Composite strain liquid preparation for improving water quality and preparation method thereof

Abstract

The invention relates to the technical field of environmental microbial preparations and water body ecological regulation and control. The invention provides a composite strain liquid preparation for improving water quality and a preparation method of the composite strain liquid preparation. The liquid preparation comprises composite strain liquid and a protective buffer agent, the compound bacteria liquid comprises four lactic acid bacteria. The total viable bacteria is greater than or equal to 1 * 10 < 10 > CFU / mL through single-plant anaerobic fermentation, low-temperature concentration and isotonic protection, and can be stabilized for 6 months at normal temperature. Practical application shows that the preparation can reduce the pH value of the algae bloom water body from gt in a short time; meanwhile, the growth of blue-green algae and green algae is remarkably inhibited, the toxicity of ammonia nitrogen is reduced, and the method has the advantages of being green, safe, low in cost and the like, and is suitable for water quality improvement and algae bloom treatment of lakes, reservoirs, landscape water bodies and aquaculture systems.

Description

Technical Field

[0001] This invention relates to the field of environmental microbial preparations and aquatic ecological regulation technology, and in particular to a compound microbial liquid preparation for improving water quality and its preparation method. Background Technology

[0002] Eutrophication and high temperatures can easily lead to algal blooms in water bodies, with excessive photosynthesis causing free carbon dioxide to accumulate. Excessive consumption can cause the pH to rise rapidly to above 9.0, leading to increased ammonia nitrogen toxicity, metal ion leaching, and stress on aquatic organisms. Current methods for controlling algal blooms and pH largely rely on chemical flocculation or the addition of acidifiers, which suffer from secondary pollution and limited buffering capacity. Beneficial microorganisms, such as lactic acid bacteria, can produce organic acids through metabolism, compete for nutrients, and inhibit pathogenic algae, providing a green and sustainable solution for water remediation. However, existing lactic acid bacteria preparations are mostly in single-strain or solid form, resulting in poor activity and ease of handling. Summary of the Invention

[0003] The purpose of this invention is to provide a compound bacterial liquid preparation for improving water quality and its preparation method. The preparation method has a precise strain ratio, is ready to use in liquid form, has strong buffering capacity, and can effectively control the increase in water pH caused by algal blooms.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a compound bacterial liquid preparation for improving water quality, the liquid preparation comprising a compound bacterial solution and a protective buffer, wherein the compound bacterial solution comprises *Lactobacillus pentophilus* (…). Secundilactobacillus pentosiphilus Lactobacillus bruneri ( Lentilactobacillus buchneri ), Pediococcus pentosaceus ( Pediococcus pentosaceus ) and acid-resistant lactobacillus ( Lactobacillus acetotolerans The ratio of viable counts of *Lactobacillus pentosophilus*, *Lactobacillus bruneri*, *Pediococcus pentosophilus*, and acid-fast *Lactobacillus* is 37-41:21-25:21-25:13-17; the total viable count in the compound bacterial solution is ≥1×10⁻⁶. 10 CFU / mL.

[0005] Preferably, the protective buffer comprises glycerol, trehalose, sodium alginate, and calcium lactate; the amount of glycerol added is 2-5% of the total mass of the compound bacterial solution, the amount of trehalose added is 1-3% of the total mass of the compound bacterial solution, the amount of sodium alginate added is 0.5-1% of the total mass of the compound bacterial solution, and the amount of calcium lactate added is 0.1-0.3% of the total mass of the compound bacterial solution.

[0006] The present invention also provides a method for preparing the compound microbial strain liquid formulation, comprising the following steps: (1) Fermentation of Lactobacillus pentosophilus, Lactobacillus brunelli, Pediococcus pentosophilus and Lactobacillus acid-fast bacteria were carried out separately to obtain fermentation broths of different strains; (2) Concentrate the fermentation broth of different strains separately, adjust the pH value, let stand, and then mix the fermentation broth of different strains according to the ratio of live bacteria to obtain a compound bacterial solution. (3) Add a protective buffer to the compound bacterial solution, stir and filter to remove flocculent impurities and obtain a liquid preparation.

