Method for fermenting and culturing microorganisms by using catering sewage

Abstract

The invention discloses a method for culturing microorganisms by fermentation of restaurant sewage, and belongs to the technical field of biological fermentation. According to the scheme, the method comprises the following steps: restaurant sewage pretreatment: solid-liquid separation, ultrasonic-assisted oxidation and gravity settling; preparing a culture medium; inoculating and fermenting; and post-processing the product. Experiments show that the product can be efficiently applied to microbial fertilizers, pesticides and feed additives. The problems of pollutant residues, infectious microbe control and product activity in catering sewage resource utilization are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fermentation engineering, specifically a method for fermenting and culturing microorganisms by utilizing restaurant sewage, and particularly to a method for fermenting and culturing microorganisms by using restaurant sewage to prepare a culture medium. Background Art

[0002] Since the 1980s, my country's rapid economic growth has driven the continued expansion of the catering industry, and catering wastewater treatment has also entered a period of rapid development. However, despite the continuous iteration of treatment facilities and technologies, many urgent issues remain. Large quantities of improperly treated catering wastewater, mixed with other types of wastewater, are discharged directly into rivers, lakes, and other natural water bodies. Harmful substances such as oils, polycyclic aromatic hydrocarbons, and plant and animal proteins, once present in the wastewater, can cause persistent organic pollution once they seep into groundwater. For example, in a southern city, illegal discharge of wastewater from surrounding restaurants resulted in groundwater levels of carcinogens such as benzopyrene exceeding the permitted levels by tens of times. This pollution is extremely persistent, making it extremely difficult to control and restore the ecosystem, often requiring decades or even longer. Currently, the treatment of catering wastewater has become a major challenge for both catering and wastewater treatment companies, seriously impacting their sustainable development. Especially with the introduction of the national environmental protection tax and the increasing attention paid to environmental issues by a series of environmental protection policies, the traditional high-energy consumption and high-pollution treatment methods have become unsustainable, and the market urgently needs an environmentally friendly and green catering wastewater treatment method.

[0003] Currently, catering wastewater treatment technology faces multiple significant bottlenecks. In the pollutant separation process, traditional solid-liquid separation technology and gravity sedimentation processes are not effective in removing difficult-to-degrade organic matter such as oils and polycyclic aromatic hydrocarbons, which account for approximately 30%-40% of catering wastewater. The chemical oxygen demand (COD) removal rate can only reach 50-60%, which is far below the industry's ideal standard. This inefficient early treatment allows a large amount of residual pollutants to enter the subsequent microbial culture process, causing extremely serious bacterial contamination problems. The bacterial rate generally exceeds 20%, seriously interfering with the normal metabolic activities of the target bacteria and greatly affecting their activity. In terms of culture medium preparation, existing processes mostly rely on chemical sterilization methods such as high temperature and high pressure. Although they can kill common microorganisms to a certain extent, they have limited effect on heat-resistant spores, and spore residues often occur. Moreover, this type of sterilization method lacks a long-term antibacterial mechanism, making it difficult to ensure the purity of the culture medium during subsequent use. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for culturing microorganisms by fermenting restaurant wastewater, comprising the following steps:

[0005] Step 1: Pretreatment of catering wastewater

[0006] a. Solid-liquid separation: remove large particles of residue;

[0007] b. Ultrasonic-assisted oxidation: adding oxidants to the wastewater and ultrasonically treating it;

[0008] c. Gravity sedimentation: let it stand, add flocculant and adjust pH to obtain the supernatant;

[0009] Step 2: Culture medium preparation

[0010] a. Liquid culture medium: Mix the supernatant obtained in step 1 with water and add the following components:

[0011] Soybean meal, cornmeal, glucose, peptone, yeast powder, manganese sulfate, potassium dihydrogen phosphate, magnesium sulfate, nano-titanium dioxide loaded humic acid;

[0012] b. Solid medium: Add the following components to the supernatant obtained in step 1 and water:

[0013] Bran, cornmeal, soybean meal, manganese sulfate, potassium dihydrogen phosphate, magnesium sulfate, nano-titanium dioxide loaded humic acid;

[0014] c. Sterilization;

[0015] Step 3: Fermentation

[0016] a. Inoculation: The target strain is inoculated into a liquid or solid culture medium, wherein the strain is selected from: Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Beauveria bassiana, Aspergillus oryzae, Streptomyces rocheri, Bacillus licheniformis, Bacillus megaterium, Bacillus silicate, yeast, Trichoderma, one or more species;

[0017] b. Liquid culture or solid culture;

[0018] Step 4: Product post-processing

[0019] a. The fermentation broth is spray-dried to prepare bacterial powder, or directly mixed with organic fertilizer raw materials, potassium dihydrogen phosphate, urea, monoammonium phosphate, potassium sulfate, ammonium molybdate, ferrous sulfate, to prepare microbial fertilizers, pesticides or feed additives;

[0020] b. The solid culture is crushed and cyclone separated to prepare spore powder.

[0021] Preferably, the method for culturing microorganisms by fermenting restaurant wastewater comprises the following steps:

[0022] Step 1: Pretreatment of catering wastewater

[0023] a. Solid-liquid separation: Use a vibrating screen or drum screen with a pore size of 1-5 mm to remove large particles of residue (such as vegetable leaves and bone residue);

[0024] b. Ultrasonic-assisted oxidation: Add a composite oxidant (sodium persulfate 0.01-0.1%, potassium ferrate 0.005-0.02%) to the wastewater and ultrasonically treat for 20-40 minutes at a frequency of 20-60kHz and a power of 100-300W;

[0025] c. Gravity sedimentation: Let stand for 2-4 hours, add a flocculant (polyaluminum chloride or polyacrylamide, 1-200 mg / L) and adjust the pH to 6.5-7.5 to obtain the supernatant;

[0026] Step 2: Culture medium preparation

[0027] a. Liquid culture medium: Mix the supernatant obtained in step 1 with water at a ratio of 1:0-10 and add the following components (mass percentage):

[0028] Soybean meal powder 0.01-0.075%, cornmeal 0.01-0.05%, glucose 0.01-0.03%, peptone 0.001-0.005%, yeast powder 0.001-0.005%, manganese sulfate 0.0005-0.005%, potassium dihydrogen phosphate 0.0005-0.005%, magnesium sulfate 0.0005-0.005%, nano-titanium dioxide loaded humic acid (TiO2-HA) 0.001-0.01%;

[0029] b. Solid medium: Mix the supernatant obtained in step 1 with water at a ratio of 1:0-10 and add the following components (by mass percentage):

[0030] Bran 0.3-1.2%, cornmeal 0.2-0.8%, soybean meal 0.2-0.6%, manganese sulfate 0.0005-0.005%, potassium dihydrogen phosphate 0.0005-0.005%, magnesium sulfate 0.0005-0.005%, TiO2-HA 0.001-0.01%;

[0031] c. Sterilization: 121±2℃ for 30 minutes (liquid) or 120 minutes (solid);

[0032] Step 3: Fermentation

[0033] a. Inoculation: The target strain is inoculated into the culture medium, wherein the strain is selected from: Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Beauveria bassiana, Aspergillus oryzae, Streptomyces rocheri, Bacillus licheniformis, Bacillus megaterium, Bacillus silicate, yeast, Trichoderma;

[0034] b. Liquid culture: culture temperature 35-40°C, ventilation ratio 0.5-1.5, rotation speed 60-1000 rpm, culture for 20-72 hours;

[0035] c. Solid culture: temperature 28-32 ° C, regular stirring (once every 6-12 hours), culture for 24-96 hours;

[0036] Step 4: Product post-processing

[0037] a. The fermentation broth is spray-dried to prepare bacterial powder (viable count 5 billion -500 billion / g), or directly mixed with organic fertilizer raw materials, potassium dihydrogen phosphate, urea, monoammonium phosphate, potassium sulfate, ammonium molybdate, ferrous sulfate, to prepare microbial fertilizers, pesticides or feed additives;

[0038] b. The solid culture is crushed and cyclone separated to prepare spore powder.

