Bacterium agent for treating high-pollution and low-dissolved-oxygen shallow lake and application thereof

Abstract

The application belongs to the field of environmental microorganism technology, and discloses a bacterial agent suitable for high-pollution low-dissolved-oxygen shallow lake treatment and application. The bacterial agent comprises Pseudomonas putida NBNZ-3340 (preservation number CCTCC NO: M 2023405), Brevibacillus agri NBNZ-3730 (preservation number CCTCC NO: M 2023406) and Brevibacillus parabrevis NBNZ-3745 (preservation number CCTCC NO: M 2023407). The composite bacterial agent has the functions of low-temperature resistance, low-oxygen resistance, efficient removal of nitrogen in shallow lake and other environmental water bodies and sediment, promotion of growth of submerged plants and enhancement of stress resistance by comprehensively utilizing the different nitrogen treatment characteristics of the three strains, and can be used for purification treatment of shallow lake, aquaculture and other water bodies and sediment.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to bacterial agents and their applications suitable for treating shallow lakes with high pollution and low dissolved oxygen. Background Technology

[0002] Shallow lakes, with an average depth of less than 2 meters, have smaller volumes compared to deep lakes. This results in a larger sediment-water contact area, more frequent material exchange, and easier resuspension and release of nutrients and organisms from sediments due to external disturbances. Furthermore, they have a weaker capacity to dilute externally introduced nutrients and are more sensitive to human activities. Currently, most successful global cases of eutrophication lake remediation have occurred in deep lakes. Domestic efforts to remediate major large-scale shallow eutrophic lakes (such as Taihu Lake and Chaohu Lake) have not achieved the expected results.

[0003] Ammonia nitrogen is one of the main pollutants causing eutrophication in lakes. The accumulation of ammonia nitrogen often leads to the proliferation of algae, resulting in reduced dissolved oxygen and even mass mortality of aquatic organisms. The decomposition of these organisms further deteriorates water quality, ultimately creating a vicious cycle. Because shallow lakes are easily disturbed, have difficult-to-establish ecosystems, and recover slowly, the removal of pollutants such as ammonia nitrogen primarily relies on in-situ remediation. Building upon traditional chemical in-situ remediation methods, the core of current in-situ remediation technology is ecological restoration, which uses a combination of microbial agents and submerged plants to adsorb and decompose pollutants in river and lake water and sediment, thereby rapidly restoring the aquatic ecosystem.

[0004] Heterotrophic nitrifying-aerobic denitrifying bacteria can simultaneously remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen under oxygen conditions, possessing enormous commercial application potential. Currently, microbial remediation technologies commonly use various heterotrophic nitrifying-aerobic denitrifying bacterial preparations to remove pollutants such as ammonia nitrogen from industrial and domestic wastewater and sediment. However, compared to wastewater treatment processes where ammonia nitrogen concentrations are high and artificial aeration is possible, in in-situ remediation of lakes faces limitations. These limitations include slow water flow, low dissolved oxygen levels, low concentrations of nutrients (such as ammonia nitrogen) for bacterial growth, and the suitability of large-scale aeration equipment. Under non-continuous aeration conditions, heterotrophic nitrifying-aerobic denitrifying bacterial preparations often exhibit low growth activity, weak colonization and reproduction capabilities after application, failing to effectively remove nitrogen. Currently, there are no nitrogen-removing bacterial agents or applications specifically developed for the characteristics of shallow lake environments.

[0005] Meanwhile, the loose texture of the bottom sediment in shallow lakes makes it easy for submerged plants to resuspend during planting, hindering their rooting and survival. Currently used microbial remediation agents mostly focus on treating the bottom sediment and purifying the water, lacking targeted products or related application technologies for the survival and synergistic growth of submerged plants, thus failing to effectively form a virtuous cycle of joint treatment by microorganisms and submerged plants. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a microbial compound agent, which includes: *Pseudomonas alloputida* NBNZ-3340 (accession number CCTCC NO: M 2023405), *Brevibacterium frigoritolerans* NBNZ-3730 (accession number CCTCC NO: M 2023406), and *Peribacillus simplex* NBNZ-3745 (accession number CCTCC NO: M 2023407).

