A compound microbial agent for treating nitrogen and phosphorus in water bodies and application thereof

Abstract

This invention belongs to the field of wastewater treatment, specifically relating to a compound microbial agent for treating nitrogen and phosphorus in water and its application. The compound microbial agent comprises Bacillus subtilis ACCC10627 and Bacillus megaterium ATCC14581 in a weight ratio of 3-5:1. This invention achieves simultaneous and efficient removal of nitrogen and phosphorus from water through the complementary and synergistic metabolism of the two bacilli. This compound microbial agent provides a more competitive technical solution for achieving nitrogen and phosphorus standards in water treatment and has significant engineering application value.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment, specifically relating to a compound microbial agent for treating nitrogen and phosphorus in water and its application. Background Technology

[0002] With the continued advancement of urbanization, wastewater treatment has become a crucial issue in global environmental protection. The excessive discharge of nutrients such as nitrogen and phosphorus has attracted widespread social attention. Nitrogen and phosphorus are common nutrients in water bodies, but excessive nitrogen and phosphorus emissions can cause serious water pollution problems. Major sources of nitrogen include agricultural activities, industrial wastewater, and urban sewage. In agriculture, the widespread use of chemical fertilizers and pesticides has led to the excessive release of nitrogen compounds. Agricultural runoff and agricultural wastewater are also significant sources of nitrogen. Industrial wastewater contains large amounts of nitrogen compounds, such as ammonia nitrogen and nitrates.

[0003] The main pathways for phosphorus emissions include agriculture, industry, and wastewater discharge. In agriculture, the use of fertilizers and pesticides leads to the accumulation of phosphorus in the soil, which then enters water bodies through runoff. Soil erosion in farmland also washes phosphorus-containing soil particles into rivers and lakes. Phosphorus in industrial wastewater mainly comes from processes such as chemical manufacturing and food processing, some of which contain high concentrations of phosphorus compounds. Wastewater discharge is also a significant source of phosphorus pollution, especially untreated domestic sewage, which has relatively high phosphorus content.

[0004] Water pollution, as a component of environmental pollution, remains poorly controlled. Traditional wastewater treatment technologies mainly include physical, chemical, and biological methods. Physical and chemical methods can effectively remove particulate pollutants from wastewater, but they are costly and prone to causing secondary pollution. Biological methods are less effective at removing nitrogen and phosphorus pollutants from wastewater. All traditional wastewater treatment methods have various limitations.

[0005] The literature by Li Xue et al. (Li Xue, Liu Shufeng, Yan Yumeng, et al. Effects of compound Bacillus on water quality and bacterial community structure in crucian carp aquaculture) used Bacillus megaterium (A0), Bacillus coagulans (BC), and Bacillus subtilis (BS) as research subjects to explore the effects of compound Bacillus A0+BC and A0+BS on the improvement of nutrients N and P in crucian carp aquaculture water. High-throughput sequencing was used to analyze changes in bacterial community structure. The results showed that compound Bacillus A0+BS had a significant effect on NH4+. + -N, NO2 - -N, NO3 - -N and TP showed the most significant degradation effects, with degradation rates of 46.3%, 76.3%, 35.6%, and 80.3%, respectively; the compound Bacillus A0+BC had no degradation effect on TP.

[0006] However, the removal rates of TN and TP by the aforementioned compound Bacillus in aquaculture water were only about 46% and 80%, respectively, which is insufficient to meet increasingly stringent wastewater discharge standards. Developing a stable compound microbial agent with efficient simultaneous nitrogen and phosphorus removal capabilities remains a pressing technical problem to be solved in this field. Summary of the Invention

[0007] In view of this, the present invention provides a compound microbial agent for treating nitrogen and phosphorus in water and its application. The compound microbial agent comprises Bacillus subtilis ACCC 10627 and Bacillus megaterium ATCC 14581 in a weight ratio of 3-5:1. The two strains are complementary in function and have a synergistic effect, which can efficiently remove nitrogen and phosphorus pollution from water.

[0008] The technical solution of the present invention is as follows:

[0009] A compound microbial agent for treating nitrogen and phosphorus in water includes Bacillus subtilis ACCC 10627 and Bacillus megaterium ATCC 14581; the weight ratio of Bacillus subtilis ACCC 10627 powder to Bacillus megaterium ATCC 14581 powder is 3-5:1.

[0010] Furthermore, the weight ratio of Bacillus subtilis ACCC 10627 bacterial powder to Bacillus megaterium ATCC 14581 bacterial powder is 3.5-4.5:1.

[0011] Furthermore, the weight ratio of Bacillus subtilis ACCC 10627 bacterial powder to Bacillus megaterium ATCC 14581 bacterial powder is 4:1.

[0012] This invention discloses a method for preparing a compound microbial agent for treating nitrogen and phosphorus in water, comprising the following steps:

[0013] S1, Activation

[0014] Activation culture medium was prepared and autoclaved at 121 °C for 20 min. Freeze-dried Bacillus subtilis ACCC 10627 and Bacillus megaterium ATCC 14581 were inoculated into the culture medium. Bacillus subtilis was incubated at 32 °C for 24 h and Bacillus megaterium was incubated at 30 °C for 24 h to obtain activated pure culture slant cultures.