[0039] Preferably, the ultrasonic treatment frequency in step 1b is 40 kHz, the power is 200 W, and the treatment time is 30 minutes.

[0040] Preferably, the composite oxidant is a mixed solution of 0.05% sodium persulfate and 0.01% potassium ferrate.

[0041] Preferably, the preparation method of the nano-titanium dioxide loaded humic acid (TiO2-HA) is as follows: humic acid and nano-TiO2 are mixed in a mass ratio of 1:10, stirred at pH 5-6 for 2 hours, and centrifuged to dry.

[0042] Preferably, the bacterial species in step 3a is Bacillus subtilis or Streptomyces rocherii.

[0043] Preferably, the pH value of the liquid culture medium is 6.8-7.5.

[0044] Preferably, the amount of the organic fertilizer raw material added in step 4a is 50-99.99%.

[0045] Preferably, the components of the microbial fertilizer in step 4a include 0-60% monoammonium phosphate and 0-50% potassium sulfate.

[0046] Preferably, the COD removal rate of the supernatant after gravity sedimentation in step 1c is ≥85%.

[0047] Preferably, the ventilation ratio of the liquid culture in step 3b is 1.0 and the rotation speed is 200 rpm.

[0048] Preferably, the stirring frequency of the solid culture in step 3c is once every 12 hours.

[0049] Beneficial effects of the present invention:

[0050] This invention significantly improves microbial culture efficiency through the synergistic effects of ultrasonic oxidation pretreatment and nano-antibacterial materials: COD removal rate increases from the traditional 60% to over 85%, the contaminant bacteria rate is reduced to below 5%, and the viable count of target bacteria exceeds 30 billion / mL (compared to ≤15 billion / mL using traditional methods). The photocatalytic antibacterial mechanism of nano-TiO2-HA significantly enhances storage stability (6-month survival rate ≥90%). Furthermore, the product can be directly used in the agricultural and food industries, increasing rice yields by 18% and preventing microbial diseases by ≥90%, with overall performance far exceeding existing technologies. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0053] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified. Among them, the Bacillus subtilis involved has been disclosed in Chinese patent CN202111479660.9, Bacillus thuringiensis has been disclosed in Chinese patent CN202011277047.4, Bacillus amyloliquefaciens has been disclosed in Chinese patent CN202111629296.X, Beauveria bassiana has been disclosed in Chinese patent CN202410293234.3, Aspergillus oryzae has been disclosed in CN201911400210.9, and Streptomyces rocheri has been disclosed in Chinese patent CN2018 10357031.0, Bacillus licheniformis has been disclosed in Chinese patent CN202010481276.1, Bacillus megaterium has been disclosed in Chinese patent CN201910734015.3, Bacillus silicate (Bacillus circulans) has been disclosed in Chinese patent CN201611266952.3, yeast has been disclosed in Chinese patent CN202311065243.9, and Trichoderma has been disclosed in Chinese patent CN201910717110.2.

[0054] Example 1: Bacillus subtilis liquid fermentation

[0055] Step 1: Preprocessing

[0056] The restaurant wastewater is passed through a vibrating screen (pore size 3 mm) to remove residue;

[0057] 0.05% sodium persulfate and 0.01% potassium ferrate were added and ultrasonicated at 40 kHz for 30 minutes;

[0058] After standing for 3 hours, 50 mg / L of polyaluminium chloride was added and the pH was adjusted to 7.0. The COD removal rate of the supernatant was 87%.