[0007] Another objective of this invention is to provide the application of the aforementioned microbial compound agent. This compound agent is specifically suitable for the restoration of shallow lakes under conditions of low temperature, low oxygen, and low nutrients, and simultaneously possesses denitrification and submerged plant growth promotion and stress resistance capabilities. To achieve the above objectives and other related objectives, the technical solution adopted by this invention is as follows:

[0008] The applicant selected three strains from the strain library. After mixing these strains to prepare a mixed bacterial agent, the agent can withstand low temperature, low oxygen, and low nutrient conditions, and simultaneously possesses the ability to denitrify and promote the growth of submerged plants and resist stress.

[0009] The three strains are as follows:

[0010] Pseudomonas alloputida NBNZ-3340, this strain was deposited at the China Center for Type Culture Collection on March 24, 2023, with the classification name: Pseudomonas sp.NBNZ-3340, accession number CCT CC NO: M2023405, address: Wuhan University, Wuhan, China.

[0011] The cold-resistant short bacillus *Brevibacterium frigoritolerans* NBNZ-3730 was deposited at the China Center for Type Culture Collection (CCTCC) on March 24, 2023. Its classification is *Brevibacterium sp.*NBNZ-3730*, accession number is CCTCC NO: M2023406, and its address is Wuhan University, Wuhan, China.

[0012] Peribacillus simplex NBNZ-3745, this strain was deposited at the China Center for Type Culture Collection on March 24, 2023, with the classification name: Peribacillus sp.NBNZ-3745, accession number CCTC C NO: M2023407, address: Wuhan University, Wuhan, China.

[0013] A microbial compound agent, comprising: *Pseudomonas salloputida* NBNZ-3340 (CCTCC NO: M 2023405), *Brevibacterium frigoritolerans* NBNZ-3730 (CCTCC NO: M 2023406), and *Peribacillus simplex* NBNZ-3745 (CCTCC NO: M 2023407), wherein the effective bacterial content ratio of the agent is 1:0.01–10:0.01–10.

[0014] Preferably, the compound microbial agent described above contains an effective bacteria content greater than or equal to 2 × 10⁻⁶. 8 cfu / g;

[0015] The above-mentioned microbial compound inoculants are used in the treatment of sewage in shallow lakes.

[0016] The above-mentioned microbial compound inoculants are used to promote the growth and stress resistance of submerged plants in shallow lakes.

[0017] The preferred application described above is that the compound microbial agent is directly sprayed into the lake without aeration.

[0018] In the above-described applications, preferably, the shallow lake has a water temperature of 10–40℃, a water depth of 0.5–2 meters, and a dissolved oxygen range of 0–10 mg / L in the bottom sediment and surface water.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1) This invention utilizes *Pseudomonas aeruginosa* NBNZ-3340 to achieve a >95% removal rate of ammonia nitrogen in low-temperature and normal-temperature lake waters under non-aerated low-oxygen conditions.

[0021] 2) This invention utilizes cold-resistant short bacillus NBNZ-3730 and simple spore-forming bacillus NBNZ-3745, achieving a nitrate nitrogen removal rate of >50% in both low-temperature and normal-temperature lake waters under non-aerated low-oxygen conditions.

[0022] 3) This invention uses a compound bacterial agent of *Pseudomonas aeruginosa* NBNZ-3340, *Bacillus thuringiensis* NBNZ-3730, and *Bacillus simplex* NBNZ-3745. Under non-aerated low-oxygen conditions, the removal rate of ammonia nitrogen in lake water at low and normal temperatures is >80%, and the removal rate of nitrate nitrogen is >60%.

[0023] 4) The present invention uses the above-mentioned microbial compound agent to achieve a removal rate of >48% of ammonia nitrogen in highly polluted sediment of urban shallow lakes under non-aerated low-oxygen conditions.

[0024] 5) The present invention applies the above-mentioned microbial compound inoculant, which increases the growth of Vallisneria natans by >150% under non-aerated low-oxygen conditions, and increases the survival rate and stress resistance of Vallisneria natans by >200% in highly polluted sediment.

[0025] 6) This invention utilizes single strains and complex bacterial communities to efficiently degrade ammonia nitrogen pollution in shallow lake water and sediment under low oxygen conditions, and effectively promotes the growth and stress resistance of submerged plants, thus having high application value. Attached Figure Description

[0026] Figure 1 Morphological results of *Pseudomonas aeruginosa* NBNZ-3340 on LB medium, according to an embodiment of the present invention.

[0027] Figure 2 The morphological results of the cold-resistant short bacillus NBNZ-3730 provided according to an embodiment of the present invention on LB medium.

