Method for enhancing yellow serofluid treatment by hermetia illucens through cellulose degrading bacteria
By adding corn stalk powder to yellow slurry water and using cellulose to degrade bacteria, the problem that black soldier flies are difficult to deal with cellulose in yellow slurry water is solved, and the treatment efficiency and survival rate are improved.
Patent Information
- Application Number
- CN202411902762.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-03-07
AI Technical Summary
The prior art is difficult to effectively treat cellulose in yellow slurry water, resulting in inefficient treatment of black soldier fly.
Help the black soldier fly treat cellulose by adding corn stalk powder to yellow slurry water and degrading bacteria with cellulose, such as Bacillus cereus and Lactobacillus plantarum.
The black soldier flies' treatment ability to treat cellulose in corn stalk powder has been significantly improved, the survival rate of black soldier flies has been improved, and the treatment effect on yellow slurry water has been enhanced.
Smart Images
Figure CN119563766A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, relates to a technology for treating wastewater by using microorganisms, and specifically relates to a method for treating yellow pulp water by using cellulose-degrading bacteria to enhance black soldier flies. Background Art
[0002] Yellow pulp water, also known as soy water, is organic wastewater generated during the production of soy products. Yellow pulp water contains a large amount of water-soluble protein, amino acids and other nutrients. It is a high-concentration and highly biodegradable wastewater. Because it is rich in nutrients, if it is directly discharged into rivers and lakes, it will cause serious pollution to the environment.
[0003] Black soldier fly, also known as shiny flat-horned soldier fly, is a saprophytic insect of the family Smilocyprinus in the order Diptera. Black soldier fly larvae like to live in moist silt, turf, bark, animal and plant debris, or in pig sties, cow sheds, chicken sheds, and garbage dumps. They feed inside the feed during the day and move on the surface of the feed at night. They feed on fresh pig manure, cow manure, chicken manure, rotten plants and animals, as well as agricultural and sideline product scraps, tofu dregs, rice bran, wheat bran, and distiller's grains. Using insects as bioconverters to treat agricultural waste has been a hot topic in recent years, and black soldier flies have become the most popular resource conversion insect due to their high conversion efficiency and pollution degradation ability. However, current research focuses on the treatment of agricultural solid waste such as livestock and poultry manure by black soldier flies, and very few studies have been conducted on the treatment of liquid waste generated in agriculture or the food industry.
[0004] In order to solve the above problems encountered in the treatment of yellow pulp water, a method for treating yellow pulp water by using cellulose-degrading bacteria to enhance black soldier flies was developed. Summary of the invention
[0005] The purpose of the present invention is to provide a method for treating yellow pulp water by using cellulose degrading bacteria to enhance black soldier flies.
[0006] The present invention is achieved through the following technical solutions: A method for enhancing the use of black soldier flies to treat yellow pulp water by using cellulose degrading bacteria comprises the steps of adding corn stalk powder to the yellow pulp water to prepare sterile food, adding a microbial preparation prepared by using cellulose degrading bacteria, and then feeding black soldier flies to achieve the treatment of the yellow pulp water.
[0007] Furthermore, the cellulose degrading bacteria is Bacillus cereus Bacillus cereus, It comes from China General Microbiological Culture Collection Center, and the strain number is bio-00015.
[0008] The cellulose degrading bacteria is Lactobacillus plantarum Lactobacillus plantarum, It comes from China General Microbiological Culture Collection Center, and the strain number is bio-120907.
[0009] The sterile food is prepared by adding 1-2 kg of corn stalk powder to every 10 kg of yellow pulp water.
[0010] The preparation method of the microbial preparation comprises the following steps: reviving the cellulose-degrading bacteria, using LB broth liquid culture medium to expand the culture, preparing the strain fermentation liquid, and concentrating the liquid to obtain the microbial preparation.
[0011] The preparation method of the microbial preparation comprises the following steps: sterilizing LB broth liquid culture medium in a high pressure sterilizer at 115-123° C. for 15-25 minutes as a culture medium for the strain, then adding cellulose degrading bacteria at a 1% inoculation amount, culturing overnight on a horizontal shaker at 37° C. and 180 rpm to obtain a strain fermentation liquid, centrifuging at 5000 rpm for 5 minutes, discarding the supernatant, resuspending the bacterial precipitate with a sterile PBS buffer, and concentrating to obtain a strain working liquid.
