Method for treating pitaya branch waste by combining hermetia illucens larvae with complex microbial inoculants and application of pitaya branch waste

Abstract

The invention relates to the field of environmental protection and resource utilization, in particular to a method for treating pitaya branch waste by combining hermetia illucens larvae with a complex microbial inoculant and application. By adding the chicken manure, the utilization efficiency of the black soldier flies on pitaya branch wastes is improved, and an optimal pitaya branch and chicken manure compounding ratio is found; several strains of functional bacteria are screened from the transformation substrate and are verified to be capable of promoting the transformation of the black soldier flies on the mixture of the pitaya branches and the chicken manure; three strains with different functional advantages are further selected to prepare the complex microbial inoculant, so that the conversion efficiency of the hermetia illucens can be obviously improved, and a new method is provided for the treatment of pitaya branches by a hermetia illucens conversion technology.

Description

Technical Field

[0001] The invention relates to the field of environmental protection and resource utilization, and relates to a method and application of treating pitaya branch waste by using black soldier fly larvae in combination with a composite bacterial agent. Background Art

[0002] At present, the main methods for dealing with waste pitaya branches are composting, incineration, and use as livestock and poultry feed, which often harm the environment and the nutritional value of pitaya branches is not highly utilized. There is currently no reasonable method to deal with discarded pitaya branches, and new biotechnology is needed to solve this problem.

[0003] Pitaya branches are similar to cacti, with high pectin and fiber content. Pitaya is mainly grown in Hainan Province, with a large planting area. The main processing method is natural anaerobic composting, which has a long cycle, great impact on the environment and low economic value.

[0004] As a saprophagous resource-type insect, soldier flies have a wide range of food sources and grow fast, and have great advantages in waste treatment. They can convert organic waste such as restaurant leftovers, food processing by-products, and livestock and poultry manure into biomass for a variety of applications, such as high content of crude protein and crude fat. Through further processing, they can be used as high-value-added products such as protein feed and biodiesel, thereby increasing economic benefits and solving social problems. Summary of the invention

[0005] The purpose of the present invention is to solve the problems of low efficiency and great environmental harm of traditional treatment methods of pitaya branch waste, and provide a method for treating pitaya branch waste by using black soldier fly larvae in combination with a composite bacterial agent, so as to solve the problem of resource utilization of pitaya branch waste.

[0006] Another object of the present invention is to provide the application of the above method in treating pitaya branch waste.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] The applicant screened thermostable cellulose and pectin-degrading bacteria using a high temperature (50°C) culture medium, and eventually obtained strain C5. 16srDNA alignment and tree analysis showed that C5 was Bacillus atrophaeus.

[0009] The Bacillus atrophaeus C5 was sent to the China Center for Type Culture Collection for preservation on October 22, 2024, with classification name: Bacillus atrophaeus C5, preservation number: CCTCCNO: M20242291, address: Wuhan University, Wuhan, China.

[0010] The invention discloses an application of Bacillus atrophaeus C5 in treating pitaya branch waste, wherein the application process is combined with black soldier flies.

[0011] Application of Bacillus atrophaeus C5 in the preparation of microbial agent for treating pitaya branch waste.

[0012] A composite microbial agent for treating pitaya branch waste in combination with black soldier flies comprises: Bacillus atrophaeus C5, Bacillus subtilis BSF-CL and Lactobacillus buchneri L3-9; the Bacillus subtilis has a preservation number of CCTCC NO: M2014222, and the Lactobacillus buchneri has a preservation number of CCTCC NO: M2018511.

[0013] The above-mentioned microorganisms and, preferably, the effective bacterial concentration ratio of the Bacillus atrophaeus C5, Bacillus subtilis BSF-CL and Lactobacillus buchneri L3-9 is 1-4:1-4:1-4.

[0014] The best ratio is 4:1:1.

[0015] The above-mentioned application, specifically, includes the following processing steps:

[0016] The method for treating pitaya branch waste by using black soldier fly larvae in combination with microbial agents comprises the steps of inoculating black soldier fly larvae and microbial agents into a mixture of pitaya branch waste and chicken manure; the mass ratio of the pitaya branch waste to chicken manure is 1-4:1-4.

[0017] In the above-mentioned application, preferably, the microbial agent is Bacillus atrophaeus C5 or a composite microbial agent containing C5.