[0028] Figure 3 Morphological results of *Bacillus subtilis* NBNZ-3745 on LB medium according to an embodiment of the present invention. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited to the scope of the embodiments. Variations that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention are all within the scope of protection of the present invention.

[0030] Example 1:

[0031] Screening and functional evaluation of low-temperature resistant denitrifying bacteria

[0032] The culture medium and its components are as follows:

[0033] LB liquid medium for strain activation: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, pH 7.0–7.5.

[0034] LB solid medium for strain activation: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 15 g / L agar powder, pH 7.0–7.5.

[0035] The strains were screened using TD liquid medium: 4.78 g / L disodium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, 3.416 g / L sodium acetate, 0.191 g / L ammonium chloride, 0.361 g / L potassium nitrate, 0.05 g / L ethylenediaminetetraacetic acid, 5 mg / L calcium chloride, 2.2 mg / L zinc sulfate, 5.06 mg / L manganese chloride, 5 mg / L ferrous sulfate, 1.1 mg / L ammonium molybdate, 1.57 mg / L copper sulfate, 1.61 mg / L cobalt chloride, pH 7.4.

[0036] The strains were screened using TD solid medium: 4.78 g / L disodium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, 3.416 g / L sodium acetate, 0.191 g / L ammonium chloride, 0.361 g / L potassium nitrate, 0.05 g / L ethylenediaminetetraacetic acid, 5 mg / L calcium chloride, 2.2 mg / L zinc sulfate, 5.06 mg / L manganese chloride, 5 mg / L ferrous sulfate, 1.1 mg / L ammonium molybdate, 1.57 mg / L copper sulfate, 1.61 mg / L cobalt chloride, 15 g / L agar powder, pH 7.4.

[0037] After the above culture media are prepared, they are all autoclaved at 121℃ for 30 minutes before use.

[0038] Initial screening and evaluation: Strains from the resource bank of Hubei Provincial Engineering Research Center for Biological Pesticides were inoculated onto LB agar plates using the streak method and incubated overnight at 30°C. Single colonies were then transferred to TD agar plates for screening using the spot inoculation method and incubated overnight at 30°C. This process was repeated 5 times. The fastest-growing strains were selected, streak-purified, and then preserved. A total of 11 strains were obtained after initial screening.

[0039] Denitrification performance verification: The functional strains obtained from the initial screening were inoculated into LB liquid medium and activated overnight at 30℃ and 220 rpm with shaking. After washing the activated bacterial solution with sterile physiological saline, 1% was inoculated into TD liquid medium and allowed to stand for 2-14 days at room temperature and 10℃ (the specific time depends on the bacterial growth status). The control group was inoculated with the same volume of sterile physiological saline. After the reaction, the ammonia nitrogen and nitrate nitrogen contents in the blank TD medium and the bacterial supernatant were determined according to the methods described in GB7479-87 and HJ / T346-2007, and the removal rate was calculated. Combined with the absorbance of the bacterial solution at a wavelength of 600 nm (considered as biomass), the denitrification performance of each strain was comprehensively evaluated. As shown in Table 1, strains NBNZ-3340, NBNZ-3730, and NBNZ-3745 showed faster growth rates and better denitrification performance under various conditions.

[0040] Table 1. Denitrification data of functional strains.

[0041]

[0042]

[0043] Example 2:

[0044] Biological identification of denitrifying strains

[0045] The above-mentioned strains were identified using 16S rDNA sequence alignment. Genomic DNA was extracted from the strains using existing techniques, and this DNA was used as a template to amplify the strain's 16S rDNA using a pair of universal primers (27F, 1492R). The upstream primer was 27F (5'-AGAGTTTGATCCTGGCTCA-3'), and the downstream primer was 1492R (5'-GGTTACCTTGTTACGACTT-3'). The 16S rRNA coding gene sequence was obtained after PCR amplification and sequencing. The sequenced 16S rRNA gene sequence was then compared and analyzed using the EzBioCloud Database (https: / / www.ezbiocloud.net / ) for bacterial identification.

[0046] Morphological observation showed that strain 3340 was a Gram-negative bacterium, such as Figure 1The colony morphology shown is round, with neat edges, a smooth and moist surface, and a raised center. Based on 16S rRNA gene sequence typing, this strain was identified as *Pseudomonas alloputida*. It was ultimately named *Pseudomonas alloputida* NBNZ-3340, and was deposited at the China Center for Type Culture Collection (CCTCC) on March 24, 2023, with the classification name *Pseudomonas sp. NBNZ-3340*, accession number CCTCC NO: M2023405, address: Wuhan University, Wuhan, China.

