Method for promoting hermetia illucens to convert chicken manure by utilizing uric acid degrading bacteria

Abstract

The invention discloses a method for promoting hermetia illucens to convert chicken manure by utilizing uric acid degrading bacteria, belongs to the technical field of microorganisms, and solves the problems that in the prior art, when the hermetia illucens treats the chicken manure, the hermetia illucens grow slowly due to too high uric acid content in the chicken manure, and the hermetia illucens sand needs to be fermented again. The invention relates to a method for promoting hermetia illucens to convert chicken manure by using uric acid degrading bacteria, which comprises the following steps: inoculating the uric acid degrading bacteria into an LB broth liquid culture medium, culturing overnight on a horizontal shaking table under the conditions of 37 DEG C and 180 rpm, centrifugally concentrating strain fermentation liquor to obtain a strain working solution, adding the strain working solution into the chicken manure, and treating the chicken manure by using hermetia illucens larvae which are fattened for 1-5 days. When the hermetia illucens treats the chicken manure, uric acid in the chicken manure can be remarkably reduced, growth and development of the hermetia illucens can be promoted, the process steps can be shortened, the problem that the hermetia illucens need to be fermented again is solved, and the production efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a method for promoting the conversion of chicken manure by black soldier fly using uric acid-degrading bacteria. Background Art

[0002] The black soldier fly, scientifically known as Hermetia illucens, also called the shiny black soldier fly, is a saprophagous insect belonging to the genus Hermetia in the family Stratiomyidae of the order Diptera. The larvae of the black soldier fly prefer to live in moist mud, turf, bark, plant and animal residues, or in pig houses, cattle houses, chicken houses, and garbage dumps. They feed inside the feed during the day and move to the surface of the feed at night, feeding on fresh pig manure, cow manure, chicken manure, rotten plants and animals, as well as agricultural by-products such as bean curd residue, rice bran, wheat bran, and distiller's grains. Using insects as biological converters to treat agricultural waste has been a hot topic in recent years, and the black soldier fly has become the most popular resource-converting insect due to its high conversion efficiency and pollution degradation ability.

[0003] Chickens obtain protein through food intake, and nitrogen in the protein is converted into amino acids during digestion. Amino acids in the chicken body are metabolized into nitrogen compounds such as urea and uric acid. Different from mammals, chickens do not excrete nitrogen through urea but mainly through uric acid. Uric acid in chicken manure has a dual effect on the growth and development of the black soldier fly. High concentrations of uric acid can have a toxic effect on the black soldier fly, inhibiting its growth, while appropriate amounts of uric acid can provide a nitrogen source for the black soldier fly through microbial decomposition, which is helpful for its growth. Therefore, if the uric acid contained in chicken manure is not properly treated, it will pose a threat to the environment and insects. When fermenting chicken manure to reduce the uric acid content in chicken manure, there are limitations such as long time consumption and large land occupation, and it will also have an impact on the environment.

[0004] To solve the above problems existing in the prior art, a method for promoting the conversion of chicken manure by black soldier fly using uric acid-degrading bacteria has been developed. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for promoting the conversion of chicken manure by black soldier fly using uric acid-degrading bacteria, so as to solve the problems existing in the prior art, such as slow growth of black soldier fly and the need for re-fermentation of insect frass when the black soldier fly processes chicken manure due to the excessive uric acid content in chicken manure.

[0006] The present invention is achieved by the following technical solutions: A method for promoting the conversion of chicken manure by black soldier fly using uric acid-degrading bacteria, the steps are as follows: inoculate uric acid-degrading bacteria into LB broth liquid medium, and culture overnight at 37°C and 180 rpm on a horizontal shaker. Centrifuge and concentrate the strain fermentation broth to obtain a strain working solution, add it to chicken manure, and use black soldier fly larvae that have been fattened for 1-5 days to process the chicken manure.

[0007] Furthermore, the uric acid-degrading bacterium is Enterococcus faecalis.

[0008] The addition amount of the strain working solution is 0.1 mL of the strain working solution per 1000 g of chicken manure.

[0009] The uric acid-degrading bacterium is obtained according to the following steps: (1) Sampling: Surface-sterilize the black soldier fly larvae, dissect and take out the intestinal contents under sterile conditions, and place them in sterile physiological saline for treatment; (2) Microbial culture: Dilute the intestinal contents, spread them on the LB broth medium supplemented with uric acid, and perform constant-temperature culture. Select single colonies with consistent morphology and transfer them to a new medium for culture; (3) Screening of high-performance strains: Expand and culture the strains obtained from the culture in LB medium, inoculate the bacterial liquid into the high-uric acid medium, and select the strains that can degrade uric acid fastest by detecting the change in substrate concentration. After domestication, the desired strains are obtained.