[0012] The present invention uses cellulose-degrading bacteria to enhance the ability of black soldier flies to treat yellow slurry water and corn stalk powder, wherein the mixing of yellow slurry water and corn stalk powder has the following advantages: corn stalk powder can absorb and reduce the water content of yellow slurry water to adapt to the growth of black soldier flies; after adding corn stalk powder, black soldier flies will not be exposed to a pure liquid environment all the time, thereby improving the survival rate of black soldier flies; corn stalk powder itself is also a kind of waste, and corn stalk powder is also treated while treating yellow slurry water. However, yellow slurry water contains a large amount of water-soluble proteins, amino acids and other nutrients, which is conducive to the breeding of black soldier flies; however, corn stalk powder is rich in substances such as lignin wrapped in cellulose and hemicellulose, and cellulose has multiple glucose units, which are connected by β-1,4-glycosidic bonds, so that the plant cell wall has a rigid structure, so the cellulose in corn stalk powder is difficult for black soldier flies to utilize. The invention selects Bacillus cereus and Lactobacillus plantarum with cellulose degradation ability to help black soldier flies to process cellulose contained in raw materials, thereby enhancing the effect of black soldier flies in processing yellow pulp water and corn stalk powder.
[0013] Compared with the prior art, the present invention has the following advantages: cellulose-degrading bacteria can significantly improve the black soldier fly's ability to process cellulose in corn stalk powder, and the corn stalk powder adjusts the water content of yellow pulp water to adapt to the growth of black soldier flies, so that the black soldier flies will not be exposed to a pure liquid environment all the time, thereby improving the survival rate of the black soldier flies. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 It is the transparent circle of Bacillus cereus on Congo red medium; Figure 2 is the degradation effect of Bacillus cereus on filter paper strips; Figure 3 The curve of cellulase activity of Bacillus cereus changing with time; Figure 4 The dry matter reduction rate after culturing for 15 days according to the method of Examples 6-8; Figure 5 The bioconversion rate after 15 days of cultivation according to the method of Examples 6-8. DETAILED DESCRIPTION
[0015] The present invention is further described below by way of examples and drawings.
[0016] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels, among which yellow pulp water was obtained from Huainan Bailan Group, black soldier fly eggs were purchased from Yuqi Ecological Breeding Factory in Fuyang City, Anhui Province, and corn straw powder was purchased from the farmers' market. Example
[0017] Preparation of microbial inoculants Strain 1: Bacillus cereus Bacillus cereus It comes from China General Microbiological Culture Collection Center, and the strain number is bio-00015.
[0018] Species 2: Lactobacillus plantarum Lactobacillus plantarum It comes from China General Microbiological Culture Collection Center, and the strain number is bio-120907.
[0019] S1.1. Resuscitate the purchased strain according to the instructions provided by the preservation center, expand the culture using LB broth liquid culture medium, and then preserve it using the glycerol seed preservation method.
[0020] S1.2. Add 20 mL of LB broth liquid culture medium into a 50 mL conical flask, seal it with tissue culture sealing film, sterilize it in an autoclave at 121°C for 20 min, and store it in a refrigerator as the culture medium for the strain.
[0021] S1.3. Add strain 1 and strain 2 at a 1% inoculum rate to the culture medium prepared in step S1.2, and culture overnight on a horizontal shaker at 37°C and 180 rpm to obtain strain fermentation broth 1 and strain fermentation broth 2.
[0022] S1.4. Take 1 mL of the fermentation broth 1 and 2 obtained in step S1.3 and centrifuge at 5000 rpm for 5 min. Discard the supernatant and resuspend the bacterial pellet in 1 mL of sterile PBS buffer. Use a UV spectrophotometer to measure the absorbance A1 and A2 at a wavelength of 600 nm, using sterile PBS as a control.
[0023] S1.5, according to the absorbance measured, the fermentation broth of strain 1 and strain 2 were concentrated to OD 600is 1, recorded as strain working solution 1 and strain working solution 2. Example
[0024] The method for preparing sterile culture medium and sterile black soldier flies is as follows: S2.1. Collect 1 g of black soldier fly eggs and place them in a 1.5 ml EP tube.
[0025] S2.2. Add 30 mL of fruit fly food (which can be used as black soldier fly food) to a 200 mL pudding bottle, seal it with tissue culture sealing film, sterilize it in an autoclave at 121°C for 20 min, and store it in a refrigerator as a developmental culture medium for black soldier fly eggs.
