Culture method of bacillus pasteurii and application of bacillus pasteurii in sewage treatment

Abstract

The invention relates to a culture method of bacillus pasteurii and application of the bacillus pasteurii in sewage treatment.The culture method comprises the steps that the bacillus pasteurii is sequentially subjected to activation culture, seed culture and fermentation culture, and the fermentation culture is that seed liquid activated after seed culture is inoculated into a fermentation culture medium to be subjected to two-stage culture, the first fermentation stage is carried out at a first temperature, the second fermentation stage is carried out at a second temperature, and the first temperature is 2 DEG C higher than the second temperature. Through the temperature difference design of 2 DEG C of two-stage fermentation culture, the first fermentation stage promotes rapid proliferation of thalli, the second fermentation stage induces efficient synthesis of EPS and urease, dynamic balance of thalli growth and product synthesis is achieved, and the problem that the product synthesis efficiency of a traditional constant-temperature process is low is solved. The bacillus pasteurii fermentation liquor obtained by the method disclosed by the invention has high-expression EPS and urease activity, so that the biological flocculation and mineralization consolidation effects can be improved, and the bacillus pasteurii fermentation liquor is well suitable for high-density eel culture sewage treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a method for culturing Bacillus pasteurianus and application thereof in sewage treatment. Background Art

[0002] Eel farming produces large amounts of manure, leftover bait, and dead algae, leading to high concentrations of organic matter, nitrogen and phosphorus nutrients, and suspended particulate matter in the water. Improper treatment can easily lead to eutrophication and black, smelly sediment. Among traditional treatment methods, microbial methods have become a key technology for eel farming wastewater treatment due to their targeted degradation of organic matter and nitrogen and phosphorus pollutants, high process flexibility, low operating costs, and environmental friendliness. However, single microbial methods have low treatment efficiency and produce loose, easily disintegrated flocs.

[0003] In the microbial treatment system, Bacillus pasteurianus has shown special application value in aquaculture wastewater treatment due to its unique physiological characteristics and metabolic functions. During the fermentation process, Bacillus pasteurianus can secrete extracellular polymers (EPS, the main components of which are polysaccharides, proteins, etc.) and metabolize to produce urease: on the one hand, EPS absorbs suspended particles in wastewater through charge neutralization and bridging, prompting them to form flocs. At the same time, urease decomposes urea to produce CO3 2- , and Ca in water 2+ The combination forms calcium carbonate precipitate, further strengthening the structural stability of the flocs; on the other hand, the continued growth of the strain can utilize organic matter such as proteins and lipids in the feces, simultaneously reducing the COD and ammonia nitrogen concentrations in the water body and avoiding secondary pollution.

[0004] However, the Bacillus pasteurianus obtained using the current traditional culture process has the problems of low EPS production and low urease activity, which makes the flocculation and mineralization effects unstable. Therefore, its application in aquaculture wastewater treatment is very limited and concentrated in specific scenarios.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a culture method of Bacillus pasteurianus and application of the same in sewage treatment.

[0007] The technical solution adopted in the present invention is:

[0008] On one hand, the present invention provides a method for culturing Bacillus pasteurianus, comprising sequentially performing activation culture, seed culture, and fermentation culture on Bacillus pasteurianus, wherein the fermentation culture comprises inoculating a seed liquid activated after the seed culture into a fermentation medium for a two-stage culture, wherein the first fermentation stage is performed at a first temperature, and the second fermentation stage is performed at a second temperature, wherein the first temperature is 2°C higher than the second temperature.

[0009] The present invention proposes a method for culturing Bacillus pasteurianus. Through a two-stage fermentation culture with a 2°C temperature difference design, the first fermentation stage promotes rapid bacterial proliferation, while the second fermentation stage induces efficient synthesis of EPS and urease, achieving a dynamic balance between bacterial growth and product synthesis, and solving the problem of low product synthesis efficiency in traditional constant temperature culture processes.

[0010] Preferably, the first temperature is 29-31°C.