[0047] Strain 3730 is a Gram-positive bacterium, such as Figure 2 The colony morphology shown is round with rough edges and surface. Based on 16S rRNA gene sequence typing, this strain was identified as *Brevibacterium frigoritolerans*. It was ultimately named *Brevibacterium frigoritolerans* NBNZ-3730, and was deposited at the China Center for Type Culture Collection (CCTCC) on March 24, 2023. Its classification name is *Brevibacterium sp.* NBNZ-3730*, accession number is CCTCC NO: M2023406, and its address is Wuhan University, Wuhan, China.

[0048] Strain 3745 is a Gram-positive bacterium, such as Figure 3 The colonies shown are round, with neat edges, and a smooth, moist, and plump surface. Based on 16S rRNA gene sequence typing, this strain is identified as *Peribacillus simplex*. It is ultimately named *Peribacillus simplex* NBNZ-3745. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on March 24, 2023, with the classification name: *Peribacillus sp.* NBNZ-3745, accession number: CCTCC NO: M2023407, address: Wuhan University, Wuhan, China.

[0049] Example 3:

[0050] Application of denitrifying strains in the removal of ammonia nitrogen and nitrate nitrogen pollutants in water bodies

[0051] After activating and culturing *Pseudomonas aeruginosa* NBNZ-3340, *Brugia cerevisiae* NBNZ-3730, and *Bacillus simplex* NBNZ-3745 in LB medium, the bacteria were collected by centrifugation and prepared into cultures with a bacterial count of 1×10⁻⁶. 8 Liquid bacterial agent at cfu / mL.

[0052] The aquaculture water used in the experiment was collected from the surface water of the company's own fishpond. After water quality testing, the concentrations of ammonia nitrogen and nitrate nitrogen in the water were adjusted to 25 mg / L, and the pH was set to 7.0.

[0053] The lake water used in the experiment was collected from a shallow lake in Wuhan, Hubei Province. After water quality testing, the concentrations of ammonia nitrogen and nitrate nitrogen in the water were adjusted to 10 mg / L, and the pH was set to 7.0.

[0054] Take 2L of aquaculture or lake water and place it in an Erlenmeyer flask. Add 1mL of bacterial solution (experimental group) or blank culture medium (control group) to each flask. Perform three treatments: aquaculture water is left to stand at room temperature of 25℃ for 3 days, lake water is left to stand at room temperature of 25℃ for 7 days, and lake water is left to stand at 10℃ for 14 days. Then, test the ammonia nitrogen and nitrate nitrogen content of the water according to the methods described in national standards GB7479-87 and HJ / T346-2007.

[0055] The results showed that, under non-aeration conditions, compared with the blank control group, the corrected removal rate of ammonia nitrogen by *Pseudomonas allogeneia* NBNZ-3340 in the aquaculture water treatment was 99.9%, but the corrected removal rate of nitrate nitrogen was 29.2%.

[0056] The corrected removal rate of nitrate nitrogen by the cold-resistant short bacillus NBNZ-3730 was 86.4%, but the corrected removal rate of ammonia nitrogen was 104.2% (meaning that ammonia nitrogen was basically not removed, or may have increased slightly).

[0057] The corrected removal rate of nitrate nitrogen by Bacillus simplex NBNZ-3745 was 83.5%, but the corrected removal rate of ammonia nitrogen was 99.6% (meaning that ammonia nitrogen was basically not removed).

[0058] The results showed that, under non-aeration conditions, compared with the blank control group, the corrected removal rate of ammonia nitrogen by *Pseudomonas allogeneia* NBNZ-3340 in the room temperature treatment of lake water was 96.5%, but the corrected removal rate of nitrate nitrogen was 25.7%.

[0059] The cold-resistant short bacillus NBNZ-3730 had a corrected removal rate of 75.1% for nitrate nitrogen, but a corrected removal rate of 100.7% for ammonia nitrogen (meaning that ammonia nitrogen was basically not removed).

[0060] The corrected removal rate of nitrate nitrogen by Bacillus simplex NBNZ-3745 was 67.1%, but the corrected removal rate of ammonia nitrogen was 97.6% (meaning that ammonia nitrogen was basically not removed).