[0007] Preferably, the fermentation method described in step (1) is anaerobic or microanaerobic fermentation.

[0008] Preferably, the fermentation temperature in step (1) is 30~32℃ and the pH value is 5.8~6.2.

[0009] Preferably, the concentration temperature in step (2) is 2~6℃, the concentration is carried out by tangential filtration of ceramic membrane, the concentration factor is 8~10 times, and isotonic washing is performed after concentration.

[0010] Preferably, the pH value adjusted in step (2) is 4.0~4.5, the settling temperature is 2~6℃, and the settling time is 1.5~2.5h.

[0011] Preferably, the stirring time in step (3) is 10~20 min; and the filter screen used for filtration has a pore size of 0.45 mm.

[0012] This invention also provides the application of the compound microbial liquid preparation in water quality improvement and algal bloom control.

[0013] The present invention also provides a method for using the compound microbial liquid preparation, comprising the following steps: applying the compound microbial liquid preparation into a body of water to achieve a final viable count of 5 × 10⁻⁶. 5 ~1×10 6 CFU / mL; repeat every 7-10 days during peak algal bloom periods.

[0014] Beneficial effects

[0015] The product of this invention can achieve rapid pH buffering: within 4 to 6 hours after addition, the pH of the algal bloom water body is reduced from 9.2±0.1 to 8.2±0.2, and stabilized at 7.8–8.3 within 48 hours.

[0016] Algae suppression and ammonia nitrogen reduction: Organic acids and bacteriocins inhibit the growth of cyanobacteria (Microcystis spp.) and green algae (Chlorella spp.) by 30-60%; lower pH reduces nitrogen levels. –N content decreased by >80%.

[0017] Storage resistance: ≥80% viable bacteria retention rate at room temperature (25°C) for 6 months, and ≥90% viable bacteria retention rate at 4°C for 12 months.

[0018] Green and safe: All strains belong to BSL-1, and their metabolites do not cause secondary pollution, making them suitable for drinking water sources, fishery waters, and urban landscape waters. Attached Figure Description

[0019] Figure 1 Using data from typical eutrophic tributaries as a reference, a trend curve (double Y-axis) is presented showing that the pH decreased from 9.1 to 7.8 and the total algae density decreased to 40% within 72 hours after addition, illustrating the water quality buffering and algae suppression effects of this preparation. Detailed Implementation

[0020] This invention provides a compound bacterial liquid preparation for improving water quality. The liquid preparation comprises a compound bacterial solution and a protective buffer. The compound bacterial solution includes *Lactobacillus pentosophilus*, *Lactobacillus bruneri*, *Pediococcus pentosophilus*, and acid-fast lactobacillus. The viable count ratio of *Lactobacillus pentosophilus*, *Lactobacillus bruneri*, *Pediococcus pentosophilus*, and acid-fast lactobacillus is 37-41:21-25:21-25:13-17. The total viable count in the compound bacterial solution is ≥1×10⁻⁶. 10 CFU / mL.

[0021] In this invention, the preferred ratio of viable counts of Lactobacillus pentosophilus, Lactobacillus brunelli, Pediococcus pentosophilus, and acid-fast Lactobacillus is 39:23:23:15.

[0022] In this invention, the protective buffer preferably includes glycerol, trehalose, sodium alginate, and calcium lactate; the amount of glycerol added is preferably 2-5% of the total mass of the compound bacterial solution, more preferably 3-4%; the amount of trehalose added is preferably 1-3% of the total mass of the compound bacterial solution, more preferably 2%; the amount of sodium alginate added is preferably 0.5-1% of the total mass of the compound bacterial solution, more preferably 0.7-0.8%; and the amount of calcium lactate added is preferably 0.1-0.3% of the total mass of the compound bacterial solution, more preferably 0.2%.

[0023] The present invention also provides a method for preparing the compound microbial strain liquid formulation, comprising the following steps: (1) Fermentation of Lactobacillus pentosophilus, Lactobacillus brunelli, Pediococcus pentosophilus and Lactobacillus acid-fast bacteria were carried out separately to obtain fermentation broths of different strains; (2) Concentrate the fermentation broth of different strains separately, adjust the pH value, let stand, and then mix the fermentation broth of different strains according to the ratio of live bacteria to obtain a compound bacterial solution. (3) Add a protective buffer to the compound bacterial solution, stir and filter to remove flocculent impurities and obtain a liquid preparation.