[0011] Preferably, the first fermentation stage is carried out for no more than 12 hours.

[0012] Preferably, the second fermentation stage is carried out for 16-24 hours.

[0013] Preferably, the inoculation amount of the fermentation culture is 0.5-2%.

[0014] Preferably, the fermentation medium comprises: 18-22 g / L yeast extract, 0.8-1.2 g / L urea, 13-18 g / L molasses, 0.2-0.6 g / L magnesium sulfate, 0.005-0.015 g / L nickel chloride, and pH 8.0-9.0.

[0015] Another aspect of the present invention provides an application of the Bacillus pasteurianus fermentation liquid obtained by the culturing method described in the above technical solution in the treatment of high-density eel aquaculture wastewater.

[0016] The Bacillus pasteurianus fermentation broth obtained by the method of the present invention has significant biological activity. Its highly expressed extracellular polymers and urease activity can enhance the biological flocculation and mineralization consolidation effects, and is well suitable for the treatment of high-density eel farming wastewater, thus filling the technical gap of Bacillus pasteurianus in the treatment of eel farming wastewater.

[0017] Preferably, the method comprises the following steps: filtering the eel breeding wastewater by grid, adding calcium salt to the filtered wastewater, mixing evenly, adding the Bacillus pasteurianus fermentation liquid, stirring and mixing intermittently and then standing to obtain clarified water.

[0018] Preferably, the amount of the Bacillus pasteurianus fermentation broth is 0.05-0.5% of the volume of the filtered sewage, and the concentration of calcium salt in the filtered sewage is 0.05-0.1 mol / L.

[0019] Preferably, after the standing, the method further comprises: preparing the mineralized solidified sludge into an organic fertilizer base material, and recycling the clarified water body after treating it in an artificial wetland system. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the flocculation and mineralization effects of different amounts of fermentation liquid in treating eel farming wastewater in Example 2;

[0021] Figure 2 Microscopic effects of bioflocculation in Example 3 (right picture) and the control group (left picture) are shown. DETAILED DESCRIPTION

[0022] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. Instead, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0023] On one hand, the present invention provides a method for culturing Bacillus pasteurianus, comprising sequentially performing activation culture, seed culture, and fermentation culture on Bacillus pasteurianus, wherein the fermentation culture comprises inoculating a seed liquid activated after seed culture into a fermentation medium for a two-stage culture, wherein the first fermentation stage is performed at a first temperature, and the second fermentation stage is performed at a second temperature, wherein the first temperature is 2°C higher than the second temperature.

[0024] Existing studies have shown that during the growth of Bacillus pasteurianus, the urease activity at different stages is not consistent. Usually, the urease activity of Bacillus pasteurianus will rise rapidly in the early stage of culture (logarithmic growth phase), which is attributed to the rapid reproduction of bacteria and vigorous metabolic activity. Afterwards, due to the reduction of nutrients and the accumulation of metabolites, the urease activity slowly decreases. The change pattern of EPS needs to be combined with specific culture conditions, and there are few related studies. Therefore, those skilled in the art usually improve the overall level by optimizing the urease activity in the early stage of culture. Based on kinetic studies, the inventors found that a 2°C temperature difference design was used to achieve a "proliferation-synthesis" metabolic switch. The first fermentation stage focused on bacterial growth (but the product synthesis activity was low), and the second fermentation stage accelerated product synthesis. The final product synthesis was significantly improved compared to a single constant temperature process. It should be noted that if the temperature difference is too high (the first temperature is too high or the second temperature is too low), it is easy to destroy the metabolic balance, which is not conducive to the improvement of EPS production and urease activity.