[0015] S2, Expansion Cultivation

[0016] Prepare seed liquid culture medium and sterilize at 121 ℃ for 20 min; pick activated single colonies and inoculate them into shake flasks for culture; Bacillus subtilis was cultured at 32 ℃ and 180 r / min for 20 h; Bacillus megaterium was cultured at 30 ℃ and 160 r / min for 22 h to obtain highly active seed liquids.

[0017] S3, Fermentation Culture

[0018] Prepare the fermentation medium and sterilize it at 121 °C for 30 min; inoculate the two seed cultures into the fermentation system at an inoculation rate of 5% (v / v) and ferment them separately.

[0019] Fermentation conditions for Bacillus subtilis: culture temperature 33 ℃, aeration rate 1:0.8 vvm, stirring speed 200 r / min, fermentation for 44 h, and fermentation was terminated when the spore formation rate was ≥95%.

[0020] Fermentation conditions for Bacillus megaterium: culture temperature 30 ℃, aeration rate 1:0.7 vvm, stirring speed 160 r / min, fermentation for 46 h, and fermentation was terminated when the spore formation rate was ≥93%.

[0021] S4, bacterial liquid concentration, protective agent compounding and spray drying powder production

[0022] The two fermentation broths were concentrated by tubular centrifugation at 6000 r / min for 10 min to collect high-concentration bacterial sludge. A heat-resistant spore protectant was added to the bacterial sludge and stirred evenly. Pure bacterial powder was prepared by spray drying at an inlet air temperature of 160 ℃ and an outlet air temperature of 70 ℃ to obtain pure Bacillus subtilis powder and pure Bacillus megaterium powder, respectively, with a single-strain powder viable cell content ≥80 billion CFU / g.

[0023] S5. Preparation of compound microbial agents

[0024] Accurately weigh the Bacillus subtilis powder and Bacillus megaterium powder at a fixed weight ratio of 3-5:1; then add the carrier excipients, which consist of diatomaceous earth, corn starch, and wheat bran; put all materials into a three-dimensional mixer and mix in a closed container for 35 minutes until uniformly mixed to obtain the compound microbial agent.

[0025] Preferably, the activation culture medium formulation in step S1 is: 5 g / L peptone, 3 g / L yeast extract, 1 g / L glucose, 5 g / L sodium chloride, 15 g / L agar, pH 7.1.

[0026] Preferably, the seed liquid culture medium formulation in step S2 is: glucose 15 g / L, peptone 8 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, pH 7.1.

[0027] Preferably, the fermentation medium formula in step S3 is: 20 g / L corn flour, 10 g / L soybean meal, 10 g / L glucose, 3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 1 g / L calcium carbonate, pH 7.2.

[0028] Preferably, the heat-resistant spore protectant in step S4 consists of 3% seaweed oligosaccharide and 2% skim milk powder by mass of the bacterial sludge.

[0029] Preferably, in step S5, the carrier additives have the following fixed mass fractions: diatomaceous earth 55%, corn starch 18%, wheat bran 12%, and the total amount of active bacteria powder added is 15%.

[0030] The present invention also provides the application of the above-mentioned composite microbial agent in wastewater treatment.

[0031] The beneficial effects achieved by this invention are as follows:

[0032] This invention provides a composite microbial agent comprising Bacillus subtilis ACCC10627 and Bacillus megaterium ATCC 14581 in a weight ratio of 3-5:1. Bacillus subtilis ACCC 10627 heterotrophically nitrifies and assimilates phosphorus in water, decomposing organic nitrogen and organic phosphorus, thus removing nitrogen and phosphorus from wastewater. Bacillus megaterium ATCC 14581 produces high levels of phosphatase, effectively dissolving insoluble organic phosphorus in water, and also possesses nitrate assimilation and denitrification properties, making it a classic strain for phosphorus removal in wastewater and restoration of eutrophic rivers. This invention achieves simultaneous and efficient removal of nitrogen and phosphorus from water through the metabolic complementarity and synergistic effect of Bacillus subtilis ACCC 10627 and Bacillus megaterium ATCC 14581, with significantly better results than single strains. This composite microbial agent provides a more competitive technical solution for achieving nitrogen and phosphorus standards in water treatment and has significant engineering application value. Detailed Implementation

[0033] The invention can be better understood through the following embodiments. It will then be readily understood by those skilled in the art that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0034] Bacillus subtilis ACCC 10627, Bacillus megaterium ATCC 14581, and Bacillus megaterium ACCC 11011 are commercially available strains that can be purchased.

[0035] In the following examples, Bacillus subtilis ACCC 10627, Bacillus megaterium ATCC 14581, and Bacillus megaterium ACCC 11011 were purchased from Shanghai Fuxiang Biotechnology Co., Ltd.

[0036] Bacillus megaterium CCTCC AB 207465 was purchased from the China Center for Type Culture Collection.