[0024] In this invention, the fermentation method described in step (1) is preferably anaerobic or microanaerobic fermentation.

[0025] In this invention, the fermentation temperature in step (1) is preferably 30~32℃, more preferably 31℃, and the pH value is preferably 5.8~6.2, more preferably 6.0.

[0026] In this invention, the concentration temperature in step (2) is preferably 2~6℃, more preferably 4℃, and the concentration is preferably carried out by tangential filtration of ceramic membrane. The concentration ratio is preferably 8~10 times, more preferably 9 times, and the concentration is preferably followed by isotonic washing.

[0027] In this invention, the pH value adjusted in step (2) is preferably 4.0~4.5, more preferably 4.2~4.3, the settling temperature is preferably 2~6℃, more preferably 4℃, and the settling time is preferably 1.5~2.5h, more preferably 2h.

[0028] In this invention, the stirring time in step (3) is preferably 10 to 20 minutes, and more preferably 15 minutes; the filter screen aperture used in the filtration is preferably 0.45 mm.

[0029] This invention also provides the application of the compound microbial liquid preparation in water quality improvement and algal bloom control.

[0030] The present invention also provides a method for using the compound microbial liquid preparation, comprising the following steps: applying the compound microbial liquid preparation into a body of water to achieve a final viable count of 5 × 10⁻⁶. 5 ~1×10 6 CFU / mL; repeat every 7-10 days during peak algal bloom periods.

[0031] In this invention, the final viable count is preferably 7 × 10⁻⁶. 5 ~8×10 5 The CFU / mL, and the preferred frequency of repetition is once every 8 to 9 days.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] (1) Lactobacillus pentosophila, Lactobacillus brunelli, Pediococcus pentosophila and Lactobacillus acidophilus were stored in glycerol tubes at -80°C. After thawing, they were inoculated into modified MRS liquid medium (each liter of modified MRS liquid medium contains: 10g tryptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g ammonium citrate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate) and anaerobic cultured at 30°C for 16h to enter the logarithmic phase. (2) The activated Lactobacillus pentosophila, Lactobacillus brunelli, Pediococcus pentosophila, and acid-fast Lactobacillus were fermented at 30℃ and pH 6.2, respectively, with dissolved oxygen controlled at <0.2 mg / L during fermentation; the viable count at harvest reached 1×10⁻⁶. 10 CFU / mL or higher.

[0035] (3) The fermentation broths of different strains were concentrated 8 times by tangential filtration with ceramic membrane at 6℃. After isotonic washing, the pH value was adjusted to 4.5. After standing at 6℃ for 1.5h, the fermentation broths of different strains were mixed according to the ratio of live bacteria. A protective buffer was added, and after stirring for 10min, the mixture was filtered through a filter with a pore size of 0.45mm to obtain the liquid preparation.

[0036] Factory specifications: pH 4.2±0.2; Total viable bacteria ≥ 1×10⁻⁶ 10 CFU / mL; the content of miscellaneous bacteria meets the requirements of GB 20287–2006.

[0037] Note: The ratio of viable bacteria of Lactobacillus pentosophilus, Lactobacillus brucelli, Pediococcus pentosophilus and Lactobacillus acid-fast bacillus in the liquid preparation is 37:21:25:13; The protective buffer includes glycerol, trehalose, sodium alginate, and calcium lactate; the amount of glycerol added is 2% of the total mass of the compound bacterial solution, the amount of trehalose added is 3% of the total mass of the compound bacterial solution, the amount of sodium alginate added is 1% of the total mass of the compound bacterial solution, and the amount of calcium lactate added is 0.1% of the total mass of the compound bacterial solution.