[0025] In addition, the present invention is not particularly limited to the method of activation culture and seed culture. In an embodiment of the present invention, for example, activation culture comprises the following steps: the pasteurian Bacillus strain (purchased from China General Microorganism Collection Center, deposit number CGMCC 1.3687) is inoculated onto an LB solid culture medium plate (containing tryptone 10g / L, yeast powder 5g / L, NaCl10g / L, agar 15g / L, pH 7.2), and cultured at 30°C for 24h; seed culture comprises the following steps: picking an activated pasteurian Bacillus single colony and inoculating it into 50mL liquid seed culture medium (containing yeast extract 8-12g / L, urea 0.2-0.8g / L, molasses 5-10g / L, magnesium sulfate 0.2-0.6g / L, pH 8.0-9.0), shaking and culturing at 30°C and 200rpm for 24h until the bacterial solution becomes thick (OD 600 =2.0).

[0026] Preferably, the first temperature is 29-31°C. In the present invention, the first temperature is controlled to be 29-31°C, which is suitable for bacterial growth and provides a cell factory for the subsequent large-scale synthesis of products. For example, the first temperature can be any value of 29°C, 29.5°C, 30°C, 30.5°C, 31°C, etc.; for further example, when the first temperature is selected from the above values, the second temperature can be any value of 27°C, 27.5°C, 28°C, 28.5°C, 29°C, etc.

[0027] In the present invention, if the first fermentation stage lasts significantly longer than 12 hours, the consumption of nutrients and the entry of the microorganism into the stable phase may cause partial cell death or product degradation, thereby affecting the product synthesis efficiency of the second fermentation stage.

[0028] Preferably, the second fermentation stage is carried out for 16-24 hours. In the present invention, if the second fermentation stage is less than 16 hours, the product synthesis is incomplete; if the second fermentation stage is more than 24 hours, the product may be degraded.

[0029] Preferably, the inoculum size of the fermentation culture is 0.5-2%. In the present invention, for example, the inoculum size of the fermentation culture can be any value among 0.5%, 0.8%, 1%, 1.3%, 1.5%, 1.8%, 2%, etc., without particular limitation.

[0030] Preferably, the fermentation medium comprises: 18-22 g / L yeast extract, 0.8-1.2 g / L urea, 13-18 g / L molasses, 0.2-0.6 g / L magnesium sulfate, 0.005-0.015 g / L nickel chloride, pH 8.0-9.0. In the present invention, yeast extract serves as a nitrogen source and growth factor, urea provides a nitrogen source and urease substrate, molasses is a low-cost carbon source and provides trace elements, magnesium sulfate maintains enzyme activity, and nickel chloride activates urease activity. These components synergistically promote bacterial proliferation and target product synthesis. Combined with the two-stage fermentation culture, the EPS yield can reach 9.2 g / L and the urease activity can reach 102 U / mL.

[0031] On the other hand, the present invention provides an application of a Bacillus pasteurian fermentation broth obtained by the cultivation method of the above-mentioned technical solution in the treatment of high-density eel farming wastewater. The Bacillus pasteurian fermentation broth obtained by the method of the present invention contains high EPS and urease activity, and can well treat the water quality characteristics of high-density eel farming wastewater with high organic matter, high ammonia nitrogen, high suspended solids and total phosphorus, and the two are highly compatible. First, Bacillus pasteurian can utilize organic matter such as protein and lipids in eel farming wastewater to simultaneously reduce COD and ammonia nitrogen; second, the EPS in the fermentation broth absorbs suspended particles such as leftover bait through charge neutralization and bridging effect, accelerating flocculation and precipitation; third, the urease in the fermentation broth can decompose urea to produce CO3 2- , and Ca 2+ The combination can form calcium carbonate precipitation, which not only strengthens the floc structure, but also co-precipitates with phosphate to improve TP removal rate.

[0032] Preferably, the method comprises the following steps: filtering the eel farming wastewater by grid, adding calcium salt to the filtered wastewater, mixing evenly, adding Bacillus pasteurianus fermentation liquid, stirring and mixing intermittently and then standing to obtain a clarified water body. In the present invention, grid filtration can preliminarily separate large particles of impurities to prevent large particles from wrapping around and reducing the activity of the fermentation liquid; the introduction of calcium salt simultaneously promotes CO3 2- PO4 3- The highly active Bacillus pasteurianus fermentation broth exhibits dual bioflocculation and mineralization properties, synergistically achieving efficient mineralization and consolidation of suspended solids and pollutant degradation. This method also provides valuable insights and recommendations for treating other aquaculture wastewater with similar water quality characteristics. For example, the calcium salt can be calcium chloride, calcium nitrate, or calcium acetate. The stabilization conditions can be a temperature of 25-35°C for 10-12 hours.