[0061] The results showed that, under non-aeration conditions, compared with the blank control group, the corrected removal rate of ammonia nitrogen by *Pseudomonas allogeneia* NBNZ-3340 in low-temperature treatment of lake water was 95.0%, but the corrected removal rate of nitrate nitrogen was 14.9%.

[0062] The corrected removal rate of nitrate nitrogen by the cold-resistant short bacillus NBNZ-3730 was 50.5%, but the corrected removal rate of ammonia nitrogen was 110.4% (meaning that ammonia nitrogen was basically not removed, or may have increased slightly).

[0063] The corrected removal rate of nitrate nitrogen by Bacillus simplex NBNZ-3745 was 57.0%, but the corrected removal rate of ammonia nitrogen was 106.5% (meaning that ammonia nitrogen was basically not removed, or may have increased slightly).

[0064] Example 4:

[0065] Preparation of compound microbial agents

[0066] Preparation of fermentation seed culture for the three bacterial strains: Single colonies of *Pseudomonas aeruginosa* NBNZ-3340, *Brugia cerevisiae* NBNZ-3730, and *Bacillus simplex* NBNZ-3745 were picked and inoculated into 5 mL of LB liquid medium, respectively, and cultured overnight at 30°C with shaking at 220 rpm for activation. Fresh activated bacterial cultures were then inoculated at 1% onto LB medium and cultured at 30°C with shaking at 220 rpm for 24 hours to obtain the seed culture for each of the three bacterial strains.

[0067] Fermentation culture of three strains: LB medium was used in the fermenter, filling 50% of the total volume. Seed culture was inoculated into the fermentation medium at a rate of 3% (volume percentage). The fermentation temperature was 30℃, dissolved oxygen was 10%, and the stirring speed was 200 r / min for 48 hours. After fermentation, samples were taken for microscopic examination, and the bacterial count was calculated using the dilution-spreading method. The bacterial count of the fermentation broth was higher than 2 × 10⁻⁶. 8 cfu / mL.

[0068] Preparation of the compound microbial agent: The liquid compound microbial agent is prepared by mixing the fermentation broths of the above three strains at a bacterial count ratio of 1:0.01-10:0.01-10, and adding preservatives such as 0.5% potassium sorbate. The total bacterial count is greater than 2×10⁻⁶. 8 CFU / mL. This compound bacterial agent liquid product is a yellowish-brown suspension, odorless, with a starchy aroma, and should be stored at 4°C. It has a shelf life of one year.

[0069] The compound bacterial powder is prepared by spray-drying (using soluble starch as the spray carrier) or vacuum freeze-drying the fermentation broth of the above three bacterial strains to obtain bacterial powder, then mixing them at a bacterial content ratio of 1:0.01-10:0.01-10, and adding excipients such as diatomaceous earth, bentonite, and kaolin. The total bacterial content is greater than 2×10⁻⁶. 8 CFU / g. This compound microbial agent powder is a pale yellow powder, odorless, and should be stored in a cool, dry place. It has a shelf life of one year.

[0070] Example 5:

[0071] Application of compound bacterial agents in the removal of ammonia nitrogen and nitrate nitrogen pollutants in water bodies

[0072] The aquaculture water used in the experiment was collected from the surface water of the company's own fishponds. After water quality testing, the concentrations of ammonia nitrogen and nitrate nitrogen in the water were adjusted to 25 mg / L, and the pH was set to 7.0.

[0073] The lake water used in the experiment was collected from a shallow lake in Wuhan, Hubei Province. After water quality testing, the concentrations of ammonia nitrogen and nitrate nitrogen in the water were adjusted to 10 mg / L, and the pH was set to 7.0.

[0074] As described in Example 4, four compound bacterial powders with different ratios were prepared by using effective bacterial contents of *Pseudomonas aeruginosa* NBNZ-3340: *Brevibacterium frostbite* NBNZ-3730: *Bacillus simplex* NBNZ-3745 in the ratios of 1:1:1, 1:0.01:0.01, 1:0.01:10, or 1:10:0.01. These experimental groups were named bacterial powders A, B, C, and D.

[0075] Take 10L of aquaculture or lake water and place it in an plexiglass container. Add 1g of compound bacterial powder in different proportions to each container (experimental group) or perform a blank treatment (control group). After the aquaculture water is left to stand at room temperature of 25℃ for 3 days, the lake water is left to stand at room temperature of 25℃ for 7 days, and the lake water is left to stand at 10℃ for 14 days. Then, test the ammonia nitrogen and nitrate nitrogen content of the water according to the methods described in national standards GB7479-87 and HJ / T346-2007.