[0037] Example 1

[0038] A compound microbial agent for treating nitrogen and phosphorus in water and its preparation method thereof, comprising the following steps:

[0039] S1, Activation

[0040] Activation culture media were prepared and autoclaved at 121 °C for 20 min. Freeze-dried Bacillus subtilis ACCC 10627 and Bacillus megaterium ATCC 14581 were inoculated into the culture media. The culture medium formulation was: 5 g / L peptone, 3 g / L yeast extract, 1 g / L glucose, 5 g / L sodium chloride, 15 g / L agar, pH 7.1. Bacillus subtilis was incubated at 32 °C for 24 h, and Bacillus megaterium was incubated at 30 °C for 24 h to obtain activated pure culture slant cultures.

[0041] S2, Expansion Cultivation

[0042] A seed culture medium was prepared with the following formula: glucose 15 g / L, peptone 8 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, pH 7.1, and sterilized at 121 ℃ for 20 min. Activated single colonies were picked and inoculated into shake flasks for culture. The culture conditions for *Bacillus subtilis* were 32 ℃, 180 r / min, and 20 h; the culture conditions for *Bacillus megaterium* were 30 ℃, 160 r / min, and 22 h, respectively, to obtain highly active seed cultures.

[0043] S3, Fermentation Culture

[0044] Prepare the fermentation medium with the following formula: 20 g / L corn flour, 10 g / L soybean meal, 10 g / L glucose, 3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, and 1 g / L calcium carbonate, pH 7.2, and sterilize at 121 ℃ for 30 min. Inoculate the two seed cultures into the fermentation system at an inoculation rate of 5% (v / v) and ferment them separately.

[0045] Fermentation conditions for Bacillus subtilis: culture temperature 33 ℃, aeration rate 1:0.8 vvm, stirring speed 200 r / min, fermentation for 44 h, and fermentation was terminated when the spore formation rate was ≥95%.

[0046] Fermentation conditions for Bacillus megaterium: culture temperature 30 ℃, aeration rate 1:0.7 vvm, stirring speed 160 r / min, fermentation for 46 h, and fermentation was terminated when the spore formation rate was ≥93%.

[0047] S4, bacterial liquid concentration, protective agent compounding and spray drying powder production

[0048] The two fermentation broths were concentrated by tubular centrifugation at 6000 r / min for 10 min, and high-concentration bacterial sludge was collected. 3% seaweed oligosaccharide and 2% skim milk powder (by weight of the bacterial sludge) were added to the bacterial sludge as heat-resistant preservatives for spores, and the mixture was stirred evenly. Pure bacterial powder was prepared by spray drying at an inlet air temperature of 160 ℃ and an outlet air temperature of 70 ℃, yielding pure Bacillus subtilis powder and pure Bacillus megaterium powder, respectively, with a single-strain powder viable cell content ≥80 billion CFU / g.

[0049] S5. Preparation of compound microbial agents

[0050] Accurately weigh the Bacillus subtilis powder and Bacillus megaterium powder at a weight ratio of 4:1; then add carrier excipients, with the total mass of the compound microbial agent being 100%. The fixed mass fraction of the carrier excipients is: diatomaceous earth 55%, corn starch 18%, wheat bran 12%, and the total amount of active microbial powder added is 15%; put all materials into a three-dimensional mixer and mix in a closed container for 35 minutes until uniformly mixed to obtain a compound microbial agent with both high efficiency in denitrification and organophosphorus degradation.

[0051] Example 2

[0052] A compound microbial agent for treating nitrogen and phosphorus in water.

[0053] Compared with Example 1, the difference is that the weight ratio of Bacillus subtilis powder to Bacillus megaterium powder is 3.5:1.

[0054] Example 3

[0055] A compound microbial agent for treating nitrogen and phosphorus in water.

[0056] Compared with Example 1, the difference is that the weight ratio of Bacillus subtilis powder to Bacillus megaterium powder is 4.5:1.

[0057] Example 4

[0058] A compound microbial agent for treating nitrogen and phosphorus in water.

[0059] Compared with Example 1, the difference is that the weight ratio of Bacillus subtilis powder to Bacillus megaterium powder is 3:1.

[0060] Example 5

[0061] A compound microbial agent for treating nitrogen and phosphorus in water.

[0062] Compared with Example 1, the difference is that the weight ratio of Bacillus subtilis powder to Bacillus megaterium powder is 5:1.

[0063] Comparative Example 1

[0064] The difference from Example 1 is that only one strain—Bacillus subtilis ACCC 10627—was used.

[0065] S1, Activation

[0066] An activation culture medium was prepared and autoclaved at 121 °C for 20 min. Freeze-dried Bacillus subtilis ACCC10627 was then inoculated into the medium. The culture medium formulation was: 5 g / L peptone, 3 g / L yeast extract, 1 g / L glucose, 5 g / L sodium chloride, 15 g / L agar, pH 7.1. Bacillus subtilis was incubated at 32 °C for 24 h to obtain activated pure culture slant agar.

