Compound micro-ecological preparation for improving black bottom of aquaculture pond and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QINGDAO SHANGDE BIOTECH
- Filing Date
- 2024-11-22
- Publication Date
- 2026-05-12
AI Technical Summary
Hydrogen sulfide is generated at the bottom of aquaculture ponds by sulfate-reducing bacteria, causing the pond bottom to turn black and affecting the health of fish, shrimp and crabs. Moreover, existing technologies are not able to effectively control the volatilization of hydrogen sulfide and assess the effectiveness of bacterial strains.
Using H2S-producing seaweed Shewanella XWS as an indicator bacterium, functional strains such as Priestella gigantea GBW-PM6 were screened out. Combined with Bacillus subtilis, Bacillus cereus and Bacillus amyloliquefaciens, a compound microecological preparation was made. Utilizing the principle of Fe2+ forming FeS precipitation, the thickness of black and odorous bottom sediment and the hydrogen sulfide content in the floating water were reduced.
It effectively reduces the thickness of black and odorous bottom sediment and the hydrogen sulfide content in the floating water, improves water quality, enhances the health of farmed animals, has a wide range of applications, and is simple to operate and low in cost.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial application technology, specifically relating to a compound microecological preparation for improving the black bottom of aquaculture ponds and its application. Background Technology
[0002] Most farmed aquatic animals are fed feed with high protein content. Only about 25% of this protein is converted into nutrients for the fish and shrimp; the rest is excreted into the water and sinks to the bottom through excrement and uneaten feed. The sulfur-containing amino acids, mainly methionine, cysteine, and cystine, are desulfurized by microorganisms to produce sulfates. Other sulfur-containing organic compounds include sulfanilic acids and sulfides, while sulfur-containing inorganic compounds include sulfates and sulfites. These sulfates are converted into hydrogen sulfide (H2S) by sulfate-reducing bacteria (SRB). H2S reacts with iron and manganese in the water to form black sulfide suspended particles / precipitates such as FeS and MnS, causing the pond bottom to turn black. The blackening of the bottom sediment is mainly caused by sulfate-reducing bacteria, and the black color is also an important indicator of the presence of H2S in the pond. When the concentration of H2S in water is higher than 0.1~0.2 mg / L, it can cause breathing difficulties, slow growth, weakened vitality and disease resistance in fish, shrimp and crabs, and even cause acute poisoning and death in animals.
[0003] In some aging ponds that are not dredged for years, uneaten feed and feces from the aquaculture process continuously accumulate. Without timely bottom sediment improvement, this leads to a thick layer of black, foul-smelling sediment, the continuous release of toxic and harmful substances, excessive H2S levels in the floating water, and poisoning of farmed animals. Microorganisms are the main driving force for sulfur cycle metabolism and are closely coupled with the carbon, nitrogen, and phosphorus cycles. Therefore, screening functional strains with H2S control effects and developing microecological preparations with sulfur removal capabilities have broad application prospects in improving the black bottom of aquaculture ponds. Summary of the Invention
[0004] The purpose of this invention is to provide a compound microecological preparation for improving the black bottom of aquaculture ponds and its application. This invention utilizes *Shewanella xWS*, an H2S-producing seaweed, as an indicator bacterium, and cleverly uses H2S and Fe... 2+ Based on the principle of FeS black precipitate formation, functional strains with H2S control effects were screened. These strains and functional carriers (adjuvants) were then compounded to create a granular composite microecological preparation. This preparation was applied in small, repeated applications to aquaculture ponds with deep, black, odorous sediment layers that are prone to H2S production. This effectively reduced the thickness of the black, odorous sediment and the H2S content in the floating water. This invention solves the problem of accurately assessing the effectiveness of hydrogen sulfide-controlling functional strains due to the volatility of H2S during the screening process.
[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solution:
[0006] This invention provides a strain of *Priestella megaterium* GBW-PM6, which is classified and named *Priestella megaterium* (… Priestia megaterium (), deposited at the China General Microbiological Culture Collection Center, with accession number: CGMCC No.32334.
