95-100℃, cool it to 90-95℃, and keep it at this temperature for 2-4 hours until the iodine test is qualified to obtain a liquefied liquid. The amount of acid-resistant α-high temperature amylase added is 30-50 U / g corn starch raw material powder. S2. Filter 75-95% of the total volume of the liquefied liquid to remove the filter residue and obtain a clear sugar solution. Mix the remaining liquefied liquid with the sugar solution at a volume ratio of 1:(3-5). Dilute the total sugar concentration of the mixture with water to 10-16% (w / v) to obtain a diluted solution. Add a nitrogen source to make the total nitrogen volume concentration in the diluted solution reach 0.06-0.2% to prepare a culture medium. Sterilize the culture medium at 115-125℃ for 20-30 minutes and then cool it down to 30-40℃. S3. Inoculate the sterilized culture medium with Aspergillus niger spores to achieve a spore concentration of 250,000 to 550,000 spores / mL. Ferment the medium at 30 to 40°C to prepare a fermentation broth. Filter the fermentation broth through a plate and frame filter, then through a multi-layer membrane filter. Concentrate the filtrate 25 to 40 times at 70 to 90°C in an MVR concentration system to obtain the fermentation carbon source.

5. The carbon-based bacterial mixture for aquaculture according to claim 1, characterized in that, The ratio of viable counts of Bacillus tegmentata, Bacillus coagulans, and Bacillus licheniformis in the complex microbial community is (0.5~1.5):(1~3):(6~8).

6. The carbon-based bacterial mixture for aquaculture according to claim 1, characterized in that, The viable count of the *Bacillus tekirae* is 500 million to 1.5 billion CFU / mL; the viable count of the *Bacillus coagulans* is 1 billion to 3 billion CFU / mL; and the viable count of the *Bacillus licheniformis* is 6 billion to 8 billion CFU / mL.

7. The carbon-based bacterial mixture for aquaculture according to claim 1, characterized in that, The method for preparing the composite microbial community includes: mixing activated Bacillus tekirae, Bacillus coagulans, and Bacillus licheniformis according to the proportion of live bacteria to obtain the composite microbial community.

8. A method for preparing a carbon-based bacterial mixture for aquaculture as described in any one of claims 1 to 7, characterized in that, The fermentation carbon source and the composite microbial community are mixed in a certain proportion to obtain a carbon-bacterial mixture for aquaculture.

9. The application of the carbon-based bacterial mixture for aquaculture as described in any one of claims 1 to 7 in the field of aquaculture.

10. The application according to claim 9, characterized in that, The carbon-bacterial mixture is added to the aquaculture water, and the final concentration of the fermented carbon source in the aquaculture water is 1.5~2 mL / m³. 3 The final concentration of the complex microbial community in the aquaculture water is 0.15~0.20 g / m³. 3 The carbon-bacterial mixture is added every 8 to 13 days.

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