An autotrophic denitrifying microbial agent and its preparation method and application

Abstract

The present invention discloses an autotrophic denitrifying microbial agent and its preparation method and application, which belong to the field of microbial technology; the invention comprises the following steps: (1) first-level seed fermentation culture: inoculating the bacterial strain into a mother liquid culture medium for fermentation culture to obtain a microbial yeast liquid; (2) second-level immobilized continuous culture: inoculating the microbial yeast liquid prepared in step (1) into a continuous flow liquid culture medium in a fermentation tank for immobilized continuous culture until the number of viable bacteria in the bacterial liquid reaches 10 8 Fermentation is complete when the concentration of granules / mL or higher is exceeded. A continuous flow liquid culture medium is continuously added to the fermenter and an equal volume of bacterial liquid is discharged to obtain an autotrophic denitrifying microbial inoculum. The fermenter is charged with iron sulfide compound particles and electron shuttle particles. The preparation method provided by the present invention enables high-density and rapid cultivation of mixed autotrophic denitrifying bacteria. This method is not susceptible to contamination by other bacteria, is low-cost, and can be industrialized.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a method for rapidly culturing autotrophic denitrifying bacteria and an application thereof in treating nitrogen-containing sewage. Background Art

[0002] As the country attaches more importance to the environment, sewage discharge standards continue to rise, and the requirements for total nitrogen removal are also increasing to alleviate environmental problems such as eutrophication of water bodies. Conventional sewage treatment methods often use the denitrification process to convert residual nitrate nitrogen into harmless nitrogen gas through biochemical reactions, which escapes from the water, ultimately achieving effective treatment of total nitrogen. Compared with conventional denitrification methods that require the addition of carbon sources, autotrophic denitrification technology for removing nitrates from water is a new biological denitrification technology. Common methods include sulfur autotrophic denitrification and iron autotrophic denitrification. These two types of microorganisms use sulfur and sulfide and iron and iron compounds as electron donors, respectively, to reduce nitrates to nitrogen gas.

[0003] However, currently, autotrophic microorganisms grow slowly and are difficult to cultivate, resulting in long startup times and difficulty in large-scale promotion of autotrophic denitrification applications. Therefore, research and development to achieve rapid proliferation of autotrophic denitrifying microorganisms is of great significance. Conventional culture uses liquid culture media for amplification, but it is unable to efficiently utilize solid sulfur compounds and iron compounds, resulting in significant waste. Currently, there are no reports on the continuous fermentation process of sulfur\iron autotrophic microorganisms. Based on this, this patent has developed a method for rapid growth of autotrophic microorganisms and the preparation of engineered bacterial agents, which can achieve rapid proliferation of autotrophic microorganisms and effectively improve the denitrification efficiency of wastewater treatment. Summary of the Invention

[0004] In view of this, the main purpose of the present invention is to provide a method for preparing an autotrophic denitrifying microbial agent. The preparation method provided by the present invention can achieve high-density culture of mixed autotrophic denitrifying bacteria; streamline the culture process, solve the problem of difficulty in proliferating autotrophic denitrifying bacteria, and solve the problems of conventional immobilized culture requiring the addition of carriers, which increases costs and causes contamination by other strains and long culture times. This method is not easily contaminated by foreign bacteria, has low cost, and can be used for industrial production.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] A method for preparing an autotrophic denitrifying microbial agent comprises the following steps:

[0007] (1) Primary seed fermentation culture: inoculating the bacterial strain into the mother liquid culture medium for fermentation culture to obtain a microbial yeast solution; wherein the bacterial strain includes sulfur autotrophic denitrifying microorganisms and iron autotrophic denitrifying microorganisms;

[0008] (2) Secondary immobilized continuous culture:

[0009] The microbial yeast liquid prepared in step (1) was inoculated into the continuous flow liquid culture medium of the fermentation tank and cultured continuously until the number of viable bacteria in the liquid reached 10 8 The fermentation is complete when the continuous flow liquid culture medium is continuously added into the fermentation tank and the equal volume of bacterial liquid is discharged to obtain the autotrophic denitrifying microbial agent;

[0010] The fermentation tank is added with ferrous sulfide compound particles and electron shuttle particles; the temperature of the immobilized continuous culture is 30-37° C., and the pH is 6.5-7.2.