[0038] Example 2

[0039] (1) Lactobacillus pentosophilus, Lactobacillus brunelli, Pediococcus pentosophilus and Lactobacillus acid-fast were stored in glycerol tubes at -80°C; after thawing, they were inoculated into modified MRS liquid medium and anaerobic cultured at 30°C for 16 h to enter the logarithmic phase. (2) The activated Lactobacillus pentosophila, Lactobacillus brunelli, Pediococcus pentosophila, and acid-fast Lactobacillus were fermented at 32℃ and pH 5.8, respectively, with dissolved oxygen controlled at <0.2mg / L during fermentation; the viable count at harvest reached 1×10⁻⁶. 10 CFU / mL or higher.

[0040] (3) The fermentation broths of different strains were concentrated 10 times by tangential filtration with ceramic membrane at 2℃. After isotonic washing, the pH value was adjusted to 4.0. After standing at 2℃ for 2.5h, the fermentation broths of different strains were mixed according to the ratio of live bacteria. A protective buffer was added, and after stirring for 20min, the mixture was filtered through a filter with a pore size of 0.45mm to obtain a liquid preparation.

[0041] Factory specifications: pH 4.2±0.2; Total viable bacteria ≥ 1×10⁻⁶ CFU / mL; the content of miscellaneous bacteria meets the requirements of GB 20287–2006.

[0042] Note: The ratio of viable bacteria of Lactobacillus pentosophilus, Lactobacillus brunelli, Pediococcus pentosophilus and acid-fast Lactobacillus in the liquid preparation is 41:25:21:17; The protective buffer includes glycerol, trehalose, sodium alginate, and calcium lactate; the amount of glycerol added is 5% of the total mass of the compound bacterial solution, the amount of trehalose added is 1% of the total mass of the compound bacterial solution, the amount of sodium alginate added is 0.5% of the total mass of the compound bacterial solution, and the amount of calcium lactate added is 0.3% of the total mass of the compound bacterial solution.

[0043] Example 3

[0044] (1) Lactobacillus pentosophilus, Lactobacillus brunelli, Pediococcus pentosophilus and Lactobacillus acid-fast were stored in glycerol tubes at -80°C; after thawing, they were inoculated into modified MRS liquid medium and anaerobic cultured at 30°C for 16 h to enter the logarithmic phase. (2) The activated Lactobacillus pentosophila, Lactobacillus brunelli, Pediococcus pentosophila, and acid-fast Lactobacillus were fermented at 31℃ and pH 6.0, respectively, with dissolved oxygen controlled at <0.2 mg / L during fermentation; the viable count at harvest reached 1×10⁻⁶. 10 CFU / mL or higher.

[0045] (3) The fermentation broths of different strains were concentrated 9 times by tangential filtration with ceramic membrane at 4℃. After isotonic washing, the pH value was adjusted to 4.2. After standing at 4℃ for 2 hours, the fermentation broths of different strains were mixed according to the ratio of live bacteria. A protective buffer was added, and after stirring for 15 minutes, the mixture was filtered through a filter with a pore size of 0.45 mm to obtain a liquid preparation.

[0046] Factory specifications: pH 4.2±0.2; Total viable bacteria ≥ 1×10⁻⁶ CFU / mL; the content of miscellaneous bacteria meets the requirements of GB 20287–2006.

[0047] Note: The ratio of viable bacteria of Lactobacillus pentosophilus, Lactobacillus brunelli, Pediococcus pentosophilus, and Lactobacillus acid-fast bacillus in the liquid preparation is 39:23:23:15; The protective buffer includes glycerol, trehalose, sodium alginate, and calcium lactate; the amount of glycerol added is 3% of the total mass of the compound bacterial solution, the amount of trehalose added is 2% of the total mass of the compound bacterial solution, the amount of sodium alginate added is 0.8% of the total mass of the compound bacterial solution, and the amount of calcium lactate added is 0.2% of the total mass of the compound bacterial solution.

[0048] Test case

[0049] Lake algal bloom control: During the microcystin bloom outbreak in a 5-ha eutrophic lake, apply 1L of the preparation per 150m³. 3 The entire lake was sprayed. After 24 hours, the surface pH dropped from 9.1 to 8.2; after 72 hours, chlorophyll a decreased by 42%.

[0050] Factory-style recirculating aquaculture: Comparative experiment system loading 1×10 6 With CFU / mL formulation, after 30 days of operation, the system pH was controlled at 7.8-8.1, and ammonia nitrogen ≤ 0.3 mg / L, resulting in a 12% increase in shrimp larvae survival rate.