[0018] Preferably, the inoculation amount of the microorganisms is 1-5%; the ratio of the soldier flies to the mixture is 5-10 soldier flies: 10g of the mixture.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention screened out for the first time the high temperature resistant Bacillus atrophaeus C5 that can degrade cellulose and pectin

[0021] (1) The present invention utilizes black soldier flies to transform pitaya branch waste, and greatly improves the utilization efficiency of black soldier flies on pitaya branch waste by compounding with Wenchang chicken manure, and finds the optimal compounding ratio of the black soldier flies to transform pitaya branches and the Wenchang chicken manure mixed matrix.

[0022] (2) The present invention further improves the transformation effect of black soldier flies on the mixed matrix of pitaya branches and Wenchang chicken manure through the combined transformation of functional microorganisms and black soldier flies, and the crude fiber and pectin content are significantly reduced.

[0023] (3) The method provided by the present invention for bioconversion of pitaya branches and Wenchang chicken manure using black soldier fly larvae in combination with functional microorganisms greatly improves the reduction and resource utilization of agricultural organic waste, and also provides an important link for the development of Hainan pitaya economic circular agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of soldier fly transformation of mixtures of pitaya branches and Wenchang chicken manure in different proportions.

[0025] Figure 2 This is a comparison chart of the moisture content of the residues before and after soldier fly transformation of a mixture of pitaya branches and Wenchang chicken manure in different proportions.

[0026] Figure 3 Schematic diagram of the alignment results of the 16srDNA sequences of C5 (Bacillus atrophaeus) and C14 (Bacillus tequilensis).

[0027] Figure 4 This is a comparison of the crude fat and crude protein content in the black soldier fly's body after it transformed into a mixture of pitaya branches and Wenchang chicken manure in different proportions.

[0028] Figure 5 The growth of soldier flies in pitaya and Wenchang chicken manure transformed by single bacteria and synergistically with soldier flies changes over time.

[0029] Figure 6 The degradation of cellulose and total pectin by single bacteria and soldier flies in the transformation of pitaya and Wenchang chicken manure. DETAILED DESCRIPTION

[0030] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention. The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

[0031] The present invention relates to Lactobacillus buchneri L3-9, with a deposit number of CCTCC NO: M2018511, which has been disclosed in CN109730033A, and is referred to as L3-9 in the present invention. Bacillus subtilis BSF-CL, with a deposit number of CCTCC NO: M2014222, which has been disclosed in CN106854627A, and is referred to as BSF-CL in the present invention.

[0032] Embodiment 1:

[0033] The effect of black soldier flies on pitaya branches and Wenchang chicken manure:

[0034] First, in order to design the experiment more reasonably and evaluate the experimental results, it is necessary to test the physical and chemical components of pitaya branches and Wenchang chicken manure, including moisture content, crude protein, crude fat, pectin and cellulose content. In order to evaluate the transformation effect of black soldier flies on pitaya branches and Wenchang chicken manure, 6 treatment groups R-1 to R-6 were set up according to different ratios. The experiment adopted a 300g mixed feed conversion system, and 300 6-day-old black soldier fly larvae of the same size and good growth were added to each treatment, and three replicates were made in each group. During the transformation process, the ambient temperature was controlled at 28±2℃ and the humidity was 50%~70%( Figure 1 ).

[0035] The experiment ended when the first prepupa appeared in each barrel. The insect materials were separated and dried at 60°C to constant weight. The weights before and after drying were recorded, and various conversion indicators were calculated.

[0036] A small amount of sample was taken and dried at 60℃, then ground by a grinder and passed through a 40-mesh sieve to determine the moisture content, crude fat, crude protein, pectin, cellulose and other components.

[0037] Table 1 Physicochemical components of pitaya branches and chicken manure

[0038]

[0039]

[0040] Table 2 Experimental design of transformation of pitaya branches and chicken manure combined with soldier flies

[0041]

[0042] After the transformation, the weight gain, material reduction rate, bioconversion rate and feed conversion ratio of the soldier fly larvae were measured, and the optimal compound ratio was obtained by combining various indicators.

[0043] (1)

[0044] (2)

[0045] (3)

[0046] Table 3 Development time, survival rate and larval production of BSF fed with a mixture of pitaya branches and chicken manure

[0047]

[0048]

[0049] Note: The same letters after the numbers in the same column indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate extremely significant difference (P<0.01).