[0011] The present invention can produce 10 viable bacteria in the bacterial solution after culturing in the fermentation tank for 72-96 hours according to the culture conditions. 8 / mL, at this time, the continuous flow liquid culture medium can be continuously introduced (no need to add bacteria again), and an equal volume of cultured bacterial liquid can be discharged at the same time.

[0012] Furthermore, in step (1), the sulfur autotrophic denitrifying microorganisms include one or more of denitrifying Thiobacillus, Thiobacillus thioreductus, Naples Thiobacillus, Thiobacillus thiooxidans, Thiobacillus ferrooxidans, novel Thiobacillus, Intermediate Thiobacillus, and Metabolic Incomplete Thiobacillus; the iron autotrophic denitrifying microorganisms include one or more of nitrite-reducing bacteria, halophilic cocci, Acinetobacter, Stenotrophomonas maltophilia, Microbacterium, and Pseudomonas.

[0013] Furthermore, in step (1), the mother liquid culture medium formula is: potassium nitrate 0.15-0.25g / L, magnesium chloride 0.04-0.06g / L, dipotassium hydrogen phosphate 0.15-0.25g / L, potassium dihydrogen phosphate 0.15-0.25g / L, sodium bicarbonate 0.5-0.8g / L, and a trace element solution 2-5ml / L mixed with 0.1-1g / L each of EDTA, magnesium sulfate heptahydrate, copper sulfate pentahydrate, ferrous sulfate heptahydrate, zinc sulfate, cobalt chloride hexahydrate, and manganese chloride tetrahydrate.

[0014] Furthermore, in step (1), the culture temperature is 35-37°C, the rotation speed is 140-200 r / min, the culture time is 24-36h, and the concentration reaches 10 9 cfu / ml or above.

[0015] Furthermore, in step (2), the formula of the continuous flow liquid culture medium is: ammonium sulfate 0.9-1.0 g / L, glucose 1.0-1.5 g / L, dipotassium hydrogen phosphate 0.1-0.2 g / L, magnesium sulfate heptahydrate 0.2-0.3 g / L, anhydrous calcium chloride 0.15-0.2 g / L, potassium dihydrogen phosphate 0.1-0.15 g / L, potassium bicarbonate 2-2.5 g / L, potassium nitrate 0.2-0.25 g / L, and magnesium chloride 0.05-0.06 g / L.

[0016] Furthermore, in step (2), the inoculation amount of the microbial yeast solution is 5-15 v / v% (volume ratio of the microbial yeast solution to the fermentation tank). If the amount of microbial yeast solution added is too low, the reproduction speed will be affected, and if the amount added is too high, the cost will increase accordingly.

[0017] Furthermore, in step (2), the size of the iron sulfide compound particles is 3-7 mm, and the amount added is 30-50% of the volume of the fermentation tank. The iron sulfide compound particles are specifically at least one of pyrite and pyrrhotite; the electron shuttle particles are 3-5 mm tourmaline particles, and the amount added is 5% of the volume of the fermentation tank.

[0018] In the present invention, tourmaline is selected as the electron shuttle, which can increase the growth rate of autotrophic denitrifying microorganisms and reduce the growth of miscellaneous bacteria, especially heterotrophic bacteria.

[0019] In the present invention, the electron shuttle only serves to increase the electron transfer capacity of the autotrophic denitrifying microorganisms in the reaction and is not lost during the reaction process. Adding too much electron shuttle will increase the cost. Adding too little electron shuttle and the concentration is too low, and it cannot be completely mixed with the sulfide iron compound particles, which affects the ability of the autotrophic denitrifying microorganisms to obtain electrons.

[0020] Furthermore, in step (2), the daily dosage of the continuous flow liquid culture medium is 1 / 4-1 / 6 of the volume of the fermentation tank.