[0007] This invention also provides a compound microecological preparation for improving the black bottom of aquaculture ponds, wherein the compound microecological preparation contains the following components: a live bacteria content of not less than 1.00 × 10⁻⁶. 10 The *Priestella giantiformis* GBW-PM6 strain with a CFU / g concentration of not less than 2.00 × 10⁻⁶ viable cells. 9 CFU / g Bacillus subtilis, viable bacterial count not less than 5.00×10⁻⁶ 9 The CFU / g of Bacillus cereus and the viable bacterial count are not less than 1.50 × 10⁻⁶. 10 CFU / g of Bacillus amyloliquefaciens and functional vectors.
[0008] Furthermore, the Bacillus subtilis used is Bacillus subtilis with accession number CGMCC No. 19824 ( Bacillus subtilis S2; The Bacillus cereus selected is Bacillus cereus with preservation number CGMCC No. 10052 ( Bacillus cereus J19; The Bacillus amyloliquefaciens used is the Bacillus amyloliquefaciens with the preservation number CGMCC No. 10011 ( Bacillus amyloliquefaciens HFJ-7.
[0009] Furthermore, the functional carrier, by weight percentage of the compound microecological preparation, contains 5% to 10% spore germination activator, 30% to 40% zeolite powder, 15% to 20% attapulgite, and the remainder is red staphylocumulus powder.
[0010] Furthermore, the spore germination activator contains 60% L-alanine, 40% fructose, and the zeolite powder is clinoptilolite powder with a particle size of not less than 150 mesh, and the attapulgite is extremely small granular attapulgite with a diameter of 0.5 to 1.0 mm.
[0011] Furthermore, the compound microecological preparation is a round particle with a diameter of 2-3 mm, which is obtained by mixing the strain with the carrier, adding water, rolling the particles with a granulator, and then drying them in a fluidized bed at a low temperature of 50-60℃.
[0012] The present invention also provides the application of the aforementioned Priestella gigantea GBW-PM6 in improving the black bottom of aquaculture ponds.
[0013] This invention also provides the application of the aforementioned compound microecological preparation in improving the black bottom of aquaculture ponds.
[0014] Furthermore, when the compound microecological preparation is applied to black bottom ponds, it is used once every 3 days in the early stage, with 200g~300g applied per mu of water surface. After one month, it is used once every 5 days, with 200g~300g applied per mu of water surface.
[0015] Furthermore, the thickness of the black mud on the black bottom of the pond gradually decreased, and the amount of hydrogen sulfide floating in the water decreased significantly.
[0016] Compared with the prior art, the present invention has the following advantages and beneficial technical effects:
[0017] (1) The strains of the compound microecological agent described in this invention are indigenous bacteria isolated from the aquaculture environment. After initial screening under aerobic conditions using microplate and secondary screening under microaerobic conditions using test tubes, the obtained strains can function in both water bodies with good dissolved oxygen and microaerobic conditions with poor oxygen, making them applicable to a wider range of scenarios.
[0018] (2) The screening of strains and evaluation of product efficacy in the compound microecological preparation of the present invention were carried out using *Shewanella algae* (…). Shewanella algae The indicator method was used, with H2S-producing seaweed *Shewanella xanthophyte* (XWS) as the indicator bacterium. After adding SIM medium containing ferrous salts to microplates or test tubes, 1% XWS was inoculated, followed by the addition of the bacterial suspension of the strain to be screened. The mixture was incubated at 30°C. A light black turbidity began to appear after 2-3 hours, and the black precipitate was observed after 4-6 hours. The microplate method was used to screen aerobic / aerobic sulfur-controlling bacteria, while the test tube method was used to screen microaerobic / anaerobic sulfur-controlling bacteria. This method has the advantages of strong targeting, simple operation, short time consumption, and low cost.
[0019] (3) In addition to functional microorganisms, this invention also incorporates components that promote the germination of spore-forming bacteria, thereby improving the efficiency of spore activity. Furthermore, zeolite powder and attapulgite are both porous carriers, promoting the local adsorption of small molecules such as H2S and increasing the contact concentration between probiotics and H2S. Although the strong dispersibility of zeolite powder is not conducive to granulation, attapulgite has a certain degree of viscosity, which can overcome this problem. No additional binder needs to be added during the granulation process, facilitating release after settling to the bottom of the water. After granulation, the mixture is dried at a low temperature of 50~60℃ in a fluidized bed, which facilitates the preservation of live bacteria.