[0076] As shown in Table 2, under non-aeration conditions, compared to the result in Example 3 where a single strain could only remove ammonia nitrogen or nitrate nitrogen, the microbial compound agent containing three strains with an effective bacterial content ratio in the range of 1:0.01 to 10:0.01 to 10 all showed significant denitrification effects on water bodies, simultaneously removing both ammonia nitrogen and nitrate nitrogen. Among them, the increased content of *Pseudomonas aeruginosa* NBNZ-3340 helped remove ammonia nitrogen pollutants, while the increased content of *Bacillus thuringiensis* NBNZ-3730 and / or *Bacillus simplex* NBNZ-3745 was beneficial for the removal of nitrate nitrogen pollutants. Different proportions of the agent can be prepared and applied according to different pollution environments.

[0077] The compound microbial agent achieved a corrected removal rate of 87.3% for ammonia nitrogen and 79.1% for nitrate nitrogen in aquaculture water.

[0078] The corrected removal rate for ammonia nitrogen in lake water reached 85.1%, and the corrected removal rate for nitrate nitrogen reached 72.5%.

[0079] In lake water at low temperature (10℃), the corrected removal rate for ammonia nitrogen reached 80.3%, and the corrected removal rate for nitrate nitrogen reached 62.4%.

[0080] Table 2. Denitrification data of compound bacterial agents in different water bodies.

[0081]

[0082] Example 6:

[0083] Application of compound microbial agents in the removal of ammonia nitrogen pollutants from highly polluted sediment under laboratory conditions

[0084] As described in Example 4, four different ratios of compound bacterial powder were prepared by mixing *Pseudomonas aeruginosa* NBNZ-3340, *Brugia cerevisiae* NBNZ-3730, and *Bacillus simplex* NBNZ-3745 in a ratio of 1:1:1, 1:0.01:0.01, 1:0.01:10, or 1:10:0.01. These experimental groups were named bacterial powders A, B, C, and D.

[0085] The overlying water and bottom sediment used in the experiment were collected from a shallow lake in Wuhan, Hubei Province. The bottom sediment was mainly composed of grayish-black silt rich in organic matter and nutrients, formed by pollutants from domestic and industrial sources deposited at the bottom of the riverbed through physical, chemical, and biological processes. The total nitrogen content of the sediment was 2934.8 mg / kg, the ammonia nitrogen content was 162.2 mg / kg, and the water content was 46.0%, classifying it as highly polluted sediment. The survival rate of submerged plants was less than 20%. Based on previous experimental verification, under aeration conditions, commercially available bottom-improving microbial agents containing Bacillus and yeast can be used for the remediation of this type of sediment; without aeration, commercially available bottom-improving microbial agents are basically unable to perform normal remediation functions.

[0086] Experiment 1: Both the experimental group and the blank control group used acrylic glass buckets with a diameter of 15cm and a height of 50cm. A 10cm high layer of bottom mud was laid on top, and a water depth of 30cm was covered. The buckets were placed in a greenhouse with a light condition of 14h:10h cycle and an indoor temperature of 25℃. No aeration equipment was used during the experiment.

[0087] Experiment 2: Following the setup of Experiment 1, a 10cm high layer of bottom mud was laid in the bucket, with only a small amount of water on top. The bottom mud was monitored daily to keep it moist, while other conditions remained the same.

[0088] In Experiment 1 and Experiment 2 above, the experimental groups were given bacterial powder A four times, once every 7 days. Each time, 0.1g of bacterial agent was dissolved and mixed in a small amount of top water and added to the tank.

[0089] The blank control group in Experiment 1 and Experiment 2 above was given 0.1g of sterile excipient powder four times, once every 7 days.

[0090] The experiment did not require aeration equipment. After 60 days of operation, sediment from each group was collected, and the ammonia nitrogen content of the sediment was tested in accordance with the national standard HJ / 634-2012.

[0091] Experimental results show that, under non-aeration conditions, when the microbial compound agent was added to the highly polluted sediment of shallow urban lakes and treated for 60 days, in Experiment 1 with overlying water, the corrected removal rate of ammonia nitrogen in the sediment by compound agent A reached 48.8% compared to the blank control group; in Experiment 2 without overlying water, the corrected removal rate of ammonia nitrogen in the sediment by compound agent A reached 83.4% compared to the blank control group.