[0067] S2, Expansion Cultivation

[0068] A seed culture medium was prepared with the following formula: glucose 15 g / L, peptone 8 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, pH 7.1, and sterilized at 121 ℃ for 20 min. Activated single colonies were picked and inoculated into shake flasks for culture. Bacillus subtilis was cultured at 32 ℃ and 180 r / min for 20 h to obtain a highly active seed culture.

[0069] S3, Fermentation Culture

[0070] Prepare the fermentation medium with the following formula: 20 g / L corn flour, 10 g / L soybean meal, 10 g / L glucose, 3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, and 1 g / L calcium carbonate, pH 7.2, and sterilize at 121 ℃ for 30 min; inoculate the seed culture into the fermentation system at a rate of 5% (v / v).

[0071] Fermentation conditions for Bacillus subtilis: culture temperature 33 ℃, aeration rate 1:0.8 vvm, stirring speed 200 r / min, fermentation for 44 h, and fermentation was terminated when the spore formation rate was ≥95%.

[0072] S4, bacterial liquid concentration, protective agent compounding and spray drying powder production

[0073] The fermentation broth was concentrated by tubular centrifugation at 6000 r / min for 10 min, and high-concentration bacterial sludge was collected. 3% seaweed oligosaccharide and 2% skim milk powder (by weight of the bacterial sludge) were added to the bacterial sludge as heat-resistant preservatives for the spores, and the mixture was stirred evenly. Pure bacterial powder was prepared by spray drying at an inlet air temperature of 160 ℃ and an outlet air temperature of 70 ℃, yielding pure Bacillus subtilis bacterial powder with a live bacteria content ≥80 billion CFU / g.

[0074] S5, Add carrier excipients

[0075] Bacillus subtilis pure powder was added to a carrier excipient. The carrier excipient had a fixed mass fraction of 55% diatomaceous earth, 18% corn starch, and 12% wheat bran, with a total active bacterial powder addition of 15%. All materials were added to a three-dimensional mixer and mixed in a closed container for 35 minutes until homogeneous.

[0076] Comparative Example 2

[0077] The difference from Example 1 is that only one strain was used - Bacillus megaterium ATCC 14581.

[0078] S1, Activation

[0079] An activation culture medium was prepared and autoclaved at 121 °C for 20 min. Freeze-dried Bacillus megaterium ATCC14581 was then inoculated into the medium. The culture medium consisted of: 5 g / L peptone, 3 g / L yeast extract, 1 g / L glucose, 5 g / L sodium chloride, 15 g / L agar, and pH 7.1. The Bacillus megaterium was incubated at 30 °C for 24 h to obtain an activated pure culture slant.

[0080] S2, Expansion Cultivation

[0081] A seed culture medium was prepared with the following formula: glucose 15 g / L, peptone 8 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, pH 7.1, and sterilized at 121 ℃ for 20 min. Activated single colonies were picked and inoculated into shake flasks for culture. Bacillus megaterium was cultured at 30 ℃ and 160 r / min for 22 h to obtain a highly active seed culture.

[0082] S3, Fermentation Culture

[0083] Prepare the fermentation medium with the following formula: 20 g / L corn flour, 10 g / L soybean meal, 10 g / L glucose, 3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, and 1 g / L calcium carbonate, pH 7.2, and sterilize at 121 ℃ for 30 min; inoculate the seed culture into the fermentation system at a rate of 5% (v / v).

[0084] Fermentation conditions for Bacillus megaterium: culture temperature 30 ℃, aeration rate 1:0.7 vvm, stirring speed 160 r / min, fermentation for 46 h, and fermentation was terminated when the spore formation rate was ≥93%.

[0085] S4, bacterial liquid concentration, protective agent compounding and spray drying powder production

[0086] The fermentation broth was concentrated by tubular centrifugation at 6000 r / min for 10 min, and high-concentration bacterial sludge was collected. 3% seaweed oligosaccharide and 2% skim milk powder (by weight of the bacterial sludge) were added to the bacterial sludge as heat-resistant preservatives for the spores, and the mixture was stirred evenly. Pure bacterial powder was prepared by spray drying at an inlet air temperature of 160 ℃ and an outlet air temperature of 70 ℃ to obtain pure Bacillus megaterium powder with a viable bacterial content ≥80 billion CFU / g.

[0087] S5, Add carrier excipients

[0088] Bacillus megaterium powder is added to a carrier excipient. The carrier excipient has a fixed mass fraction of 55% diatomaceous earth, 18% corn starch, and 12% wheat bran, with a total active bacterial powder addition of 15%. All materials are added to a three-dimensional mixer and mixed in a closed container for 35 minutes until homogeneous.

[0089] Comparative Example 3

[0090] Compared with Example 1, the only difference is that Bacillus megaterium ACCC 11011 is used instead of Bacillus megaterium ATCC 14581.

[0091] S1, Activation

[0092] Activation culture medium was prepared and autoclaved at 121 °C for 20 min. Freeze-dried Bacillus subtilis ACCC 10627 and Bacillus megaterium ACCC 11011 were inoculated into the culture medium. The culture medium formulation was: 5 g / L peptone, 3 g / L yeast extract, 1 g / L glucose, 5 g / L sodium chloride, 15 g / L agar, pH 7.1. Bacillus subtilis was incubated at 32 °C for 24 h, and Bacillus megaterium ACCC 11011 was incubated at 30 °C for 24 h to obtain activated pure culture slant cultures.