[0033] Preferably, the amount of Bacillus pasteurianus fermentation broth is 0.05-0.5% of the volume of the filtered sewage, and the concentration of calcium salt in the filtered sewage is 0.05-0.1 mol / L. In the present invention, adding 0.05-0.5% of Bacillus pasteurianus fermentation broth to sewage has a better sewage treatment effect.

[0034] Preferably, after the standing, the process further comprises: converting the mineralized solidified sludge into an organic fertilizer base material, treating the clarified water body in an artificial wetland system, and recycling it. In the present invention, a closed loop of resource utilization is achieved through sludge → organic fertilizer, supernatant → reuse.

[0035] Example 1

[0036] A method for culturing Bacillus pasteurianus comprises sequentially performing activation culture, seed culture, and fermentation culture on Bacillus pasteurianus, specifically comprising the following steps:

[0037] S1. Activation culture: Inoculate the Bacillus pasteurianus strain onto an LB solid medium plate (containing 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar, pH 7.2) and culture at 30°C for 24 h.

[0038] S2. Seed culture: A single colony of activated Bacillus pasteurianus was inoculated into 50 mL of liquid seed culture medium (containing 10 g / L yeast extract, 0.5 g / L urea, 7.5 g / L molasses, 0.5 g / L magnesium sulfate, pH 8.5) and cultured with shaking at 30°C and 200 rpm for 24 h.

[0039] S3. Fermentation culture: The activated seed liquid was inoculated into a fermentation medium (containing 20 g / L yeast extract, 1 g / L urea, 15 g / L molasses, 0.5 g / L magnesium sulfate, 0.01 g / L nickel chloride, pH 8.5) at an inoculum size of 1% and cultured in two stages. The first fermentation stage was cultured at 30° C. and 200 rpm for 12 h, and the second fermentation stage was cultured at 28° C. and 100 rpm for 24 h (i.e., culture was continued until the 36th h) to obtain a Bacillus pasteurianus fermentation broth.

[0040] Example 2

[0041] The invention discloses an application of the Bacillus pasteurianus fermentation liquid obtained in Example 1 in the treatment of high-density eel farming wastewater, comprising the following steps: filtering 1 L of eel farming wastewater (water quality: COD 450 mg / L, SS 250 mg / L, TP 36.5 mg / L) through a 1 mm grid; adding calcium chloride to the filtered wastewater to a value of 0.05 mol / L, mixing the mixture uniformly; then adding 0.05%, 0.1%, 0.25%, and 0.5% of the volume of the filtered wastewater, respectively; mixing the mixture intermittently with stirring for 30 minutes, and then standing the mixture at 30°C for 12 hours to allow flocculation and precipitation to obtain clarified water.

[0042] Example 3

[0043] The invention discloses an application of the Bacillus pasteurianus fermentation liquid obtained in Example 1 in the treatment of high-density eel farming wastewater, comprising the following steps: filtering 100 L of eel farming wastewater (water quality: COD 450 mg / L, SS 250 mg / L, TP 36.5 mg / L) through a 1 mm grid, adding calcium chloride to the filtered wastewater to a concentration of 0.05 mol / L, mixing the mixture uniformly, adding 0.1% of the volume of the filtered wastewater to the Bacillus pasteurianus fermentation liquid, stirring and mixing the mixture intermittently for 30 minutes, standing the mixture at 30°C for 12 hours to allow flocculation and precipitation, preparing an organic fertilizer base material from the mineralized solidified sludge, and treating the clarified water in an artificial wetland system for recycling.