[0020] In summary, the compound microecological preparation provided by this invention can generate huge economic benefits and has broad application prospects and promotional value in the purification and restoration of black bottoms in aging aquaculture ponds. Attached Figure Description
[0021] Figure 1This refers to the isolation and purification of pond pathogens during the screening of H2S-producing indicator bacteria in Example 1;
[0022] Figure 2 This is the initial TCBS plate used in Example 1 to screen Shewanella algae XWS;
[0023] Figure 3 This refers to the process in Example 1 where the culture system turned black after inoculation with indicator bacteria XWS;
[0024] Figure 4 The microplate method was used in Example 1 to evaluate the sulfur control effect of different strains;
[0025] Figure 5 Example 2 uses the test tube method to evaluate the sulfur control effect of different strains;
[0026] Figure 6 This is the 16S rDNA phylogenetic tree of GBW-PM6 in Example 3;
[0027] Figure 7 The colony and cell morphology (stained with crystal violet) of GBW-PM6 in Example 3;
[0028] Figure 8 This is a comparison of H2S content between the control group and the zeolite powder group in Example 5;
[0029] Figure 9 This refers to the appearance of the compound microecological preparation particles in Example 6;
[0030] Figure 10 It is the black bottom mud of the shrimp pond in Example 6;
[0031] Figure 11 This is a side view of the changes in black mud after using a compound microecological preparation once in Example 6;
[0032] Figure 12 This is a positive view of the changes in black mud after using the compound microecological preparation once in Example 6;
[0033] Figure 13 This is the H2S situation in the floating water after using the compound microecological preparation once in Example 6;
[0034] Figure 14 The color of the black mud after using the compound microecological preparation three times in Example 6. Detailed Implementation
[0035] The technical solution of the present invention will be further described in detail with reference to the following specific examples.
[0036] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.
[0037] The Bacillus subtilis (Bacillus subtilis) of this invention Bacillus subtilis S2 was derived from sludge from aquaculture ponds in Jiaozhou, Qingdao, and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 15, 2020, with accession number CGMCC No. 19824. It has the functions of improving algal communities, flocculating organic matter, and inhibiting Vibrio.
[0038] The Bacillus cereus ( Bacillus cereus J19 was derived from the bottom sediment of sea cucumber farming areas and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 25, 2014, with the accession number CGMCCNo.10052. It possesses the ability to inhibit Vibrio parahaemolyticus and decompose organic matter in aquaculture water, thereby improving water transparency.
[0039] The Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens HFJ-7 was derived from plant rhizosphere soil and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 19, 2014, with the accession number CGMCC No. 10011. It has the functions of killing fecal coliforms, reducing ammonia nitrogen in feces, and producing surfactants to reduce the surface tension of water.
[0040] Modified SIM medium composition: tryptone 20.0 g / L, polyptone 6.0 g / L, ferric ammonium sulfate 1.0 g / L, sodium thiosulfate 1.0 g / L, solid medium supplemented with agar 3.5 g / L, pH 7.2 ± 0.2 (25℃).
[0041] Example 1: Establishment of a screening and evaluation method for hydrogen sulfide functional bacteria and initial screening of sulfur-controlling functional strains
[0042] To study the formation process of black mud in ponds, this invention utilizes an indicator bacterium that produces H2S, which shows black colonies on TCBS plates. Figures 1-2After purification, sequencing and identification confirmed it to be *Shewanella algae*, designated as indicator strain XWS (other known H2S-producing indicator strains can also be used for screening). Indicator strain XWS was cultured in LB liquid medium for 12 h as a seed culture. 100 μL of the XWS seed culture was inoculated into a test tube containing 10 mL of modified SIM medium and incubated statically at 30°C. Color changes were observed every half hour. It was found that as indicator strain XWS multiplied, the culture system gradually turned black starting at 2 h, intensified after 4 h, and became completely black after 8 h. This indicates that *Shewanella algae* is a sulfate-reducing bacterium, multiplying very rapidly under suitable nutrient conditions and producing high amounts of H2S in a very short time. Figure 3 It can be used to screen bacteria that control hydrogen sulfide.