[0093] S2, Expansion Cultivation

[0094] A seed culture medium was prepared with the following formula: glucose 15 g / L, peptone 8 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, pH 7.1, and sterilized at 121 ℃ for 20 min. Activated single colonies were picked and inoculated into shake flasks for culture. The culture conditions for Bacillus subtilis were 32 ℃, 180 r / min, and 20 h; the culture conditions for Bacillus megaterium ACCC11011 were 30 ℃, 160 r / min, and 22 h, respectively, to obtain highly active seed cultures.

[0095] S3, Fermentation Culture

[0096] Prepare the fermentation medium with the following formula: 20 g / L corn flour, 10 g / L soybean meal, 10 g / L glucose, 3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, and 1 g / L calcium carbonate, pH 7.2, and sterilize at 121 ℃ for 30 min. Inoculate the two seed cultures into the fermentation system at an inoculation rate of 5% (v / v) and ferment them separately.

[0097] Fermentation conditions for Bacillus subtilis: culture temperature 33 ℃, aeration rate 1:0.8 vvm, stirring speed 200 r / min, fermentation for 44 h, and fermentation was terminated when the spore formation rate was ≥95%.

[0098] Fermentation conditions for Bacillus megaterium ACCC 11011: culture temperature 30 ℃, aeration rate 1:0.7 vvm, stirring speed 160 r / min, fermentation for 46 h, and fermentation was terminated when the spore formation rate was ≥93%.

[0099] S4, bacterial liquid concentration, protective agent compounding and spray drying powder production

[0100] The two fermentation broths were concentrated by tubular centrifugation at 6000 r / min for 10 min, and high-concentration bacterial sludge was collected. 3% seaweed oligosaccharide and 2% skim milk powder (by weight of bacterial sludge) were added to the bacterial sludge as heat-resistant spore protectants, and the mixture was stirred evenly. Pure bacterial powder was prepared by spray drying at an inlet air temperature of 160 ℃ and an outlet air temperature of 70 ℃, respectively, yielding pure Bacillus subtilis powder and pure Bacillus megaterium ACCC 11011 powder, with a single-strain powder viable cell content ≥80 billion CFU / g.

[0101] S5, Add carrier excipients

[0102] Accurately weigh the Bacillus subtilis powder and Bacillus megaterium ACCC 11011 powder at a weight ratio of 4:1; then add carrier excipients, with the total mass of the compound microbial agent being 100%. The fixed mass fraction of the carrier excipients is: diatomaceous earth 55%, corn starch 18%, wheat bran 12%, and the total amount of active microbial powder added is 15%; put all materials into a three-dimensional mixer and mix in a closed container for 35 minutes until uniformly mixed to obtain the final product.

[0103] Comparative Example 4

[0104] Compared with Example 1, the only difference is that Bacillus megaterium CCTCC AB 207465 is used instead of Bacillus megaterium ATCC 14581.

[0105] S1, Activation

[0106] Activation culture medium was prepared and autoclaved at 121 °C for 20 min. Freeze-dried Bacillus subtilis ACCC 10627 and Bacillus megaterium CCTCC AB 207465 were inoculated into the culture medium. The culture medium formulation was: 5 g / L peptone, 3 g / L yeast extract, 1 g / L glucose, 5 g / L sodium chloride, 15 g / L agar, pH 7.1. Bacillus subtilis was incubated at 32 °C for 24 h, and Bacillus megaterium CCTCC AB 207465 was incubated at 30 °C for 24 h to obtain activated pure culture slant cultures.

[0107] S2, Expansion Cultivation

[0108] A seed culture medium was prepared with the following formula: glucose 15 g / L, peptone 8 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, pH 7.1, and sterilized at 121 ℃ for 20 min. Activated single colonies were picked and inoculated into shake flasks for culture. The culture conditions for *Bacillus subtilis* were 32 ℃, 180 r / min, and 20 h; the culture conditions for *Bacillus megaterium* CCTCC AB 207465 were 30 ℃, 160 r / min, and 22 h, respectively, to obtain highly active seed cultures.

[0109] S3, Fermentation Culture

[0110] Prepare the fermentation medium with the following formula: 20 g / L corn flour, 10 g / L soybean meal, 10 g / L glucose, 3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, and 1 g / L calcium carbonate, pH 7.2, and sterilize at 121 ℃ for 30 min. Inoculate the two seed cultures into the fermentation system at an inoculation rate of 5% (v / v) and ferment them separately.

[0111] Fermentation conditions for Bacillus subtilis: culture temperature 33 ℃, aeration rate 1:0.8 vvm, stirring speed 200 r / min, fermentation for 44 h, and fermentation was terminated when the spore formation rate was ≥95%.