[0092] The above denitrification data are corrected data based on the control group.

[0093] Example 7:

[0094] Application of compound microbial agents in the removal of ammonia nitrogen pollutants from sediments in urban shallow lakes

[0095] As described in Example 4, four different ratios of compound bacterial powder were prepared by mixing *Pseudomonas aeruginosa* NBNZ-3340, *Brugia cerevisiae* NBNZ-3730, and *Bacillus simplex* NBNZ-3745 in a ratio of 1:1:1, 1:0.01:0.01, 1:0.01:10, or 1:10:0.01. These experimental groups were named bacterial powders A, B, C, and D.

[0096] The sediment remediation experimental area was located in a shallow lake in Wuhan, Hubei Province. The lake water depth in the experimental area was 50–80 cm, and the dissolved oxygen in the surface water of the sediment ranged from 0–10 mg / L. The area of ​​the bacterial agent treatment group and the blank control group was 50 m². 2 Total area 100m 2 The entire enclosure framework was fixed in the water using steel pipes. The enclosure fabric was made of PVC, and gabions were used at the bottom edge to prevent water exchange between the inside and outside of the enclosure. Before the experiment, the existing aquatic plants within the enclosure area were removed, and a layer of highly polluted sediment collected from near the lake's sewage outlet was laid on top of the existing sediment, totaling approximately 6m². 3Highly polluted sediment. This sediment is highly polluted, with a survival rate of less than 20% for submerged plants. Based on previous experiments, commercially available sediment-improving microbial agents containing Bacillus and yeast can be used for the remediation of this type of sediment under aeration conditions; however, without aeration, these agents are largely ineffective in remediation.

[0097] The experiment was conducted from May to July 2023: the average high / low temperature in May was 26 / 18℃, with 17 rainy days; the average high / low temperature in June was 29 / 22℃, with 14 rainy days; and the average high / low temperature in July was 33 / 26℃, with 16 rainy days.

[0098] In the experimental group, bacterial powder A was added four times, once every seven days, at a dose of 10 g / m² each time. 2 The dosage was diluted with a small amount of water before addition. In the control group area, sterile excipient powder was added four times, once every 7 days, at a dose of 10g / m², following the same method and dosage as the experimental group. 2 No aeration equipment was used during the experiment. Sediment samples were collected from each treatment group every 30 days before and after the experiment and stored at -20℃. After 90 days of operation, a total of four batches of sediment samples were collected, and the ammonia nitrogen content of the sediment was tested according to the national standard HJ / 634-2012.

[0099] Experimental results show that when the microbial compound agent A is added to the highly polluted sediment of shallow urban lakes with a water depth of 50-80 cm and treated for 90 days under non-aeration conditions, the corrected removal rate of ammonia nitrogen in the sediment reaches 38.5% compared with the blank control group.

[0100] Example 8:

[0101] Application of compound microbial agents in promoting the growth of submerged plants

[0102] As described in Example 4, four different ratios of compound bacterial powder were prepared by mixing *Pseudomonas aeruginosa* NBNZ-3340, *Brugia cerevisiae* NBNZ-3730, and *Bacillus simplex* NBNZ-3745 in a ratio of 1:1:1, 1:0.01:0.01, 1:0.01:10, or 1:10:0.01. These experimental groups were named bacterial powders A, B, C, and D.

[0103] The water used in the experiment was collected from a shallow lake in Wuhan, Hubei Province. The substrate used for planting *Vallisneria natans* was commercially available aquatic plant substrate. 50L plastic storage boxes were used, each filled with 10cm of aquatic plant substrate and covered with 30cm of water. The boxes were placed in a greenhouse with a 14h:10h light cycle and an indoor temperature of 25℃. Ten *Vallisneria natans* seedlings were planted in each box. The initial seedling height and root length were trimmed to 5cm, and the fresh weight was kept relatively uniform before planting.

[0104] The experimental group received bacterial powder A four times, once every seven days, with 0.1g dissolved and mixed in a small amount of water each time. The blank control group received sterile excipient powder four times, once every seven days, at the same dosage as the experimental group. No aeration equipment was required for the experiment. After 30 days, the Vallisneria natans from each group were collected, and the plant height, root length, number of leaves, and number of tillers were measured and recorded.