[0059] Step 2: Liquid culture medium preparation

[0060] Supernatant: water = 1:2, add:

[0061] Soybean meal 0.05%, cornmeal 0.03%, glucose 0.02%, peptone 0.003%, yeast powder 0.002%, manganese sulfate 0.001%, potassium dihydrogen phosphate 0.001%, magnesium sulfate 0.001%, TiO2-HA 0.005%;

[0062] Adjust the pH to 7.0 and sterilize at 121°C for 30 minutes.

[0063] Step 3: Fermentation

[0064] Inoculate with Bacillus subtilis, culture at 37°C, aeration ratio 1.0, rotation speed 200 rpm, and culture for 48 hours;

[0065] Viable bacteria count: 32 billion / mL, miscellaneous bacteria rate 3%.

[0066] Step 4: Product application

[0067] The fermentation liquid is added with 60% organic fertilizer raw materials and 0.3% potassium dihydrogen phosphate to make microbial fertilizer, which is used for rice cultivation and increases rice production by 18%.

[0068] Example 2: Solid-state fermentation of Streptomyces rocheri

[0069] Step 1: Preprocessing

[0070] Ultrasonic treatment parameters: 50 kHz, power 150 W, time 25 min;

[0071] After standing for 4 hours without adding flocculants, the COD removal rate was 82%;

[0072] Step 2: Solid culture medium preparation

[0073] Supernatant: water = 1:5, add:

[0074] Bran 1.0%, cornmeal 0.6%, soybean meal 0.4%, TiO2-HA 0.008%;

[0075] Sterilize at 121°C for 120 minutes.

[0076] Step 3: Fermentation

[0077] Inoculate Streptomyces rocherii, culture at 35°C, stir once every 12 hours, and culture for 72 hours;

[0078] Viable spore count: 6.5 billion / g, contaminant bacteria rate 5%.

[0079] Step 4: Product application

[0080] Spore powder is added with 0.005% ammonium molybdate to make a microbial pesticide for the prevention and treatment of tomato bacterial wilt with an efficiency of 92%.

[0081] Example 3: Aspergillus oryzae liquid fermentation

[0082] Step 1: Preprocessing

[0083] Ultrasonic treatment parameters: 30 kHz, power 250 W, time 40 min;

[0084] After standing for 2.5 hours, polyacrylamide 100 mg / L, pH adjusted to 6.8, COD removal rate 78%;

[0085] Step 2: Liquid culture medium preparation

[0086] Supernatant: clean water = 1:0 (pure sewage), add:

[0087] Soybean meal 0.075%, cornmeal 0.05%, glucose 0.03%, peptone 0.005%, yeast powder 0.005%, TiO2-HA 0.01%;

[0088] Adjust the pH to 7.5 and sterilize at 121°C for 30 minutes.

[0089] Step 3: Fermentation

[0090] Inoculate Aspergillus oryzae, culture at 37°C, aeration ratio 1.5, rotation speed 300 rpm, and culture for 36 hours;

[0091] Viable bacteria count: 48 billion / mL, miscellaneous bacteria rate 2%.

[0092] Step 4: Product application

[0093] Spray-dried bacterial powder (500 billion viable bacteria / g) is used in soy sauce brewing, increasing enzyme activity by 25%.

[0094] Comparative Example 1 (based on Example 1): no ultrasonic treatment, only gravity sedimentation;

[0095] Comparative Example 2 (based on Example 1): no TiO2-HA was added;

[0096] Comparative Example 3 (based on Example 1): constant temperature fermentation (37° C.);

[0097] Comparative Example 4 (based on Example 1): the composite oxidant was replaced with single sodium persulfate (0.1%).

[0098] The test results of the products obtained in all the above examples and comparative examples are shown in Table 1.