[0010] The method for surface-sterilizing the black soldier fly larvae is to disinfect the larvae with 70% ethanol, and then wash them with sterile physiological saline or phosphate buffer solution to remove surface microorganisms.

[0011] The addition amount of uric acid is 0.02 - 0.03% of the total amount of the LB broth medium, making the uric acid reach a nearly saturated state.

[0012] The high-uric acid selective pressure medium is LB liquid medium + uric acid selective pressure: Tryptone: 10 g / L; Yeast Extract: 5 g / L; Sodium Chloride (NaCl): 10 g / L; Uric Acid: 2 g / L; pH: 7.0; The high-uric acid selective pressure medium is used to screen microorganisms that can tolerate high concentrations of uric acid or can metabolize uric acid, especially for screening some bacteria or yeasts that can decompose or tolerate uric acid. In such a selective medium, uric acid plays the role of selective pressure, allowing only those microorganisms with specific metabolic pathways (for example, enzymes that can decompose uric acid or tolerate uric acid) to grow.

[0013] For the selection of the strain that can degrade uric acid fastest, add 50 mL of autoclaved high-uric acid medium to a conical flask, add 0.1 OD600 of the bacterial liquid, take 1 mL of the culture solution every 6 hours, centrifuge it, and then use a uric acid detection kit to detect the uric acid content. After 48 h, count the uric acid degradation effects of different strains, and select the optimal strain for domestication and preservation according to the uric acid degradation amount.

[0014] In view of the problem that the growth of black soldier flies is slow due to the influence of uric acid during the process of treating chicken manure, the present invention develops a method of artificially adding a bacterial agent that can promote the degradation of uric acid, so as to achieve the purpose of improving the efficiency and output during the process of treating chicken manure with black soldier flies. If the uric acid contained in chicken manure is not properly treated, it will pose a threat to the environment. Usually, people will ferment chicken manure to reduce the environmental impact, or re-ferment the vermicompost after treatment with black soldier flies. These measures have limitations such as long time consumption and the need for additional sites. The bacterial agent for promoting uric acid degradation is obtained through the isolation of intestinal microorganisms of black soldier flies, the screening of high-performance strains, the domestication of strains, and purification and identification. The obtained strains are used for the preparation of the bacterial agent, and then used for the treatment of chicken manure.

[0015] Compared with the prior art, when the present invention treats chicken manure with black soldier flies, it can significantly reduce the uric acid in chicken manure, which can not only promote the growth and development of black soldier flies, but also shorten the process steps, solve the problem that the vermicompost needs to be re-fermented, and improve the production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a screening process diagram for the screening of high-performance strains of the present invention.

[0017] Figure 2 It is a statistical chart of larva and vermicompost parameters in the experimental example. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be further described below through examples and drawings.

[0019] The instruments, equipment, reagents and materials used in the examples are all obtained through commercial channels.

[0020] I. Bacterial Agent Screening (1) Isolation of Intestinal Microorganisms The isolation steps of intestinal microorganisms of black soldier fly larvae generally include the following main steps: sampling, culture medium selection, cultivation, isolation, identification and storage. These steps are aimed at isolating various microorganisms from the intestines of black soldier fly larvae, so as to further study their relationships with the growth of black soldier flies, waste treatment, etc. The following is a brief description of these steps: 1. Sampling Select larvae: Select healthy black soldier fly larvae as samples. Usually, larvae in the growth stage are selected to ensure the representativeness of the microbial community in their intestines.

[0021] Sterilization operation: In order to avoid external contamination, the black soldier fly larvae need to be surface-sterilized before sampling. The common method is to disinfect the larvae with 70% ethanol, and then wash them with sterile physiological saline or phosphate buffer solution to remove surface microorganisms.

[0022] 2. Intestinal Isolation Dissect the larvae: Under aseptic conditions, gently cut open the abdomen of the black soldier fly larvae with dissection tools (such as a scalpel or forceps) to expose the intestine.

[0023] Remove the intestinal contents: Take out the contents of the intestine (including bacteria and other microorganisms in the intestine) and place them in sterile physiological saline for treatment.

[0024] 3. Selection of microbial culture medium Select a selective medium: Use different selective media according to different types of microorganisms (such as bacteria, fungi, etc.). The selective medium can inhibit the growth of unwanted microorganisms and promote the isolation of target microorganisms. In this technique, the LB broth medium added with 0.025% uric acid is adopted, and the uric acid in it reaches a nearly saturated state.

[0025] 4. Cultivation and isolation Prepare the dilution: Mix the intestinal contents with sterile physiological saline or buffer solution and perform serial dilutions (such as 10-fold dilutions).

[0026] Spread the plate: Spread an appropriate amount of the dilution on the surface of the culture medium to ensure that the microorganisms are evenly distributed for the isolation of single colonies.