[0021] The second object of the present invention is to provide an autotrophic denitrifying microbial agent prepared by any of the above methods.

[0022] Specifically, the fermented bacterial agent can be directly used as a liquid bacterial agent after being discharged from the fermentation tank, or it can be used as a solid bacterial agent after centrifugal separation.

[0023] The present invention also aims to provide an application of the above-mentioned microbial agent, which specifically comprises adding the agent to a sewage treatment device or equipment, introducing sewage, leaving it for 1-3 days for exposure, and then discharging it. Subsequently, water is introduced at 40-50% of the water intake. After running for 2 days, the water intake is increased to 70-80%. After running for 2 days, water is introduced at the normal water intake to complete the strain application.

[0024] Furthermore, when the microbial agent is a liquid microbial agent, the microbial agent is added at a rate of 10-15 L / m 3 Liquid bacterial agent; when the bacterial agent is a solid bacterial agent, the bacterial agent is added in an amount of 2-5 kg ​​solid bacterial agent / m 3 .

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) The present invention simplifies the culture process and solves the problem of difficulty in proliferating autotrophic denitrifying bacteria. Conventional immobilized culture requires the addition of carriers, which increases costs and causes contamination by other bacteria species and long culture times. This method is not easily contaminated by foreign bacteria, has low cost, and can be industrialized for production.

[0027] (2) The present invention improves the utilization efficiency of solid sulfur compounds and iron compounds during the expansion process of liquid culture medium, and at the same time realizes the continuous fermentation of sulfur-iron autotrophic microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a growth curve diagram of autotrophic denitrifying microorganisms cultivated in a continuous culture mode and a discontinuous culture mode provided in an embodiment of the present invention.

[0029] Figure 2 It is the total nitrogen removal rate of the cultured autotrophic denitrifying microorganisms, conventional biochemical pond sludge (including heterotrophic denitrifying microorganisms), and conventional biochemical pond sludge + carbon source acetic acid for sewage with different nitrogen concentrations provided by the embodiments of the present invention.

[0030] Figure 3 This is a growth curve diagram of the tourmaline electron shuttle group, the no electron shuttle group, and the biochar electron shuttle group in Comparative Example 2. DETAILED DESCRIPTION

[0031] The following description sets forth numerous specific details to facilitate a thorough understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific implementations disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] Example 1 Preparation of a yeast solution containing mixed microorganisms

[0034] Mother culture medium formula: potassium nitrate 0.2 g / L, magnesium chloride 0.05 g / L, dipotassium hydrogen phosphate 0.2 g / L, potassium dihydrogen phosphate 0.2 g / L, sodium bicarbonate 0.6 g / L, and a trace element solution 3 ml / L mixed with 0.5 g / L each of EDTA, magnesium sulfate heptahydrate, copper sulfate pentahydrate, ferrous sulfate heptahydrate, zinc sulfate, cobalt chloride heptahydrate, and manganese chloride heptahydrate.

[0035] One or more of Thiobacillus denitrificans, Thiobacillus thioparus, Thiobacillus naples, Thiobacillus thiooxidans, Thiobacillus ferrooxidans, Thiobacillus novellas, Thiobacillus intermedius, and Thiobacillus perometabolis, as well as iron-autotrophic denitrifying microorganisms: Acinetobacter guillouiae, Paracoccus, Acinetobacter, Stenotrophomonas, Microbacterium sp., Pseudomonas sp) select one or more microorganisms from the bacterial species, add them into the mother liquid culture medium and culture them to obtain a microbial yeast solution; in this embodiment, Thiobacillus denitrificans, Thiobacillus thiooxidans, Thiobacillus ferrooxidans, Thiobacillus intermedia, nitrite-reducing bacteria, Acinetobacter, and Microbacterium are specifically selected; the culture conditions are: 30-37°C, 170r / min for 30h, and the concentration reaches 10 9 cfu / ml or above.