[0099] Table 1 Product Inspection

[0100]

[0101] Analysis of the above results: As can be seen from Example 1 and Comparative Example 1, the COD removal rate increased from 55% to 87%, and the number of viable bacteria doubled (150 → 32 billion / mL); the rate of foreign bacteria decreased by 17% (20% → 3%), proving that ultrasound + composite oxidants are highly effective in degrading pollutants. As can be seen from Example 1 and Comparative Example 2, the rate of foreign bacteria decreased from 25% to 3%, and the number of viable bacteria increased by 78% (180 → 32 billion / mL); the photocatalytic effect of TiO2-HA inhibited foreign bacteria and promoted the metabolism of target bacteria. As can be seen from Example 1 and Comparative Example 3, the number of viable bacteria increased by 52% (210 → 32 billion / mL) and the storage survival rate increased by 20%. Only when ultrasonic oxidation and TiO2-HA are combined can the comprehensive effect of COD removal rate ≥ 85%, foreign bacteria rate ≤ 5%, and viable bacteria count ≥ 30 billion / mL be achieved.

[0102] The present invention adopts ultrasound-assisted oxidation pretreatment: composite oxidant (sodium persulfate + potassium ferrate) cooperates with ultrasound to generate free radicals (·OH / SO4 - ·) Efficient degradation of difficult-to-decompose organic matter; Addition of nano-TiO2-HA: Humic acid-loaded nano-TiO2 continuously releases reactive oxygen species (ROS) in the culture medium, selectively inhibiting miscellaneous bacteria. In existing technologies, ultrasound is mostly used for physical fragmentation and has not been combined with potassium ferrate to degrade polycyclic aromatic hydrocarbons. The combination of humic acid and nano-TiO2 has never been used in microbial culture in this field, and there are no reports on its antibacterial mechanism. Ultimately, the viable bacterial count was increased: Example 1 achieved a viable bacterial count of 32 billion / mL (conventional methods ≤15 billion / mL); Storage stability: The 6-month survival rate was ≥90% (conventional methods ≤75%), an excellent result that exceeded our expectations. This invention solves the core problem of resource utilization of catering wastewater through the technical integration of environmental engineering, nanomaterials, and fermentation processes.

[0103] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

[0104] The above description of the present invention and its embodiments is non-limiting and is only one embodiment of the present invention. The actual application is not limited to this. In short, if a person skilled in the art is inspired by this description and, without departing from the purpose of the present invention, designs methods and embodiments similar to this technical solution without creatively designing, they shall fall within the scope of protection of the present invention.

Claims

1. A method for culturing microorganisms by fermenting restaurant wastewater, characterized in that: The following steps are involved: Step 1: Pretreatment of catering wastewater a. Solid-liquid separation: remove large particles of residue; b. Ultrasonic-assisted oxidation: adding oxidants to the wastewater and ultrasonically treating it; c. Gravity sedimentation: let it stand, add flocculant and adjust pH to obtain the supernatant; Step 2: Culture medium preparation a. Liquid culture medium: Mix the supernatant obtained in step 1 with water and add the following components: Soybean meal, cornmeal, glucose, peptone, yeast powder, manganese sulfate, potassium dihydrogen phosphate, magnesium sulfate, nano-titanium dioxide loaded humic acid; b. Solid medium: Add the following components to the supernatant obtained in step 1 and water: Bran, cornmeal, soybean meal, manganese sulfate, potassium dihydrogen phosphate, magnesium sulfate, nano-titanium dioxide loaded humic acid; c. Sterilization; Step 3: Fermentation a. Inoculation: The target strain is inoculated into a liquid or solid culture medium, wherein the strain is selected from: Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Beauveria bassiana, Aspergillus oryzae, Streptomyces rocheri, Bacillus licheniformis, Bacillus megaterium, Bacillus silicate, yeast, Trichoderma, one or more species; b. Liquid culture or solid culture; Step 4: Product post-processing a. The fermentation broth is spray-dried to prepare bacterial powder, or directly mixed with organic fertilizer raw materials, potassium dihydrogen phosphate, urea, monoammonium phosphate, potassium sulfate, ammonium molybdate, ferrous sulfate, to prepare microbial fertilizers, pesticides or feed additives; b. The solid culture is crushed and cyclone separated to prepare spore powder.