[0112] Fermentation conditions for Bacillus megaterium CCTCC AB 207465: culture temperature 30 ℃, aeration rate 1:0.7 vvm, stirring speed 160 r / min, fermentation for 46 h, and fermentation was terminated when the spore formation rate was ≥93%.

[0113] S4, bacterial liquid concentration, protective agent compounding and spray drying powder production

[0114] The two fermentation broths were concentrated by tubular centrifugation at 6000 r / min for 10 min, and high-concentration bacterial sludge was collected. 3% seaweed oligosaccharide and 2% skim milk powder (by weight of bacterial sludge) were added to the bacterial sludge as heat-resistant spore protectants, and the mixture was stirred evenly. Pure bacterial powder was prepared by spray drying at an inlet air temperature of 160 ℃ and an outlet air temperature of 70 ℃, respectively, yielding pure Bacillus subtilis powder and pure Bacillus megaterium CCTCC AB 207465 powder, with a single-strain powder viable cell content ≥80 billion CFU / g.

[0115] S5, Add carrier excipients

[0116] Accurately weigh the Bacillus subtilis powder and Bacillus megaterium CCTCC AB 207465 powder at a weight ratio of 4:1; then add carrier excipients, with the total mass of the compound microbial agent being 100%, the fixed mass fraction of the carrier excipients being: diatomaceous earth 55%, corn starch 18%, wheat bran 12%, and the total amount of active microbial powder added being 15%; put all materials into a three-dimensional mixer and mix in a closed container for 35 minutes until uniformly mixed, and the product is obtained.

[0117] Comparative Example 5

[0118] Compared with Example 1, the only difference is the change in the weight ratio of Bacillus subtilis ACCC 10627 powder to Bacillus megaterium ATCC 14581 powder. The weight ratio of Bacillus subtilis ACCC 10627 powder to Bacillus megaterium ATCC 14581 powder is 2:1.

[0119] Comparative Example 6

[0120] Compared with Example 1, the only difference is the change in the weight ratio of Bacillus subtilis ACCC 10627 powder to Bacillus megaterium ATCC 14581 powder. The weight ratio of Bacillus subtilis ACCC 10627 powder to Bacillus megaterium ATCC 14581 powder is 1:2.

[0121] Comparative Example 7

[0122] Compared with Example 1, the only difference is the change in the weight ratio of Bacillus subtilis ACCC 10627 powder to Bacillus megaterium ATCC 14581 powder. The weight ratio of Bacillus subtilis ACCC 10627 powder to Bacillus megaterium ATCC 14581 powder is 1:4.

[0123] Experiment on nitrogen and phosphorus treatment in factory wastewater

[0124] The wastewater used in the experiment was taken from the wastewater outlet of a factory. The wastewater was centrifuged at 10,000 r / min for 10 min, and the light yellow supernatant was collected.

[0125] The total nitrogen (TN) content was determined according to the alkaline potassium persulfate-ultraviolet spectrophotometric method (GB 11894-89), and the total phosphorus (TP) content was determined according to the ammonium molybdate spectrophotometric method (GB11893-89). The results were: total nitrogen (TN) 1764.73 mg / L, total phosphorus (TP) 17.85 mg / L. The TN content was diluted to 20 mg / L and the TP content was 2.5 mg / L. The results were then set aside for future use.

[0126] Take a conical flask containing 1L of wastewater and inoculate it with bacterial suspensions from Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, and Comparative Example 7 at an inoculation rate of 6% (v / v). Incubate the flasks at 25°C under 16h light per day for 6 days. Three replicates are set up for each treatment group.

[0127] After the culture was completed (day 7), 50 mL of culture medium was centrifuged at 5000 r / min for 8 min, and 5 mL of supernatant was collected. The TN and TP contents in the supernatant were measured, and the removal rate was calculated.

[0128] The results are shown in Table 1. After treating polluted water with the composite microbial agents of Examples 1, 2, and 3 of this invention, the TN removal rates were 99.8%, 99.3%, and 99.5%, respectively. After treating polluted water with agents from Comparative Examples 1, 2, 3, 4, 5, 6, and 7, the TN removal rates were 75.3%, 80.6%, 86.2%, 83.4%, 90.2%, 91.4%, and 93.4%, respectively.

[0129] After wastewater was treated with the composite microbial agents of Examples 1, 2, and 3 of this invention, the TP removal rates were 99.9%, 99.2%, and 99.4%, respectively. After wastewater was treated with the microbial agents of Comparative Examples 1, 2, 3, 4, 5, 6, and 7, the TP removal rates were 83.7%, 80.4%, 83.3%, 82.7%, 89.4%, 92.5%, and 91.1%, respectively.