[0036] Example 2 Continuous culture of microorganisms

[0037] The formula of the continuous flow liquid culture medium for bacterial fermentation is: ammonium sulfate 1.0 g / L, glucose 1.2 g / L, dipotassium hydrogen phosphate 0.15 g / L, magnesium sulfate heptahydrate 0.25 g / L, anhydrous calcium chloride 0.18 g / L, potassium dihydrogen phosphate 0.12 g / L, potassium bicarbonate 2.2 g / L, potassium nitrate 0.23 g / L, and magnesium chloride 0.055 g / L.

[0038] The microbial yeast liquid prepared in Example 1 was inoculated into the continuous flow fermentation medium at an inoculation rate of 10 v / v%.

[0039] 30% sulfur particles with a volume size of 4-6 mm and 5% tourmaline particles with a volume size of 3-5 mm were added to the fermentation tank as a fixed carrier for bacterial growth. The mixture was cultured at pH 7.2, temperature 35°C, and cultured for 80 hours until the number of viable bacteria in the bacterial solution reached 10 8Fermentation is complete when the number of viable bacteria reaches 10. 8 pc / mL or more, the continuous culture was successful.

[0040] Monitor the concentration of microorganisms in the tank and obtain the growth curve such as Figure 1 shown.

[0041] The fermented bacterial agent is centrifuged to make its moisture content 50%-70% to prepare a solid bacterial agent.

[0042] Example 3 Use of microbial agents

[0043] Potassium nitrate was used to prepare solutions with nitrogen concentrations of 20, 30, 40, 50, and 60 mg / L, and trace elements were added to simulate actual sewage. The cultured autotrophic denitrifying microbial liquid agent (dosage of 10 L / m 3 ), conventional biochemical pool sludge (containing heterotrophic denitrifying microorganisms) (dosage of 3000 mg / L), conventional biochemical pool sludge and carbon source (ammonium acetate, the dosage is based on the calculated theoretical denitrification COD), continuous water inflow, HRT = 4h. The total nitrogen results of the reactor effluent are as follows Figure 2 As shown in the figure, after cultivation, the total nitrogen removal rate of autotrophic denitrifying microorganisms for wastewater containing different nitrogen concentrations has remained above 90%, while the total nitrogen removal rate of the heterotrophic denitrifying microorganism group has gradually decreased with the increase of the concentration of nitrogen-containing wastewater. The heterotrophic denitrifying microorganism + ammonium acetate group can maintain a higher total nitrogen removal rate due to the addition of carbon source ammonium acetate, but it is still slightly lower than that of the autotrophic denitrifying microorganism group after cultivation. The results show that autotrophic denitrifying microorganisms can efficiently remove nitrogen from wastewater without adding an external carbon source.

[0044] Comparative Example 1 Discontinuous Culture of Microorganisms

[0045] The culture medium formulation was the same as for the continuous culture method. Fermentation conditions were as follows: the microbial fermentation solution prepared in Example 1 was inoculated at a 10 v / v% inoculum, 30 v / v% of sulfur particles with a particle diameter of 4-6 mm, and 5% of tourmaline particles with a volume size of 3-5 mm were added, the pH was 7.0, and the temperature was 35°C. The culture was carried out for 7 days.

[0046] The growth curve of discontinuous culture is also as follows Figure 1 As shown, the continuous culture system in Example 2 was able to maintain a relatively high bacterial liquid concentration except in the initial period; the microbial concentration in the discontinuous culture method of this example decreased significantly after 5 days.

[0047] Comparative Example 2

[0048] This example also carried out the cultivation method according to Example 2, setting up a tourmaline electron shuttle group and a group without an electron shuttle, and selecting biochar as the electron shuttle group.

[0049] Growth curves such as Figure 3 As shown in the figure, the growth curve without the electron shuttle was significantly delayed compared to the growth curve with the electron shuttle, and the peak value was also lower than that with the electron shuttle. After 14 days, the bacterial concentration decreased. When biochar was used as the electron shuttle, its growth curve was more consistent with that of the tourmaline group, with a slight delay, but the peak value was still lower than that of the tourmaline group, and it was unable to maintain a high concentration.