2. The method for culturing microorganisms by fermenting restaurant wastewater according to claim 1, characterized in that: The specific steps are: Step 1: Pretreatment of catering wastewater a. Solid-liquid separation: remove large particles of residue through a vibrating screen or drum screen with a pore size of 1-5mm; b. Ultrasonic-assisted oxidation: Add a composite oxidant to the wastewater and perform ultrasonic treatment at a frequency of 20-60kHz and a power of 100-300W for 20-40 minutes; c. Gravity sedimentation: Let stand for 2-4 hours, add flocculant and adjust pH to 6.5-7.5, and obtain the supernatant; Step 2: Culture medium preparation a. Liquid culture medium: Mix the supernatant obtained in step 1 with water at a ratio of 1:0-10, and add the following components by mass percentage: Soybean meal powder 0.01-0.075%, cornmeal 0.01-0.05%, glucose 0.01-0.03%, peptone 0.001-0.005%, yeast powder 0.001-0.005%, manganese sulfate 0.0005-0.005%, potassium dihydrogen phosphate 0.0005-0.005%, magnesium sulfate 0.0005-0.005%, nano titanium dioxide loaded humic acid 0.001-0.01%; b. Solid medium: Mix the supernatant obtained in step 1 with water at a ratio of 1:0-10, and add the following components by mass percentage: Bran 0.3-1.2%, cornmeal 0.2-0.8%, soybean meal 0.2-0.6%, manganese sulfate 0.0005-0.005%, potassium dihydrogen phosphate 0.0005-0.005%, magnesium sulfate 0.0005-0.005%, nano-titanium dioxide loaded humic acid 0.001-0.01%; c. Sterilization: 121±2℃ for 30 minutes or 120 minutes; Step 3: Fermentation a. Inoculation: The target strain is inoculated into the culture medium, wherein the strain is selected from: Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Beauveria bassiana, Aspergillus oryzae, Streptomyces rocheri, Bacillus licheniformis, Bacillus megaterium, Bacillus silicate, yeast, Trichoderma in one or more species; b. Liquid culture: culture temperature 35-40°C, ventilation ratio 0.5-1.5, rotation speed 60-1000 rpm, culture for 20-72 hours; c. Solid culture: temperature 28-32°C, regular stirring, culture for 24-96 hours; Step 4: Product post-processing a. The fermentation broth is spray-dried to prepare bacterial powder, or directly mixed with organic fertilizer raw materials, potassium dihydrogen phosphate, urea, monoammonium phosphate, potassium sulfate, ammonium molybdate, ferrous sulfate, to prepare microbial fertilizers, pesticides or feed additives; b. The solid culture is crushed and cyclone separated to prepare spore powder.

3. The method according to claim 2, characterized in that The ultrasonic treatment in step 1b is performed at a frequency of 40 kHz, a power of 200 W, and a treatment time of 30 minutes.

4. The method according to claim 2, characterized in that The composite oxidant is a mixed solution of 0.05% sodium persulfate and 0.01% potassium ferrate.

5. The method according to claim 2, characterized in that The preparation method of the nano-titanium dioxide loaded humic acid is as follows: humic acid and nano-TiO2 are mixed in a mass ratio of 1:10, stirred at a pH of 5-6 for 2 hours, and centrifuged to dry.

6. The method according to claim 2, characterized in that The bacterial species in step 3a is Bacillus subtilis or Streptomyces rocheri; and the pH value of the liquid culture medium is 6.8-7.

5.

7. The method according to claim 2, characterized in that The amount of the organic fertilizer raw material added in step 4a is 50-99.99%; the components of the microbial fertilizer in step 4a include 0-60% monoammonium phosphate and 0-50% potassium sulfate.

8. The method according to claim 2, characterized in that The COD removal rate of the supernatant after gravity sedimentation in step 1c is ≥85%; the aeration ratio of the liquid culture in step 3b is 1.0 and the rotation speed is 200 rpm.

9. The method according to claim 2, characterized in that The stirring frequency of the solid culture in step 3c is once every 12 hours.

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