[0105] Experimental results showed that, when the microbial compound agent A was used to treat Vallisneria natans under non-aeration conditions for 30 days, the relative tillering growth rate reached 177.6% and the relative fresh weight growth rate reached 198.6% compared with the blank control group.

[0106] The data are relative ratios corrected based on the blank group data.

[0107] Example 9:

[0108] Application of compound microbial agents in enhancing the stress resistance of submerged plants

[0109] As described in Example 4, four different ratios of compound bacterial powder were prepared by mixing *Pseudomonas aeruginosa* NBNZ-3340, *Brugia cerevisiae* NBNZ-3730, and *Bacillus simplex* NBNZ-3745 in a ratio of 1:1:1, 1:0.01:0.01, 1:0.01:10, or 1:10:0.01. These experimental groups were named bacterial powders A, B, C, and D.

[0110] The overlying water and bottom sediment used in the experiment were collected from a shallow lake in Wuhan, Hubei Province. The bottom sediment was mainly composed of grayish-black silt rich in organic matter and nutrients, formed by pollutants from domestic and industrial sources deposited at the bottom of the riverbed through physical, chemical, and biological processes. The total nitrogen content of the sediment was 2911.9 mg / kg, the ammonia nitrogen content was 150.9 mg / kg, and the water content was 45.2%, classifying it as highly polluted sediment. The survival rate of submerged plants was less than 20%. Based on previous experimental verification, under aeration conditions, commercially available bottom-improving microbial agents containing Bacillus and yeast can be used for the remediation of this type of sediment; without aeration, commercially available bottom-improving microbial agents are basically unable to perform normal remediation functions.

[0111] The experiment used 50L plastic storage boxes, lined with 10cm deep, heavily polluted bottom mud, and covered with 30cm of water. The boxes were placed in a greenhouse with a 14h:10h light cycle and an indoor temperature of 25℃. Each storage box contained 10 *Vallisneria natans* seedlings. The initial seedling height and root length were trimmed to 5cm, and the fresh weight was kept relatively uniform before planting.

[0112] The experimental group received bacterial powder A four times, once every seven days, with 0.1g dissolved and mixed in a small amount of water each time. The blank control group received sterile excipient powder four times, once every seven days, at the same dosage as the experimental group. No aeration equipment was required for the experiment. After 30 days, Vallisneria natans were collected from each group, and plant height, root length, number of leaves, number of tillers, and other indicators such as the activity of enzymes related to plant growth promotion and stress resistance were measured and recorded using a kit.

[0113] Experimental results showed that when Vallisneria natans was planted in highly polluted sediment (total nitrogen > 2000 mg / kg) of shallow urban lakes, and treated with the aforementioned compound microbial agent A for 30 days under non-aeration conditions, compared with the blank control group, the survival rate of Vallisneria natans increased by 225.2%, the relative tillering rate increased by 225.0%, the relative fresh weight rate increased by 172.6%, the average unit protein superoxide dismutase (SOD) activity increased by 351.1%, the average unit tissue fresh weight SOD activity increased by 291.3%, the average unit protein catalase (CAT) activity increased by 525.1%, and the average unit tissue fresh weight CAT activity increased by 460.6%.

[0114] The above data are relative ratios corrected based on the blank group data.

Claims

1. A microbial compound agent, wherein the microbial compound agent comprises: The compound microbial agent contains Pseudomonas salloputida NBNZ-3340 (accession number CCTCC NO: M 2023405), Brevibacterium frigoritolerans NBNZ-3730 (accession number CCTCC NO: M 2023406), and Peribacillus simplex NBNZ-3745 (accession number CCTCC NO: M 2023407). The ratio of effective bacteria content in the compound microbial agent is 1:0.01~10:0.01~10.

2. The compound microbial agent according to claim 1, wherein the effective bacteria content in the compound microbial agent is greater than or equal to 2 × 10⁻⁶. 8 cfu / g.

3. The application of the microbial compound agent according to claim 1 in the treatment of sewage in shallow lakes.

4. The application of the microbial compound inoculant according to claim 1 in promoting the growth and stress resistance of submerged plants in shallow lakes.

5. In the application according to claim 3 or 4, the compound microbial agent is used by directly sprinkling it into the lake without aeration.

6. The application according to claim 3 or 4, wherein the water temperature of the shallow lake is 10~40℃, the water depth is 0.5~2 meters, and the dissolved oxygen in the bottom sediment and surface water ranges from 0~10 mg / L.

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