[0044] Comparative Example 1

[0045] The main difference between this comparative example and Example 1 is that in the fermentation culture process of step S3, a constant temperature process (30° C., 200 rpm for 36 hours) is used instead of a two-stage culture. The other steps remain unchanged.

[0046] Comparative Example 2

[0047] The main difference between this comparative example and Example 1 is that in the fermentation culture process of step S3, LB liquid culture medium is used instead of fermentation medium. The other steps remain unchanged.

[0048] Comparative Example 3

[0049] The main difference between this comparative example and Example 1 is that in step S3, a constant temperature process (30°C, 200 rpm for 36 hours) is used instead of a two-stage culture, and LB liquid culture medium is used instead of a fermentation medium. The other steps remain unchanged.

[0050] Comparative Example 4

[0051] The main difference between this comparative example and Example 1 is that in the fermentation culture process of step S3, the time of the first fermentation stage is adjusted from 12 hours to 16 hours. The other steps remain unchanged.

[0052] Comparative Example 5

[0053] The main difference between this comparative example and Example 1 is that in the fermentation culture process of step S3, the second fermentation stage time is adjusted from 24 hours to 28 hours. The other steps remain unchanged.

[0054] Comparative Example 6

[0055] The main difference between this comparative example and Example 1 is that during the fermentation culture process in step S3, the temperature of the second fermentation stage is adjusted from 28° C. to 26° C. The other steps remain unchanged.

[0056] Performance Testing

[0057] (1) Investigation of bacterial growth and product synthesis kinetics in Example 1

[0058] As shown in Table 1, the OD of bacterial solution was 600 From 0.11 to 2.125, the bacteria rapidly proliferated, while the EPS and urease synthesis changed slowly. 600 The values ​​were all around 2.00, with little change. The growth rate of the bacteria slowed down, while the synthesis of EPS and urease accelerated, with EPS production increasing by 8.51 times and urease production increasing by 7.28 times. This shows that the present invention, through two-stage temperature switching culture, can promote rapid proliferation of bacteria in the first fermentation stage and simultaneously induce accelerated synthesis of EPS and urease in the second fermentation stage.

[0059] Table 1

[0060]

[0061] (2) Comparison of EPS production and urease activity between Example 1 and Comparative Examples 1-6

[0062] As can be seen from Table 2, compared with Comparative Examples 1-3, Example 1 of the present invention shows that the two-stage fermentation culture process of the present invention works synergistically with the special fermentation medium to significantly improve the EPS production and urease activity of Bacillus pasteurianus; compared with Comparative Examples 4-6, Example 1 shows that the first fermentation stage time is too long, the second fermentation stage time is too long, and the second fermentation stage temperature is too low (resulting in an increased temperature difference) will reduce the EPS production and urease activity, thereby illustrating the accuracy and necessity of the process parameters (two-stage time, temperature) of the present invention.

[0063] Table 2

[0064]

[0065] (3) Effects of different amounts of fermentation liquid on eel farming wastewater treatment in Example 2

[0066] Observe the clarified water (supernatant) after flocculation, and measure the pH, flocculation and COD changes. The results are recorded in Figure 1 and Table 3. The results show that after adding different amounts of fermentation liquid and letting it react for 12 hours, the pH value of the supernatant of the aquaculture wastewater is slightly lower than that of the control group, the removal rate of suspended particles (SS) reaches more than 92%, the COD removal rate reaches more than 86%, and the TP removal rate reaches more than 90%. Among them, the addition amount of 0.1% has the best effect, with an SS removal rate of 95.25%, a COD removal rate of 89.67%, and a TP removal rate of 92.5%. The above results show that adding 0.05-0.5% of the highly active fermentation liquid of the present invention to eel aquaculture wastewater can well adsorb suspended matter and solidify it to form dense large flocs, promote the sedimentation of the flocs, and the live bacteria in the fermentation liquid continue to grow and decompose organic matter such as proteins and lipids, thereby reducing the COD content in the water body. At the same time, Ca 2+ The salts formed with phosphate will settle in the sludge.