[0130] Table 1 Removal rates of TN and TP under different treatments

[0131] Example 1 Group 99.8 99.9 Example 2 group 99.3 99.2 Example 3 Group 99.5 99.4 Comparative Example 1 75.3 83.7 Comparative Example 2 80.6 80.4 Comparative Example 3 Groups 86.2 83.3 Comparative Example 4 Groups 83.4 82.7 Comparative Example 5 Groups 90.2 89.4 Comparative Example 6 Groups 91.4 92.5 Comparative Example 7 Groups 93.4 91.1

[0132] Under the same dosage, the treatment effects of the wastewater in Examples 1, 2, and 3 of this invention are significantly better than those of Comparative Examples 1, 2, 3, 4, 5, 6, and 7. In Examples 1, 2, and 3, the compound microbial agents *Bacillus subtilis* ACCC 10627 and *Bacillus megaterium* ATCC 14581 synergistically enhance the removal of nitrogen and phosphorus from wastewater. In contrast, Comparative Examples 1 and 2 use only a single strain, and Comparative Examples 3 and 4 change the type of *Bacillus megaterium* strain, resulting in a significant decrease in nitrogen and phosphorus removal from wastewater. Comparative Examples 5, 6, and 7, by changing the dosage ratio of *Bacillus subtilis* ACCC 10627 and *Bacillus megaterium* ATCC 14581, also show a significant decrease in nitrogen and phosphorus removal from wastewater.

[0133] The above experimental results show that the combination of Bacillus subtilis ACCC 10627 and Bacillus megaterium ATCC 14581 in this invention has a significant synergistic effect in removing nitrogen and phosphorus pollution from industrial wastewater. The combined microbial agent of Bacillus subtilis ACCC 10627 and Bacillus megaterium ATCC 14581 in this invention is significantly more effective than Bacillus subtilis ACCC 10627 alone and Bacillus megaterium ATCC 14581 alone in removing nitrogen and phosphorus pollution from industrial wastewater. At the same dosage, replacing Bacillus megaterium ATCC 14581 with Bacillus megaterium ACCC 11011 and with Bacillus megaterium CCTCC AB 207465 significantly weakens the effect in removing nitrogen and phosphorus pollution from industrial wastewater. Two specific strains, Bacillus subtilis ACCC 10627 and Bacillus megaterium ATCC 14581, exhibited a significant synergistic effect in removing nitrogen and phosphorus pollution from industrial wastewater, demonstrating a significant technical advantage. The synergistic effect of these two strains is related to the dosage ratio of the two strains.

[0134] Experiment on the treatment of nitrogen and phosphorus in artificially prepared domestic sewage

[0135] Prepare domestic sewage artificially according to Table 2.

[0136] Table 2. Composition and concentration of artificially prepared domestic sewage

[0137] Anhydrous sodium acetate 187.5 glucose 187.5 ammonium chloride 68.43 Urea 23.25 Potassium dihydrogen phosphate 24.38 Calcium chloride 18.75 Magnesium sulfate 12.5 Ferrous sulfate 19.25 boric acid 1.88 Cobalt nitrate 2.29 Zinc chloride 1.28 manganese sulfate 0.16 Copper sulfate 0.38 Sodium molybdate 0.75

[0138] Take a conical flask containing 1L of artificially prepared domestic sewage and inoculate it with bacterial suspensions from Example 1, Example 4, Example 5, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, and Comparative Example 7 at an inoculation rate of 6% (v / v). Incubate the flasks at 25°C under 16h light per day for 6 days. Three replicates are set up for each treatment group.

[0139] After the culture was completed (day 7), 50 mL of culture medium was centrifuged at 5000 r / min for 8 min, and 5 mL of supernatant was collected. The TN and TP contents in the supernatant were measured, and the removal rate was calculated.

[0140] The results are shown in Table 3. After treating polluted water with the combined microbial agents of Examples 1, 4, and 5 of this invention, the TN removal rates were 99.5%, 99.7%, and 99.3%, respectively. After treating polluted water with the microbial agents of Comparative Examples 1, 2, 3, 4, 5, 6, and 7, the TN removal rates were 72.6%, 86.2%, 88.1%, 85.2%, 89.1%, 87.3%, and 90.5%, respectively.

[0141] After treating wastewater with the combined microbial agents of Examples 1, 4, and 5 of this invention, the TP removal rates were 99.7%, 99.4%, and 99.5%, respectively. After treating wastewater with the microbial agents of Comparative Examples 1, 2, 3, 4, 5, 6, and 7, the TP removal rates were 79.6%, 82.9%, 87.2%, 89.3%, 84.2%, 90.7%, and 89.6%, respectively.

[0142] Table 3 Removal rates of TN and TP under different treatments

[0143] Example 1 Group 99.5 99.7 Example 4 group 99.7 99.4 Example 5 group 99.3 99.5 Comparative Example 1 72.6 79.6 Comparative Example 2 86.2 82.9 Comparative Example 3 Groups 88.1 87.2 Comparative Example 4 Groups 85.2 89.3 Comparative Example 5 Groups 89.1 84.2 Comparative Example 6 Groups 87.3 90.7 Comparative Example 7 Groups 90.5 89.6

[0144] Under the same dosage, the treatment effects of the artificially prepared domestic sewage in Examples 1, 4, and 5 of this invention are significantly better than those of Comparative Examples 1, 2, 3, 4, 5, 6, and 7. In Examples 1, 4, and 5, the compound microbial agents *Bacillus subtilis* ACCC 10627 and *Bacillus megaterium* ATCC 14581 synergistically enhance the removal of nitrogen and phosphorus from the artificially prepared domestic sewage. In contrast, Comparative Examples 1 and 2 used only a single strain, and Comparative Examples 3 and 4 changed the type of *Bacillus megaterium* strain, significantly reducing the removal of nitrogen and phosphorus from the artificially prepared domestic sewage. Comparative Examples 5, 6, and 7, by changing the dosage ratio of *Bacillus subtilis* ACCC 10627 and *Bacillus megaterium* ATCC 14581, significantly reduced the removal of nitrogen and phosphorus from the artificially prepared domestic sewage.