[0043] Fourteen candidate probiotic strains were selected from the strain bank and cultured in LB liquid medium for 24 hours. 125 μL of modified SIM medium, 150 μL of candidate probiotic culture, and 25 μL of XWS culture were added to a 96-well microplate. After mixing thoroughly, the wells were filled with modified SIM medium (too shallow a liquid surface will cause H2S to evaporate and dissipate easily). The plate was then placed in a 30°C incubator for static incubation, and the color change was observed every two hours.
[0044] Figure 4 The experimental results showed that after co-culturing 14 candidate probiotics with indicator bacteria XWS for 3-6 hours, 9 strains significantly inhibited H2S production. The liquid in the microplate was colorless, while the control group (containing only modified SIM medium and indicator bacteria XWS) was black, and the candidate probiotic group (which showed no effect) was also black. This indicates that these 9 uncolored strains can inhibit H2S production by XWS. These 9 strains were J19, HFJ-7, GBW-PM6, S2, YZB, LKJ, HSD, GBW-PM1, and GBW-PM2.
[0045] Example 2: Secondary screening of sulfur-controlling strains
[0046] At the bottom of aquaculture ponds, due to the water depth, the bottom sediment cannot directly contact the air, and when the sediment is thick, there are more oxygen-consuming factors such as organic matter in the bottom sediment. Therefore, the dissolved oxygen conditions at the bottom of the pond are generally poor. Thus, the selected sulfur-controlling strains need to have a good sulfur-controlling effect not only under aerobic conditions but also under microaerobic conditions with poor oxygen levels. Therefore, the test tube method was further used to verify the sulfur-controlling effect of the nine probiotic strains screened in Example 1 that have a sulfur-controlling effect under aerobic conditions under microaerobic conditions.
[0047] Nine candidate bacterial strains were inoculated into LB liquid medium at a 1% inoculum and cultured at 37°C and 200 rpm for 18 h on a shaker. XWS was inoculated into LB liquid medium at a 1% inoculum rate and cultured at 30°C and 200 rpm for 12 h. 5 mL of candidate bacterial culture was added to a test tube containing 10 mL of modified SIM liquid medium, and 150 μL of indicator strain XWS was added (for strictly anaerobic bacteria, a layer of vegetable oil was added to the surface of the liquid). The tubes were then incubated at 30°C for 3-6 h, with color changes observed every two hours.
[0048] Figure 5 Experimental results show that, under micro-oxygen conditions with a high liquid level in the test tube, J19, HFJ-7, GBW-PM6, and S2 have the function of inhibiting H2S production.
[0049] Example 3: Identification and Preservation of GBW-PM6
[0050] Using DNA from strain GBW-PM6 as a template, 16S rDNA was amplified using universal primers, and the amplified fragments were sequenced. Sequence alignment analysis of the 16S rDNA sequencing results showed that strain GBW-PM6 is similar to... Priestia megaterium The strain showed the highest homology, thus identifying it as *Priscilla megaterium*. The 16S rDNA phylogenetic tree of GBW-PM6 is shown below. Figure 6 As shown, its nucleotide sequence is as shown in SEQ ID No. 1, and the colony and cell morphology are as shown in... Figure 7 As shown.
[0051] The *Priestella giantiflora* GBW-PM6 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date was October 24, 2024. Priestia megaterium The accession number is: CGMCC No.32334.
[0052] Example 4: Verification of the mechanism of action of sulfur-controlling bacteria
[0053] Four sulfur-controlling functional bacteria, J19, HFJ-7, GBW-PM6, and S2, were inoculated into LB liquid medium at a 1% inoculum and cultured overnight (14–16 h) at 30°C and 200 rpm on a shaker. After centrifugation at 1000 rpm for 5 min, a supernatant containing metabolites but no bacterial cells was obtained. 5 mL of the fermentation broth or supernatant was added to a test tube containing 10 mL of LB liquid medium, and 1% (150 μL) of indicator bacteria XWS seed culture was inoculated. The tube was then incubated overnight at 30°C. Simultaneously, a blank control group (without XWS and functional bacteria), a negative control group (with XWS but without functional bacteria), and a control group (with XWS and common bacteria) were established. The H2S concentration in the fermentation broth was measured every 12 h using a sulfide detection kit. The results (Table 1) showed that the fermentation supernatants of strains J19, HFJ-7, GBW-PM6, and S2 reduced H2S production, with J19 showing the best results.