[0026] S2.3. Place the culture medium obtained in S2.2 into a clean bench for ultraviolet disinfection. Then wash the black soldier fly eggs collected in S2.1 with sodium hypochlorite, Dettol, alcohol, sterile PBST, and sodium hypochlorite to obtain sterile black soldier fly eggs. Place the eggs into a culture medium and culture them at 28-32°C and 60-70% relative humidity for 5-7 days to obtain sterile black soldier fly larvae. Example
[0027] The method for making sterile food mixed with yellow pulp water and straw powder is as follows: S3.1. Add 30 g of yellow pulp water and 5 g of corn stalk powder into a 200 mL pudding bottle, seal it with tissue culture sealing film, sterilize it in an autoclave at 121°C for 20 min, and store it in a refrigerator as a culture medium for black soldier flies. Example
[0028] Prepare Congo red solid medium and filter paper strip degradation medium as follows: S4.1. Congo red solid medium (g / L): K,HPO 0.5, MgSO4·7H2O 0.25, CMC-Na 2, Congo red 0.2, agar 14.0, gelatin 2.0, distilled water 1000 mL, pH 7.0. Treat in an autoclave at 121℃ for 20 min, cool to 70℃, pour into a culture dish, solidify, and store in a refrigerator.
[0029] S4.2, filter paper degradation medium (g / L): (NH4)2SO4 1.0, KH2PO4 1.0, MgSO4·7H2O 0.5, yeast extract 0.1, 2 filter paper strips / bottle, natural pH. After being treated in an autoclave at 121℃ for 20 min, the medium was stored in a refrigerator.
[0030] S4.3. Enzyme production medium (g / L): lb 20, CMC-Na 10, distilled water 1000 mL. Autoclave at 121℃ for 20 min and store in a refrigerator. Example
[0031] Verify the cellulose degradation ability of Bacillus cereus, method 1: one group uses an inoculation loop to apply the fermentation liquid 1 of the strain of Example 2 on the Congo red solid medium and place it in a 37°C incubator for 24 hours for observation, and the other group uses sterile water to apply it on the Congo red solid medium and place it in a 37°C incubator for 24 hours for observation. Method 2: one group draws the fermentation liquid 1 of the strain into a 50ml conical flask and then adds 30ml of filter paper degradation culture medium to culture at 37°C and 180 rpm, and the other group draws sterile water into a 50ml conical flask and then adds 30ml of filter paper degradation culture medium to culture at 37°C and 180 rpm. Method 3: draw 3ml of the fermentation liquid of the strain 1 into a 200ml conical flask and then adds 100ml of enzyme-producing degradation culture medium to culture at 35°C and 120 rpm. The liquid fermentation product is drawn every 12h, centrifuged at 5000rpm for 10min, and the supernatant is taken, which is the crude enzyme liquid. At this time, the crude enzyme liquid can be used to determine the cellulase activity. Take a certain amount of crude enzyme solution and sterilize it in a 121℃ high temperature and high pressure sterilizer for 30min as a blank control. Take a test tube, add 0.5mL of appropriately diluted enzyme solution and 1mL of 0.1mol / L acetic acid-sodium acetate buffer to it, and put it in a 50℃ water bath for 5min for preheating; then add a filter paper strip to the enzyme solution and buffer mixture, and perform enzymatic hydrolysis for 1h. After the reaction, add 1.5mL of DNS colorimetric solution to the test tube, and then put the test tube containing DNS colorimetric solution into boiling water for 10min. After the reaction, place it under running water for rapid cooling, and then make up 10mL with ultrapure water. Finally, measure the absorbance at 540nm, calculate the glucose content based on the absorbance, and calculate the enzyme activity value based on the grams of glucose generated. The production of 1μg glucose by 1mL of cellulase solution is defined as a filter paper enzyme activity unit, expressed as U. Filter paper enzyme activity calculation formula: U= A×M×1000 N×T Note: U represents enzyme activity, unit is U / mL; A represents glucose content on the standard curve; M represents dilution multiple; N represents sample volume; T represents reaction time.
[0032] Experimental Example 6 In a clean bench, 5 sterile black soldier flies grown for 5-7 days in Example 2 were added to the sterile food mixed with the three yellow slurry water and straw powder of the experimental example (3 groups of repetitions) and then cultured at 28-32°C and 60-70% relative humidity for 5 days.