[0027] Cultivate: Place the culture medium coated with the sample in a constant temperature incubator for cultivation. The cultivation conditions (such as temperature, humidity, oxygen conditions, etc.) are set according to the requirements of the target microorganism. The common cultivation temperature is 30°C - 37°C.

[0028] 5. Colony isolation and selection Observe the colonies: After a certain period of cultivation, observe whether there are colonies growing on the culture medium. The morphology of the colonies (such as color, size, shape) can initially judge different types of microorganisms.

[0029] Select single colonies: Select single colonies with consistent morphology from the culture medium and transfer them to a new culture medium for purification cultivation.

[0030] Screening of high-performance strains The strains screened in the above steps include various strains that can tolerate and utilize uric acid, and their abilities to degrade uric acid vary. In this technique, the above strains are screened by the degradation efficiency of the uric acid substrate in an attempt to obtain a uric acid-degrading bacterium with excellent performance. The following are the detailed steps: 1. Selection and cultivation of different strains Expand the cultivation of the single strains picked out in the above steps in the ordinary LB medium to obtain the strains for screening 2. Preparation of high-uric acid culture medium High uric acid selective pressure medium is usually used to screen microorganisms that can tolerate high concentrations of uric acid or metabolize uric acid, especially for screening some bacteria or yeasts that can decompose or tolerate uric acid. In such a selective medium, uric acid acts as a selective pressure, allowing only those microorganisms with specific metabolic pathways (e.g., enzymes that can decompose or tolerate uric acid) to grow. The specific formula is as follows: LB liquid medium + uric acid selective pressure: Tryptone: 10 g / L Yeast Extract: 5 g / L Sodium Chloride (NaCl): 10 g / L Uric acid: 2g / L pH: 7.0 3. Selection of high-performance strains Inoculate the isolated strains into the high uric acid medium, and select the strains that can degrade uric acid fastest by detecting the change of substrate concentration. The specific method is as follows: Add 50 mL of autoclaved high uric acid medium into an Erlenmeyer flask, add 0.1 OD600 of the bacterial solution obtained in step 1. Take 1 mL of the culture solution every 6 hours, centrifuge it, and then use a uric acid detection kit to detect the uric acid content. After 48 hours, count the uric acid degradation effects of different strains. Select the optimal strain for domestication and preservation according to the amount of uric acid degradation.

[0031] (3)Strain domestication The gene expression of bacteria is regulated by the external environment. For example, the uric acid degradation pathway will show increased expression in a long-term uric acid environment. After culturing and domesticating for multiple generations in a high uric acid environment, the uric acid tolerance and degradation ability of the strain can be improved, and a high-efficiency uric acid-degrading strain with excellent phenotype can be obtained.

[0032] (4)Strain purification, identification and preservation Preserve the strain obtained in the above steps with glycerol, amplify and culture it, and then perform 16s molecular identification. After sequence alignment, it is determined that this high-quality strain is Enterococcus faecalis. The process flow is as Figure 1 shown.

[0033] II. Process application verification 1. Bacterial agent preparation After resuscitating the above strains according to the instructions provided by the preservation center, they were expanded and cultured using LB broth liquid medium, and then preserved using the glycerol preservation method. Add 50 mL of LB broth liquid medium to a 150 mL conical flask, seal it with tissue culture sealing film, treat it at 121 °C for 20 min in an autoclave, and then store it in the refrigerator as the culture medium for the strains; add the strains to the prepared medium at an inoculation amount of 1% respectively, and culture them overnight at 37 °C and 180 rpm on a horizontal shaker to obtain the strain fermentation broth; take 1 mL of the strain fermentation broth obtained in the previous step, centrifuge it at 5000 rpm for 5 min, discard the supernatant, resuspend the cell pellet with 1 mL of sterile PBS buffer, and measure its absorbance A1 at a wavelength of 600 nm using an ultraviolet spectrophotometer, with sterile PBS as the control; according to the measured absorbance, concentrate the strain fermentation broth obtained using a 50 mL centrifuge tube, and concentrate the strain fermentation broth to OD 600 is 100, which are respectively recorded as the strain working solution.

[0034] 2. Larval fattening The following method was used to fatten the black soldier fly larvae: Take 1 g of the purchased black soldier fly eggs as one portion, add them to wheat bran with a water content of 70% according to the ratio of 1 g of eggs to 0.1 kg of culture medium, and fatten them at 28 - 32 °C and a relative air humidity of 60 - 70% for 1 - 5 days.

[0035] 3. Chicken manure treatment Add 500 g of fresh chicken manure (collected from Hefei Feichi Poultry Farm) to 20 cm * 15 cm * 5 cm plastic boxes, a total of 12 boxes. Six of them were subjected to heat sterilization treatment, and then the moisture content of the chicken manure was adjusted to 65%. The treatment groups were Control (heat treatment, without strain working solution), Heat (heat treatment, without working solution), Control + Ef (0.05 mL of working solution was added to the control group), Heat + Ef (heat treatment, 0.05 mL of working solution was added), and they were continuously treated for five days at room temperature. And a control without larvae was set for comparison with the uric acid in the worm cast.