[0054] Table 1. Effect of supernatant from sulfur-controlling bacteria on H2S concentration produced by indicator bacteria XWS (mg / L)
[0055]
[0056] Furthermore, the bacterial count of XWS in the experimental groups was detected by spreading LB solid medium onto the supernatant of the candidate bacteria. The results showed (Table 2) that the supernatants of strains HFJ-7, GBW-PM6, and S2 significantly inhibited the proliferation of XWS, while J19 had no significant effect on the bacterial count of XWS.
[0057] Table 2. Effects of supernatants from different candidate bacteria on *Shewanella algae* (×10⁻⁶) 8 CFU / mL
[0058]
[0059] In summary, the mechanisms of action of the four sulfur-controlling bacteria screened are as follows: J19 inhibits H2S production by XWS through its metabolites, but does not affect the proliferation activity of XWS; HFJ-7, GBW-PM6 and S2 mainly control H2S production by inhibiting the proliferation of XWS.
[0060] Example 5: Adsorption test of clinoptilolite powder on H2S and Shewanella algae
[0061] XWS was inoculated into LB liquid medium at a 1% inoculum rate and cultured at 30℃ and 200 r / min for 12 h to obtain XWS seed culture for later use. 1% of the indicator strain XWS seed culture was inoculated into two LB liquid media, one containing 1% clinoptilolite powder (150 mesh) and the other not, and cultured at 30℃ and 200 r / min for 24 h. The XWS bacterial count was then detected by plating on solid LB medium.
[0062] The results showed that the number of XWS bacteria in the control group without clinoptilolite was (1.20±0.27)×10⁻⁶. 8 The CFU / mL count was [value missing], while the XWS count in the experimental group with 1% clinoptilolite powder added was (1.67±0.25)×10 [value missing]. 7 The CFU / mL level was reduced by 86.1% compared to the control group.
[0063] After culturing indicator bacteria XWS in test tubes using LB liquid medium, a 1:1 volume of water was added to a 50% test tube, and a 1:1 volume of 2% clinoptilolite powder solution was added to another 50% test tube. After standing for 1 hour, the supernatant was collected, and the H2S concentration was determined using a sulfide test kit. The results showed that the H2S content was significantly reduced in the group with clinoptilolite powder added. Figure 8 ).
[0064] In the above experiment, the addition of clinoptilolite powder reduced the number of indicator bacteria XWS and the H2S content, indicating that it is related to the adsorption characteristics of zeolite powder.
[0065] Example 6: Preparation of sulfur-controlling compound microecological agent and its effect on post-treatment of black mud.
[0066] The sulfur-controlling compound microecological agent powder contains 5.00×10 9 CFU / g Bacillus cereus J19, 1.50×10 10 CFU / g Bacillus amyloliquefaciens HFJ-7, 1.00×10 10 CFU / g of *Priscilla megaterium* GBW-PM6 and 2.00 × 10⁻⁶ 9 CFU / g Bacillus subtilis S2, 5% spore germination activator (60% L-alanine, 40% fructose), 30% zeolite powder, and 15% attapulgite were used, filled to 100% with red staghorn powder. Using granular attapulgite with a small particle size (0.5~1.0mm) as the original carrier, 10% water was added to the sulfur-controlling composite microecological agent powder packet by weight, and the mixture was gradually transferred into a granulator at 60 r / min to form particles with a diameter of 2~3mm. After polishing for 10 min, the particles were dried in a fluidized bed at 50~60℃ to a moisture content of 5~7%, yielding brick-red composite microecological agent particles. Figure 9 ).