[0050] After the transformation, the growth indicators of soldier flies raised in different compound ratios of substrates were monitored. Table 3 shows that R-2 and R-3 have the longest transformation time, which is about 10 days from the 6-day-old larvae. With the increase of chicken manure content, the development time of the larvae gradually shortened, and the survival rate remained at a normal level. This may be due to the lack of nutrition in the single dragon fruit branch matrix, which leads to the competition between the soldier fly larvae and the low survival rate. The increase in chicken manure content significantly improved this nutritional deficiency, and the larval survival rate was greatly improved. However, due to the matrix of chicken manure itself, the material lost water too quickly, which affected the growth environment of the soldier flies, and thus the development time became shorter. We can also see that the fresh weight of the larvae in the R-3 group is the highest, and the total dry weight obtained is not significantly different from that of R-4 and R-5. In addition, the proportion of R-3 dragon fruit branch waste is high, which is more in line with our purpose of efficient waste reduction and resource utilization of this agricultural waste.

[0051] Table 4 Mass reduction, bioconversion and feed conversion efficiency of BSF-transformed dragon fruit branches, chicken manure and co-transformed mixtures

[0052]

[0053] Note: The same letters after the numbers in the same column indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate extremely significant difference (P<0.01).

[0054] From Table 4, we can see that the R-3 and R-4 groups have the highest bioconversion rates, and there is no significant difference between the two groups. This is also the most important indicator we pay attention to in bioconversion. Compared with the R-4 group, the R-3 group has obvious advantages in the utilization of the mixed matrix and the reduction of pitaya branches. Therefore, the R-3 group is the best compound ratio of the mixed matrix of pitaya branches and chicken manure for soldier flies to transform.

[0055] Table 5 Cellulose and pectin content in materials before and after conversion

[0056]

[0057] Table 5 shows the determination of cellulose and pectin content in the materials before and after transformation by soldier flies. Due to the different proportions of pitaya branches and chicken manure in each treatment group, the fiber and pectin content is also very different, which affects the degradation of cellulose and pectin in the mixed matrix by soldier flies. Figure 2 It can be seen that the R-3 group has the best cellulose degradation effect, reaching 40.2%, which is significantly different from other treatment groups, followed by R-4, R-5, and R-6, indicating that with the increase of chicken manure content, the growth performance of soldier flies is enhanced, which in turn leads to the improvement of cellulose degradation ability in the material. This phenomenon is caused by the improvement of the microbial flora of the entire material by the intestinal microorganisms of soldier flies. This also reflects the conclusion that the R-3 group (dragon fruit branch waste: chicken manure = 3:2) has the best compound ratio. The best pectin degradation effect is in the R-1 and R-2 groups. This is because these two groups have a large proportion of pitaya branches, high pectin content, and less nutrients that are easily absorbed and utilized in the matrix. However, when nutrition is deficient, soldier flies can only passively use relatively difficult-to-degrade pectin to maintain life activities. Of course, the degradation of pectin and cellulose is largely due to the important role played by microorganisms that produce cellulase and pectinase.

[0058] Figure 2 It is the determination of the moisture content in the material before and after the transformation of soldier flies. After the transformation, the moisture content in the substrate decreased, and the R-1 group had the smallest change, which is directly related to the decrease in soldier fly mortality, weak growth performance and smaller activity range. It can be clearly seen that the moisture content of the R-3 group decreased the most compared with other treatment groups, and the growth indicators of soldier flies such as total wet weight of larvae, material reduction rate, biological conversion rate and feed conversion ratio were also the best in general, which can also reflect from the side that the R-3 group has the best transformation effect of soldier flies. Figure 4 The crude protein and crude fat in the soldier fly body were detected after the transformation. From the results, the crude fat content of soldier flies in groups R-4 to R-6, which had relatively high chicken manure content, was relatively high, and there was a significant difference from other groups. The crude protein and crude fat results were just the opposite. The higher the content of pitaya branches, the higher the crude protein content. The crude protein content of the optimal compound ratio group R-3 was 43.37%, and the crude fat content was 13.46%. Because the total biomass of soldier flies obtained after transformation in the R-3 group was the highest, the total crude protein and crude fat that could be obtained were the highest, which could also obtain a higher economic added value.

[0059] Embodiment 2:

[0060] Acquisition of C5 (Bacillus atrophaeus) and C14 (Bacillus tequilensis) strains:

[0061] Screening method for thermostable cellulose-degrading bacteria:

[0062] The intestines of soldier flies fed with dragon fruit branches and Wenchang chicken manure were used as feed matrices, and the residues after transformation were used as the source for screening high-temperature resistant cellulose degrading bacteria. The soldier fly intestines were extracted and ground under sterile conditions. 1 mL of water was added as the stock solution. 1 g of the residue sample was weighed and added to 10 ml of sterile water. The sample was diluted by 10-fold gradient dilution method, and the dilution degree was 10. －3 ~10 －6 0.1 mL of each dilution was spread on Congo red medium plates, cultured at 50°C for 48 hours, and colonies with larger colonies and more obvious transparent circles were picked on the plates. The colonies were streaked and purified three times using the same medium plate tetrads. The purified strains were inoculated on Congo red medium, and the diameter of the hydrolysis circle (D) and colony diameter (d) on the medium were measured, and their ratio (D / d) was calculated. Screening method for pectin-degrading bacteria:

[0063] The same screening method for cellulose-degrading bacteria is used, except that the transparent zone staining reagent is Lugol's iodine solution. Pour the iodine solution into the culture medium and stain for 20 minutes, then pour off the dye and rinse off the excess dye with sodium chloride solution. If a transparent zone appears, it is pectin-degrading bacteria.

[0064] In this way, high temperature resistant (50°C) strains C5 and C14 were obtained, which can degrade cellulose and pectin simultaneously. The degradation rates of cellulose and pectin by C5 and C14 are shown in the following table, respectively.

[0065] Hydrolysis zone and colony diameter of cellulose-degrading bacteria

[0066]

[0067] Hydrolysis zone and colony diameter of pectin-degrading bacteria

[0068]

[0069] Furthermore, the enzymatic activities of cellulase and pectinase produced by the two bacteria were determined as follows:

[0070] Enzyme activity assay:

[0071] Drawing of standard curve: Take 0, 0.2, 0.4, 0.6, 0.8, 1.0mL of glucose standard solution (1g / L) in 10mL test tubes respectively, add water to 2mL, add 2mL of DNS reagent, mix well, boil in water bath for 5min, cool, add water to 10mL, use blank as control, measure absorbance at 540nm, and draw the standard curve with absorbance as ordinate and glucose content as abscissa.

[0072] Preparation of crude enzyme solution: inoculate the strain obtained from the primary screening into a 250mL Erlenmeyer flask containing 100mL of sterile rescreening culture medium, and culture with shaking at 180r / min and 50℃ for 48h. Centrifuge the culture solution at 4℃ and 8000r / min for 10min. The supernatant is the crude enzyme solution.

[0073] Determination of carboxymethyl cellulase activity: Take 0.5 mL of the diluted crude enzyme solution, add 1.5 mL of citric acid buffer (0.05 mol / L, pH 4.8) containing 0.5% CMC-Na, and place in a 50°C water bath for 30 minutes, and then immediately determine the sugar content according to the DNS method. The crude enzyme solution inactivated by boiling at 100°C for 10 minutes was used as the blank control group, and the other steps were the same.

[0074] Enzyme activity unit (U / mL): The amount of enzyme required to convert substrate to produce 1 umol of reducing sugar per minute.

[0075] Pectinase activity determination: The standard substance is galacturonic acid, and other methods are the same as the cellulase activity determination method.

[0076] Bacterial species identification:

[0077] Strain DNA extraction: Extract the genomic DNA of the strains according to the operating procedures of the bacterial genome extraction kit or the fungal genome extraction kit. Use Tnano-700 to detect the concentration (mg / L) and purity (A260 / A280) of the extracted DNA, and use 1.4% agarose gel electrophoresis to detect DNA integrity. PCR amplification and sequencing: Use the extracted bacterial genomic DNA as a template and use bacterial universal primers for PCR amplification. PCR products are detected using 1.2% agarose gel electrophoresis, and the results are observed using a gel imager. Positive PCR products are sent to Wuhan Qingke Biotechnology Co., Ltd. for 16S rDNA or ITS sequencing analysis. After 16srDNA alignment and tree analysis, C5 is Bacillus atrophaeus, C14 is Bacillus tequilensis, Figure 3 .

[0078] Carboxymethyl cellulose activity assay results

[0079]

[0080] In a 50℃ reaction system, the amount of enzyme required to produce 1ug of glucose from the substrate per minute is 1 enzyme activity unit (U)

[0081] Pectinase activity assay results

[0082]

[0083] In a 50°C reaction system, the amount of enzyme required to produce 1 ug of galacturonic acid from the substrate per minute is 1 enzyme activity unit (U).

[0084] The Bacillus atrophaeus C5 was sent to the China Center for Type Culture Collection for preservation on October 22, 2024, with classification name: Bacillus atrophaeus C5, preservation number: CCTCCNO: M20242291, address: Wuhan University, Wuhan, China.