[0067] Table 3

[0068]

[0069] (4) Eel farming wastewater flocculation mineralization consolidation effect and product resource utilization effect in Example 3

[0070] After flocculation and sedimentation, filter the sediment residue at the bottom and observe the solidification state of the flocculent under a microscope ( Figure 2 Compared to the control group without fermentation broth, the flocculated mineralized precipitate from Example 3, which added 0.1% fermentation broth, produced larger, more tightly bound, and firmer flocculation. The flocs also exhibited increased strength and significantly enhanced resistance to hydraulic erosion. Measurement of the moisture content of the flocs revealed that the control group had a moisture content of 68%, while the sludge from Example 3 had a moisture content of 45%, significantly reducing sludge production.

[0071] At the same time, the mineralized consolidated sludge was made into an organic fertilizer base material. The seed germination experiment showed that the germination rate was 88.5% and the plant weight increased by 10%. After the clarified water was treated by the artificial wetland system, the ammonia nitrogen content was reduced to 4.2 mg / L, the COD was reduced to 8 mg / L, the TP was 0.45 mg / L, and the total nitrogen was less than 5 mg / L, which can be recycled.

[0072] In summary, the highly active Bacillus pasteurianus fermentation broth obtained using the cultivation method of the present invention can produce EPS up to 9.2 g / L and urease activity up to 102 U / mL. When applied to eel aquaculture wastewater, it can simultaneously achieve suspended solids mineralization and consolidation (SS removal rate ≥ 92%), COD removal rate ≥ 86%, and total phosphorus removal rate ≥ 90%, and can also be used as a resource. This method has the advantages of low cost, no chemical residues or secondary pollution, and is suitable for large-scale aquaculture wastewater treatment.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing Bacillus pasteurianus, characterized in that: The method comprises sequentially performing activation culture, seed culture and fermentation culture on Bacillus pasteurianus, wherein the fermentation culture is to inoculate the seed liquid activated after the seed culture into the fermentation medium for two-stage culture, wherein the first fermentation stage is performed at a first temperature and the second fermentation stage is performed at a second temperature, and the first temperature is 2°C higher than the second temperature.

2. The method for culturing Bacillus pasteurianus according to claim 1, wherein The first temperature is 29-31°C.

3. The method for culturing Bacillus pasteurianus according to claim 1, wherein The first fermentation stage is carried out for no more than 12 hours.

4. The method for culturing Bacillus pasteurianus according to claim 1, wherein The second fermentation stage is carried out for 16-24 hours.

5. The method for culturing Bacillus pasteurianus according to claim 1, wherein The inoculum size of the fermentation culture is 0.5-2%.

6. The method for culturing Bacillus pasteurianus according to claim 1, wherein The fermentation medium comprises: 18-22 g / L yeast extract, 0.8-1.2 g / L urea, 13-18 g / L molasses, 0.2-0.6 g / L magnesium sulfate, 0.005-0.015 g / L nickel chloride, and a pH value of 8.0-9.

0.

7. Use of the Bacillus pasteurianus fermentation liquid obtained by the culturing method according to claim 1 in the treatment of high-density eel aquaculture wastewater.

8. The use according to claim 7, characterized in that The following steps are involved: The eel breeding wastewater is subjected to grid filtration, calcium salt is added to the filtered wastewater, mixed evenly, and then the Bacillus pasteurianus fermentation liquid is added, mixed intermittently, and then allowed to stand to obtain clarified water.

9. The use according to claim 8, characterized in that The dosage of the Bacillus pasteurianus fermentation liquid is 0.05-0.5% of the volume of the filtered sewage, and the concentration of calcium salt in the filtered sewage is 0.05-0.1 mol / L.

10. The use according to claim 8, characterized in that After the standing, the method further includes: preparing the mineralized solidified sludge into an organic fertilizer base material, and processing the clarified water body through an artificial wetland system for recycling.

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