[0051] As can be seen from the above embodiments, the present invention provides a compound bacterial liquid preparation for improving water quality and its preparation method. The liquid preparation includes a compound bacterial solution and a protective buffer; the compound bacterial solution includes *Lactobacillus pentosophilus*, *Lactobacillus brunelli*, *Pediococcus pentosophilus*, and acid-resistant *Lactobacillus*. The present invention achieves a total viable count ≥1×10⁻⁶ through single-strain anaerobic fermentation, low-temperature concentration, and isotonic protection. 10 It has a concentration of CFU / mL and remains stable at room temperature for 6 months. Practical applications show that this formulation can quickly lower the pH of algal bloom water from >9.0 and stabilize it at 7.8~8.3, while significantly inhibiting the growth of cyanobacteria and green algae and reducing ammonia nitrogen toxicity. It has the advantages of being green, safe, and low-cost, and is suitable for water quality improvement and algal bloom control in lakes, reservoirs, landscape water bodies, and aquaculture systems.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound microbial liquid preparation for improving water quality, characterized in that, The liquid formulation comprises a compound bacterial solution and a protective buffer. The compound bacterial solution includes *Lactobacillus pentosophila*, *Lactobacillus brucelli*, *Pediococcus pentosophila*, and acid-fast lactobacillus. The viable count ratio of *Lactobacillus pentosophila*, *Lactobacillus brucelli*, *Pediococcus pentosophila*, and acid-fast lactobacillus is 37-41:21-25:21-25:13-17. The total viable count in the compound bacterial solution is ≥1×10⁻⁶. 10 CFU / mL.

2. The compound microbial liquid preparation according to claim 1, characterized in that, The protective buffer comprises glycerol, trehalose, sodium alginate, and calcium lactate; the amount of glycerol added is 2-5% of the total mass of the compound bacterial solution, the amount of trehalose added is 1-3% of the total mass of the compound bacterial solution, the amount of sodium alginate added is 0.5-1% of the total mass of the compound bacterial solution, and the amount of calcium lactate added is 0.1-0.3% of the total mass of the compound bacterial solution.

3. The method for preparing the compound microbial liquid preparation according to claim 1 or 2, characterized in that, Includes the following steps: (1) Fermentation of Lactobacillus pentosophilus, Lactobacillus brunelli, Pediococcus pentosophilus and Lactobacillus acid-fast bacteria were carried out separately to obtain fermentation broths of different strains; (2) Concentrate the fermentation broth of different strains separately, adjust the pH value, let stand, and then mix the fermentation broth of different strains according to the ratio of live bacteria to obtain a compound bacterial solution. (3) Add a protective buffer to the compound bacterial solution, stir and filter to remove flocculent impurities and obtain a liquid preparation.

4. The preparation method according to claim 3, characterized in that, The fermentation method described in step (1) is anaerobic or microanaerobic fermentation.

5. The preparation method according to claim 3, characterized in that, The fermentation temperature in step (1) is 30~32℃ and the pH value is 5.8~6.

2.

6. The preparation method according to claim 3, characterized in that, The concentration temperature in step (2) is 2~6℃. The concentration is carried out by tangential filtration of ceramic membrane, and the concentration ratio is 8~10 times. After concentration, isotonic washing is performed.

7. The preparation method according to claim 3, characterized in that, The pH value after adjustment in step (2) is 4.0~4.5, the settling temperature is 2~6℃, and the time is 1.5~2.5h.

8. The preparation method according to claim 3, characterized in that, The stirring time in step (3) is 10~20 min; the filter screen used for filtration has a pore size of 0.45 mm.

9. The application of the compound microbial liquid preparation according to claim 1 or 2 in water quality improvement and algal bloom control.

10. The method of using the compound microbial liquid preparation according to claim 1 or 2, characterized in that, The process includes the following steps: Applying the compound microbial liquid preparation to the water body to achieve a final viable count of 5 × 10⁻⁶. 5 ~1×10 6 CFU / mL; repeat every 7-10 days during peak algal bloom periods.

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