Autotrophic denitrification microbial agent as well as preparation method and application thereof

Abstract

The invention discloses an autotrophic denitrification microbial agent as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. Comprising the following steps: (1) primary seed fermentation culture: inoculating a strain into a mother liquor culture medium for fermentation culture to obtain a microbial fermentation mother liquor; and (2) secondary immobilized continuous culture: inoculating the microbial fermentation mother liquor prepared in the step (1) into a continuous flow liquid culture medium of a fermentation tank, and carrying out immobilized continuous culture until the viable count in the bacterial liquid reaches 108 / mL or above, thereby completing the fermentation, a continuous flow liquid culture medium is continuously put into the fermentation tank, isopyknic bacterial liquid is discharged, the autotrophic denitrification microbial agent is obtained, and iron sulfide particles and electron shuttle particles are added into the fermentation tank. The preparation method provided by the invention can realize high-density rapid culture of mixed bacteria of autotrophic denitrifying bacteria, and the method is not easy to be infected with infectious microbes, is low in cost and can realize industrial production.

Description

Technical Field

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

[0002] As the country pays more attention to the environment, sewage discharge standards continue to rise, and the requirements for total nitrogen removal are also increasing to reduce 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 and ultimately achieves 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 type of biological denitrification technology. Common ones 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 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 medium for amplification, but it is impossible to efficiently utilize solid sulfur compounds and iron compounds, resulting in great waste. At present, 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 appreciation of autotrophic microorganisms and preparation of engineered bacterial agents, which can achieve rapid proliferation of autotrophic microorganisms and effectively improve the efficiency of sewage denitrification 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 difficulty in the proliferation of autotrophic denitrifying bacteria, and solve the problems of increased costs and contamination by other strains and long culture time caused by the need to add carriers for conventional immobilized culture. This method is not easy to be contaminated by foreign bacteria, has low cost, and can realize 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) is inoculated into the continuous flow liquid culture medium of the fermentation tank for fixed continuous culture until the number of viable bacteria in the bacterial liquid reaches 10 8 When the fermentation is completed, the continuous flow liquid culture medium is continuously added into the fermentation tank and the same volume of bacterial liquid is discharged to obtain the autotrophic denitrifying microbial agent;

[0010] Wherein, iron sulfide compound particles and electron shuttle particles are added into the fermentation tank; the temperature of the fixed continuous culture is 30-37° C., and the pH is 6.5-7.2.

[0011] After the fermentation tank is cultured for 72-96 hours according to the culture conditions, the number of live bacteria in the bacterial liquid can reach 10 8 / mL or more, then the continuous flow liquid culture medium can be continuously introduced (without adding 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 Thiobacillus denitrificans, Thiobacillus excreta, Thiobacillus Naples, Thiobacillus thiooxidans, Thiobacillus ferrooxidans, Thiobacillus novelta, Thiobacillus intermedia, and Thiobacillus metabolica incomplete; the iron autotrophic denitrifying microorganisms include one or more of nitrite-reducing bacteria, Halophilic Coccus, Acinetobacter, Stenotrophomonas maltophilia, Microbacterium, and Pseudomonas.

[0013] Furthermore, in step (1), the mother liquid culture medium formula is: 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 heptahydrate, and manganese chloride heptahydrate.

[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 and 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 the 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 thereof 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 thereof 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 is only used 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, and adding too little electron shuttle and the concentration is too low, and it cannot be completely mixed with the sulfide iron compound particles, affecting 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 the lower sediment can be used as a solid bacterial agent after centrifugal separation.

[0023] The purpose of the present invention is also to provide an application of the above-mentioned microbial agent, which specifically includes adding the agent to a sewage treatment device or equipment, passing sewage, leaving it for 1-3 days for air exposure and then discharging it, then introducing water at a rate of 40-50%, and after running for 2 days, increasing the water rate by 70-80%, and after running for 2 days, introducing water at a normal rate to complete the strain application.

[0024] Furthermore, when the bacterial agent is a liquid bacterial agent, the bacterial agent is added at 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 time. 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 liquid culture medium expansion process, and at the same time realizes the continuous fermentation of sulfur-iron autotrophic microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a growth curve diagram of autotrophic denitrifying microorganisms cultivated in the continuous cultivation mode and the discontinuous cultivation mode provided in the embodiments of the present invention.