[0050] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0051] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing an autotrophic denitrifying microbial agent, characterized in that: The steps include: (1) Primary seed fermentation culture: inoculating the bacterial strain into the mother liquid culture medium for fermentation culture to obtain a microbial yeast solution; wherein the bacterial strain includes sulfur autotrophic denitrifying microorganisms and iron autotrophic denitrifying microorganisms; (2) Secondary immobilized continuous culture: The microbial yeast liquid prepared in step (1) was inoculated into the continuous flow liquid culture medium of the fermentation tank and cultured continuously until the number of viable bacteria in the liquid reached 10 8 The fermentation is complete when the continuous flow liquid culture medium is continuously added into the fermentation tank and the equal volume of bacterial liquid is discharged to obtain the autotrophic denitrifying microbial agent; The fermentation tank is added with iron sulfide compound particles and electron shuttle particles; the temperature of the immobilized continuous culture is 30-37° C., and the pH is 6.5-7.2; In step (1), the mother liquid culture medium is formulated as follows: 0.15-0.25 g / L potassium nitrate, 0.04-0.06 g / L magnesium chloride, 0.15-0.25 g / L dipotassium hydrogen phosphate, 0.15-0.25 g / L potassium dihydrogen phosphate, 0.5-0.8 g / L sodium bicarbonate, and 2-5 ml / L of a trace element solution mixed with 0.1-1 g / L each of EDTA, magnesium sulfate heptahydrate, copper sulfate pentahydrate, ferrous sulfate heptahydrate, zinc sulfate, cobalt chloride hexahydrate, and manganese chloride tetrahydrate; In step (1), the culture temperature is 35-37° C. and the culture time is 24-36 h; In step (2), the formula of the continuous flow liquid culture medium is: ammonium sulfate 0.9-1.0 g / L, glucose 1.0-1.5 g / L, dipotassium hydrogen phosphate 0.1-0.2 g / L, magnesium sulfate heptahydrate 0.2-0.3 g / L, anhydrous calcium chloride 0.15-0.2 g / L, potassium dihydrogen phosphate 0.1-0.15 g / L, potassium bicarbonate 2-2.5 g / L, potassium nitrate 0.2-0.25 g / L, and magnesium chloride 0.05-0.06 g / L; In step (2), the size of the ferrous sulfide compound particles is 3-7 mm, and the amount added is 30-50% by volume. The ferrous sulfide compound particles are specifically at least one of pyrite and pyrrhotite; the electron shuttle particles are 3-5 mm tourmaline particles, and the amount added is 5% by volume.

2. The method according to claim 1, wherein: In step (1), the sulfur autotrophic denitrifying microorganisms include one or more of denitrifying Thiobacillus, Thiobacillus thioreductus, Naples Thiobacillus, Thiobacillus thiooxidans, Thiobacillus ferrooxidans, novel Thiobacillus, intermediate Thiobacillus, and incomplete Thiobacillus metabolism; the iron autotrophic denitrifying microorganisms include one or more of nitrite-reducing bacteria, halophilic cocci, Acinetobacter, Stenotrophomonas maltophilia, Microbacterium, and Pseudomonas.

3. The method according to claim 1, wherein: In step (1), the rotation speed during the culture is 140-200 r / min.

4. The method according to claim 1, wherein: In step (2), the inoculation amount of the microbial yeast solution is 5-15 v / v%.

5. The method according to claim 1, wherein: In step (2), the daily dosage of the continuous flow liquid culture medium is 1 / 4-1 / 6 of the volume of the fermentation tank.

6. The autotrophic denitrifying microbial agent prepared by the method according to any one of claims 1 to 5.

7. The use of the bacterial agent according to claim 6 in sewage treatment, characterized in that: The application is specifically as follows: after adding the bacterial agent to the sewage treatment device or equipment, the sewage is introduced, and the sewage is allowed to remain there for 1-3 days for exposure before being discharged. Subsequently, water is introduced at 40-50% of the water intake. After running for 2 days, the water intake is increased to 70-80%. After running for 2 days, water is introduced at the normal water intake to complete the bacterial strain application.

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