[0033] Experimental Example 7 The method is the same as that of Experimental Example 6 except that 1 OD of the strain working solution 1 in Experimental Example 1 is added at the same time as the sterile black soldier flies.
[0034] Experimental Example 8 The method is the same as that of Experimental Example 6 except that 1 OD of the strain working solution 2 in Experimental Example 1 is added together with the sterile black soldier flies. Example
[0035] The results of Examples 5 and 6 are shown in Figure 1 and Figure 2 , Figure 1 It is the transparent circle of Bacillus cereus on Congo red medium. Figure 2 is the degradation effect of Bacillus cereus on filter paper strips, Figure 3 The curve of cellulase activity of Bacillus cereus over time. It can be seen from the figure that Bacillus cereus has the ability to degrade cellulose and can produce a transparent circle on the Congo red medium. It also has the ability to degrade filter paper when inoculated in the filter paper degradation medium. Example
[0036] The larvae separated in Examples 6-8 were weighed, and the statistical results are shown in Table 1 Table 1 Example Average weight of fresh insects (mg) Example 6 60.9 Example 7 154.2 Example 8 61.9 Table 2 Example Average body length of fresh insects (mm) Example 6 11.2 Example 7 15.0 Example 8 11.5 It can be seen from Tables 1 and 2 that the growth of the black soldier flies added with Bacillus cereus in Example 7 is stronger than that of the sterile black soldier flies but slightly weaker than that of the conventionally grown black soldier flies, which proves that Bacillus cereus can help the black soldier flies better utilize the corn stalk powder in the yellow pulp water.
[0037] Effect Example 11 The method of Example 6-8 was followed, the culture time was 15, the larvae were separated, cleaned, dried and weighed, the residue after the insect treatment was dried and weighed, and the dry matter reduction rate and bioconversion rate were calculated. The statistical results are shown in Figure 4 and Figure 5 As can be seen from the figure, compared with Example 6, the dry matter reduction rate and bioconversion rate of 15 cultured according to the method of Example 7 are improved, but not as good as Example 8, indicating that Bacillus cereus can enhance the effect of black soldier flies on yellow pulp water and corn stalk powder compared with the control group.
Claims
1. A method for treating yellow pulp water by enhancing black soldier flies with cellulose degrading bacteria, characterized in that: The method comprises adding corn stalk powder into yellow pulp water to prepare sterile food, adding a microbial preparation prepared by cellulose degrading bacteria, and then feeding black soldier flies to achieve the treatment of yellow pulp water.
2. The method for treating yellow pulp water with cellulose-degrading bacteria-enhanced black soldier flies according to claim 1, characterized in that: The cellulose degrading bacteria is Bacillus cereus Bacillus cereus, It comes from China General Microbiological Culture Collection Center, and the strain number is bio-00015.
3. The method for treating yellow pulp water with cellulose-degrading bacteria-enhanced black soldier flies according to claim 1, characterized in that: The cellulose degrading bacteria is Lactobacillus plantarum Lactobacillus plantarum, It comes from China General Microbiological Culture Collection Center, and the strain number is bio-120907.
4. The method for treating yellow pulp water with cellulose-degrading bacteria-enhanced black soldier flies according to claim 1, characterized in that: The sterile food is prepared by adding 1-2 kg of corn stalk powder to every 10 kg of yellow pulp water.
5. The method for treating yellow pulp water with cellulose-degrading bacteria-enhanced black soldier flies according to claim 1, characterized in that: The preparation method of the microbial preparation comprises the following steps: reviving the cellulose-degrading bacteria, using LB broth liquid culture medium to expand the culture, preparing the strain fermentation liquid, and concentrating the liquid to obtain the microbial preparation.
6. The method for treating yellow pulp water with cellulose-degrading bacteria-enhanced black soldier flies according to claim 5, characterized in that: The preparation method of the microbial preparation comprises the following steps: sterilizing LB broth liquid culture medium in a high pressure sterilizer at 115-123° C. for 15-25 minutes as a culture medium for the strain, then adding cellulose degrading bacteria at a 1% inoculation amount, culturing overnight on a horizontal shaker at 37° C. and 180 rpm to obtain a strain fermentation liquid, centrifuging at 5000 rpm for 5 minutes, discarding the supernatant, resuspending the bacterial precipitate with a sterile PBS buffer, and concentrating to obtain a strain working liquid.