[0029] Figure 2 It is the total nitrogen removal rate of the cultured autotrophic denitrifying microorganisms, conventional biochemical pool sludge (including heterotrophic denitrifying microorganisms), and conventional biochemical pool 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] Many specific details are set forth in the following description to facilitate a full 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 can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation 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 the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] Example 1 Preparation of a mixed microbial yeast solution

[0034] Mother liquid culture medium formula: potassium nitrate 0.2g / L, magnesium chloride 0.05g / L, dipotassium hydrogen phosphate 0.2g / L, potassium dihydrogen phosphate 0.2g / L, sodium bicarbonate 0.6g / L, and 3ml / L of trace element solution mixed with 0.5g / 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, Thiobacillus perometabolis, and iron autotrophic denitrifying microorganisms: Acinetobacter guillouiae, Paracoccus, Acinetobacter, Stenotrophomonas, Microbacterium sp., Pseudomonas sp. sp) select one or more microorganisms from the bacterial species, put them into the mother liquid culture medium for cultivation, and obtain a microbial yeast solution; in this embodiment, denitrifying Thiobacillus, Thiobacillus thiooxidans, Thiobacillus ferrooxidans, Intermediate Thiobacillus, 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 and above.

[0036] Example 2 Continuous culture of microorganisms

[0037] The formula of continuous flow liquid culture medium for bacterial fermentation is: ammonium sulfate 1.0g / L, glucose 1.2g / L, dipotassium hydrogen phosphate 0.15g / L, magnesium sulfate heptahydrate 0.25g / L, anhydrous calcium chloride 0.18g / L, potassium dihydrogen phosphate 0.12g / L, potassium bicarbonate 2.2g / L, potassium nitrate 0.23g / L, magnesium chloride 0.055g / 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 conditions. The fermentation was carried out at a pH of 7.2, a temperature of 35°C, and a culture time of 80 h until the number of live bacteria in the bacterial solution reached 10 8Fermentation is complete when the number of viable bacteria reaches 10. 8 If the number of cells / mL is above, the continuous culture is 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 water content 50%-70% to make 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 inoculant (dosage of 10 L / m 3 ), conventional biochemical pool sludge (including heterotrophic denitrifying microorganisms) (dosage is 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 remained above 90%, while the total nitrogen removal rate of the heterotrophic denitrifying microorganism group gradually decreased with the increase of the concentration of nitrogen-containing wastewater. The heterotrophic denitrifying microorganism + ammonium acetate group was able to maintain a relatively high total nitrogen removal rate due to the addition of the carbon source ammonium acetate, but it was still slightly lower than that of the autotrophic denitrifying microorganism group after cultivation. The results showed 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 medium formula is the same as that of the continuous culture method. The fermentation conditions are as follows: the microbial yeast solution prepared in Example 1, the inoculation amount is 10 v / v%, 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 are added, the pH is 7.0, the temperature is 35°C, and the culture is carried out for 7 days.

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

[0047] Comparative Example 2

[0048] This example also carried out the cultivation in the same manner as in 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 curve Figure 3 As shown in the figure, it can be seen that without the addition of electron shuttles, the growth curve is significantly delayed compared to the growth curve with the addition of electron shuttles, and the maximum value is also lower than the maximum value with the addition of electron shuttles, and the bacterial liquid concentration decreases after 14 days. When biochar is selected as the electron shuttle, its growth curve is consistent with the change law of the tourmaline group, with a slight delay, but the maximum value is still lower than the maximum value of the tourmaline group, and it is also impossible to maintain a high concentration all the time.

[0050] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached 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) is inoculated into the continuous flow liquid culture medium of the fermentation tank for fixed continuous culture until the number of viable bacteria in the bacterial liquid reaches 10 8 When the fermentation is completed, the continuous flow liquid culture medium is continuously added into the fermentation tank and the same volume of bacterial liquid is discharged to obtain the autotrophic denitrifying microbial agent; Wherein, iron sulfide compound particles and electron shuttle particles are added into the fermentation tank; the temperature of the fixed continuous culture is 30-37° C., and the pH is 6.5-7.

2.

2. The method according to claim 1, characterized in that: In step (1), the sulfur autotrophic denitrifying microorganisms include one or more of denitrifying Thiobacillus, Thiobacillus excreta, Naples Thiobacillus, Thiobacillus thiooxidans, Thiobacillus ferrooxidans, novel Thiobacillus, intermediate Thiobacillus, and metabolically incomplete Thiobacillus; 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, characterized in that: 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 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 heptahydrate, and manganese chloride heptahydrate.

4. The method according to claim 1, characterized in that: In step (1), the culture temperature is 35-37°C, the rotation speed is 140-200r / min, and the culture time is 24-36h.

5. The method according to claim 1, characterized in that: 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.

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

7. The method according to claim 1, characterized in that: 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.

8. The method according to claim 1, characterized in that: 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.

9. The autotrophic denitrifying microbial agent prepared by the method according to any one of claims 1 to 8.

10. The use of the bacterial agent according to claim 9 in sewage treatment, characterized in that: The application is specifically to add the bacterial agent to the sewage treatment device or equipment, pass the sewage, leave it for 1-3 days of exposure and then discharge it, then add water at a water intake rate of 40-50%, after running for 2 days, increase the water intake rate to 70-80%, and after running for 2 days, add water at a normal water intake rate to complete the bacterial strain application.

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