[0145] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A compound microbial agent for treating nitrogen and phosphorus in water, characterized in that, It includes Bacillus subtilis ACCC10627 and Bacillus megaterium ATCC14581; the weight ratio of Bacillus subtilis ACCC10627 powder to Bacillus megaterium ATCC14581 powder is 3-5:

1.

2. The compound microbial agent according to claim 1, characterized in that, The weight ratio of Bacillus subtilis ACCC10627 powder and Bacillus megaterium ATCC14581 powder in the compound microbial agent is 3.5-4.5:

1.

3. The compound microbial agent according to claim 1, characterized in that, The weight ratio of Bacillus subtilis ACCC10627 powder and Bacillus megaterium ATCC14581 powder in the compound microbial agent is 4:

1.

4. The method for preparing the composite microbial agent according to claim 1, characterized in that, Includes the following steps: S1, Activation Activation culture medium was prepared and sterilized by autoclaving at 121℃ for 20 min. Freeze-dried Bacillus subtilis ACCC10627 and Bacillus megaterium ATCC14581 were inoculated into the culture medium. Bacillus subtilis was cultured at 32℃ for 24 h and Bacillus megaterium was cultured at 30℃ for 24 h to obtain activated pure culture slant cultures. S2, Expansion Cultivation Prepare seed liquid culture medium and sterilize at 121℃ for 20 min; pick activated single colonies and inoculate them into shake flasks for culture; Bacillus subtilis culture conditions are 32℃, 180 r / min, culture for 20 h; Bacillus megaterium culture conditions are 30℃, 160 r / min, culture for 22 h, to obtain highly active seed liquids; S3, Fermentation Culture Prepare the fermentation medium and sterilize it at 121℃ for 30 min; inoculate the two seed liquids into the fermentation system at an inoculation rate of 5% (v / v) and ferment them separately. Fermentation conditions for Bacillus subtilis: culture temperature 33 ℃, aeration rate 1:0.8 vvm, stirring speed 200 r / min, fermentation for 44 h, and fermentation was terminated when the spore formation rate was ≥95%. Fermentation conditions for Bacillus megaterium: culture temperature 30 ℃, aeration rate 1:0.7 vvm, stirring speed 160 r / min, fermentation for 46 h, and fermentation was terminated when the spore formation rate was ≥93%. S4, bacterial liquid concentration, protective agent compounding and spray drying powder production The two fermentation broths were concentrated by tubular centrifugation at 6000 r / min for 10 min to collect high-concentration bacterial sludge. A heat-resistant spore protectant was added to the bacterial sludge and stirred evenly. Pure bacterial powder was prepared by spray drying at an inlet air temperature of 160 ℃ and an outlet air temperature of 70 ℃ to obtain pure Bacillus subtilis powder and pure Bacillus megaterium powder, respectively, with a single-strain powder viable cell content ≥80 billion CFU / g. S5. Preparation of compound microbial agents Accurately weigh the Bacillus subtilis powder and Bacillus megaterium powder at a fixed weight ratio of 3-5:1; then add the carrier excipients, which consist of diatomaceous earth, corn starch, and wheat bran; put all materials into a three-dimensional mixer and mix in a closed container for 35 minutes until uniformly mixed to obtain the compound microbial agent.

5. The preparation method according to claim 4, characterized in that, The activation culture medium formula in step S1 is as follows: 5 g / L peptone, 3 g / L yeast extract, 1 g / L glucose, 5 g / L sodium chloride, 15 g / L agar, pH 7.

1.

6. The preparation method according to claim 4, characterized in that, The seed liquid culture medium formulation in step S2 is as follows: glucose 15 g / L, peptone 8 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, pH 7.

1.

7. The preparation method according to claim 4, characterized in that, The fermentation medium formula in step S3 is as follows: 20 g / L corn flour, 10 g / L soybean meal, 10 g / L glucose, 3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 1 g / L calcium carbonate, pH 7.

2.

8. The preparation method according to claim 4, characterized in that, In step S4, the heat-resistant spore protectant consists of 3% seaweed oligosaccharide and 2% skim milk powder by mass of the bacterial sludge.

9. The preparation method according to claim 4, characterized in that, In step S5, the carrier additives have the following fixed mass fractions: diatomaceous earth 55%, corn starch 18%, wheat bran 12%, and the total amount of active bacteria powder added is 15%.

10. The application of the composite microbial agent according to claim 1 in the treatment of nitrogen and phosphorus pollution in water bodies.