[0036] 4. Separation of worm cast and larvae Use an 8 - mesh sieve to screen the system after the treatment in the previous step to separate the larvae and the worm cast, and collect the larvae and the worm cast separately for standby 5. Parameter statistics of larvae and worm cast Detect the larvae and worm cast treated above. The specific parameters are as Figure 2As shown, it can be seen from the growth of larval weight, the reduction of waste by larvae, the bioconversion rate of larvae to organic matter, and the uric acid residue in vermicompost that after adding the microbial agent, the treatment ability of larvae to chicken manure has been significantly improved, the weight and bioconversion rate have increased significantly, and the addition of the microbial agent can significantly improve the nutrient reuse ability of black soldier fly under the condition of consuming the same solids. Through the detection of uric acid content in vermicompost, the addition of the microbial agent can quickly treat the uric acid in chicken manure. The combined effect of the microbial agent and larvae can significantly reduce the uric acid content compared with the naturally fermented chicken manure.

Claims

1. A method for promoting black soldier flies to transform chicken manure using uric acid degrading bacteria, characterized in that: The steps are as follows: inoculate uric acid degrading bacteria into LB broth liquid culture medium, culture overnight on a horizontal shaker at 37°C and 180 rpm, concentrate the strain fermentation liquid by centrifugation to obtain the strain working liquid, add it to chicken manure, and use black soldier fly larvae fattened for 1-5 days to treat chicken manure.

2. The method for promoting the conversion of chicken manure by black soldier flies using uric acid degrading bacteria according to claim 1, characterized in that: The uric acid degrading bacteria is Enterococcus faecalis.

3. The method for promoting the conversion of chicken manure by black soldier flies using uric acid degrading bacteria according to claim 1, characterized in that: The added amount of the strain working solution is 0.1 mL of the strain working solution per 1000 g of chicken manure.

4. The method for promoting the conversion of chicken manure by black soldier flies using uric acid degrading bacteria according to claim 1 or 2, characterized in that: The uric acid degrading bacteria are obtained according to the following steps: (1) Sampling: The black soldier fly larvae were surface disinfected, dissected under sterile conditions, and the intestinal contents were removed and placed in sterile saline for treatment; (2) Microbial culture: Dilute the intestinal contents and spread them on LB broth medium supplemented with uric acid for constant temperature culture. Select single colonies with consistent morphology and transfer them to new culture medium for culture. (3) Screening of high-performance strains: The cultured strains are expanded in LB medium, and the bacterial liquid is inoculated into a high-uric acid medium. By detecting changes in substrate concentration, the strains that can degrade uric acid the fastest are selected and domesticated to obtain the strains.

5. The method for promoting the conversion of chicken manure by black soldier flies using uric acid degrading bacteria according to claim 4, characterized in that: The method for disinfecting the surface of black soldier fly larvae is to disinfect the larvae with 70% ethanol and then wash them with sterile physiological saline or phosphate buffer to remove surface microorganisms.

6. The method for promoting the conversion of chicken manure by black soldier flies using uric acid degrading bacteria according to claim 4, characterized in that: The amount of uric acid added is 0.02-0.03% of the total amount of LB broth culture medium, so that the uric acid reaches a nearly saturated state.

7. The method for promoting the conversion of chicken manure by black soldier flies using uric acid degrading bacteria according to claim 4, characterized in that: The high uric acid selection pressure culture medium is LB liquid culture medium + uric acid selection pressure: Tryptone: 10 g / L; Yeast Extract: 5 g / L; Sodium chloride (NaCl): 10 g / L; Uric acid: 2 g / L; pH: 7.0; The high uric acid selection pressure culture medium is used to screen microorganisms that can tolerate high concentrations of uric acid or metabolize uric acid, especially for screening some bacteria or yeasts that can decompose or tolerate uric acid. In such a selective culture medium, uric acid acts as a selective pressure, allowing only those microorganisms with specific metabolic pathways (for example, enzymes that can decompose uric acid or tolerate uric acid) to grow.

8. The method for promoting the conversion of chicken manure by black soldier flies using uric acid degrading bacteria according to claim 4, characterized in that: The selection of the strain that degrades uric acid the fastest is to add 50 mL of high-uric acid culture medium sterilized by high pressure into a conical flask, add 0.1 OD600 bacterial solution, take 1 mL of culture solution and centrifuge it every 6 hours, and use a uric acid detection kit to detect the uric acid content. After 48 hours, the uric acid degradation effects of different strains are counted, and the optimal strain is selected for domestication and preservation according to the uric acid degradation amount.

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