[0067] On July 6, 2024, black and smelly bottom mud was collected from shrimp farming ponds in Rudong County after the shrimp harvest and drainage. Figure 10Experiment 1: A layer of black mud about 1 cm thick was laid at the bottom of a transparent wide-mouthed glass bottle. 1 g of compound microecological preparation granules was added, and tap water was gently poured in along the wall to make the water layer about 4 cm high. The bottle was then sealed with plastic wrap. After 3 days, the appearance and color of the mud were observed. The floating water at the mud-water interface was taken and its H2S content was measured using an H2S kit. At the same time, a control group without compound microecological preparation was set up.
[0068] The results showed that in the compound microecological preparation group, the surface of the black mud turned yellowish-brown, the floating water was clear, and the H2S content was <0.2 mg / L. In the control group, the black mud remained entirely black, the floating water had a thin mist, making it difficult to see the bottom through the water layer, and the H2S content was >0.5 mg / L. Figures 11-13 ).
[0069] Experiment 2: A 2-3 cm thick layer of black mud was placed at the bottom of a wide-mouthed, transparent glass bottle. On the first day, 1 g of compound microecological preparation granules was added, and tap water was gently poured in along the wall until the water level reached about 6-7 cm. The bottle was then sealed with plastic wrap and left to stand. Two days later, on the fourth day, another 1 g of compound microecological preparation granules was added, and the bottle was left to stand again. This process was repeated once more, for a total of three additions of the compound microecological preparation over 10 days. The results showed that the black mud turned yellowish-brown. Figure 14 ).
[0070] 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 strain of *Priestella megaterium* GBW-PM6, characterized in that, Its classification name is Priestella megaterium ( Priestia megaterium (), deposited at the China General Microbiological Culture Collection Center, with accession number: CGMCC No.32334.
2. A compound microecological preparation for improving the black bottom of aquaculture ponds, characterized in that, The compound microecological preparation contains the following components: a live bacteria content of not less than 1.00 × 10⁻⁶. 10 The *Priestella giantiformis* GBW-PM6 as described in claim 1, with a viable bacterial count of not less than 2.00 × 10⁻⁶ CFU / g. 9 CFU / g Bacillus subtilis, viable bacterial count not less than 5.00×10⁻⁶ 9 The CFU / g of Bacillus cereus and the viable bacterial count are not less than 1.50 × 10⁻⁶. 10 CFU / g of Bacillus amyloliquefaciens and a functional carrier; the Bacillus subtilis used was Bacillus subtilis S2 with accession number CGMCC No. 19824; the Bacillus cereus used was Bacillus cereus with accession number CGMCC No. 10052. Bacillus cereus J19; The Bacillus amyloliquefaciens used is the Bacillus amyloliquefaciens with the preservation number CGMCC No. 10011 ( Bacillus amyloliquefaciens HFJ-7; The functional carrier, by weight percentage of the compound microecological preparation, contains 5%~10% spore germination activator, 30%~40% zeolite powder and 15%~20% attapulgite, with the remainder being red staphylocide powder.
3. The compound microecological preparation for improving the black bottom of aquaculture ponds according to claim 2, characterized in that, The spore germination activator contains 60% L-alanine and 40% fructose, the zeolite powder is clinoptilolite powder with a particle size of not less than 150 mesh, and the attapulgite is extremely small granular attapulgite with a diameter of 0.5 to 1.0 mm.
4. The compound microecological preparation for improving the black bottom of aquaculture ponds according to claim 2, characterized in that, The compound microecological preparation is a round particle with a diameter of 2-3 mm. It is obtained by mixing the strain and the carrier, adding water, rolling the particles with a granulator, and then drying them in a fluidized bed at a low temperature of 50-60℃.
5. The application of the *Priestella gigantea* GBW-PM6 as described in claim 1 in improving the black bottom of aquaculture ponds.
6. The application of the compound microecological preparation according to claim 3 or 4 in improving the black bottom of aquaculture ponds, characterized in that, When the compound microecological preparation is applied to black bottom ponds, it should be applied once every 3 days in the early stage, with 200g~300g per mu of water surface. After one month, it should be applied once every 5 days, with 200g~300g per mu of water surface.
7. The application according to claim 6, characterized in that, The thickness of the black mud on the black bottom of the pond gradually decreased, and the amount of hydrogen sulfide floating in the water decreased significantly.