[0085] Embodiment 3:

[0086] Single bacteria combined with black soldier flies to treat pitaya branches and chicken manure:

[0087] In order to improve the ability of black soldier flies to treat pitaya branches and Wenchang chicken manure, two screened thermotolerant bacteria C5 (Bacillus atrophaeus) and C14 (Bacillus equilensis) with both cellulose and pectin degradation capabilities and two laboratory-preserved bacteria BSF-CL (CCTCC NO: M2014222, CN106854627A) and L3-9 (CCTCC NO: M2018511, CN109730033A) with synergistic soldier fly growth promotion were selected. A total of four strains of bacteria were set up for transformation in combination with soldier flies. The transformation matrix was the optimal compound ratio of 3:2 of pitaya branches and Wenchang chicken manure obtained in Example 1, the total weight of the mixed material was 300 g, 300 black soldier fly larvae were inoculated, the inoculation amount was 2%, and three replicates were performed in each group.

[0088] The CK group did not add any bacterial agents, and only used soldier flies for transformation.

[0089] During the transformation process, the ambient temperature was controlled at 28±2°C and the humidity was 50% to 70%. The growth of the larvae was recorded every two days. When the larvae reached the maximum weight, the transformation was completed. The larvae and the residue were separated, and the weight gain, material reduction rate, bioconversion rate, feed conversion ratio and other indicators of the soldier fly larvae were measured after sampling and drying, and various transformation indicators were calculated.

[0090] Table 6 Experimental design (60% pitaya branches and 40% chicken manure) R-3 for further co-transformation with microorganisms

[0091]

[0092] Inoculate 5-day-old soldier fly larvae and take samples every two days to monitor the growth of soldier flies. Figure 5 We can see that there is not much difference in the growth rate of soldier flies between the treatment groups in the first 4 days of transformation, but there is a significant difference in the growth rate of soldier flies between the different treatment groups in the stage of 4-8 days, especially the C5 and C14 groups grew faster. This may be because the two strains of Bacillus grow and reproduce relatively quickly, which has a short-term promotion effect on the growth of soldier flies. On the 8th day, we can clearly see that the fresh weight of soldier flies in the C5 group is the highest among all treatments, and the insect weight can reach 1.36g / 10 heads. After 10 days, the growth of soldier flies has basically stagnated or slightly decreased. The experiment is over and the insect feed is separated.

[0093] Table 7 Mass reduction, bioconversion and feed conversion efficiency of BSF-converted dragon fruit branches, chicken manure and co-converted mixtures on a dry matter basis

[0094]

[0095] Table 8 Cellulose and pectin content in the residue after conversion

[0096]

[0097]

[0098] Table 9 Degradation of cellulose and total pectin by single bacteria and soldier flies in transforming pitaya waste

[0099]

[0100] Note: The same letters after the numbers in the same column indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate extremely significant difference (P<0.01).

[0101] Table 7 is a statistical result of the material reduction rate, bioconversion rate and feed conversion ratio after the transformation by soldier flies. Obviously, L3-9 has the best transformation indicators, with a bioconversion rate of 8.33%, an increase of 20.55% compared to the CK group, followed by BSL-CL bacteria, with a bioconversion rate of 7.86%, an increase of 13.74% compared to the CK group. The highest material reduction rate was in the BSL-CL treatment group, which was 41.48%, followed by the C14 group, which reached 40.31%. In the efficient treatment of organic waste, waste reduction is also an extremely important issue. In addition, cellulose degradation is also an important indicator of our concern. Table 8 is the determination of the cellulose and pectin content in the residue after the four functional bacteria cooperated with soldier flies to transform the pitaya mixed matrix. Figure 6 From the degradation effect, it can be seen that C5 has the highest cellulose degradation rate, reaching 28.82%, followed by BSF-CL and L3-9, which are 21.14% and 19.78% respectively. However, the addition of microorganisms did not significantly improve the degradation effect of pectin. The best effect was in the C14 group, which was 77.12%, and there was no significant difference with other treatment groups including the CK group. C5 and BSF-CL belong to the genus Bacillus, which produce many types of enzymes with strong ability. They can produce cellulase to promote cellulose degradation. Lactic acid bacteria (L3-9) are frequently mentioned in the literature to promote the growth of soldier flies. This may be because the metabolites produced by lactic acid bacteria can provide the nutrients required by soldier flies and promote their growth and metabolic activities. In addition, lactic acid bacteria may also produce some enzyme substances that help decompose cellulose, making it easier to be degraded by soldier flies.

[0102] Embodiment 4:

[0103] From the experimental results of Example 3, it can be seen that the three functional bacteria C5, BSF-CL and L3-9 have different functional advantages in the process of coordinating the transformation of pitaya branches and chicken manure mixed matrix by soldier flies. These three strains of bacteria are now used to prepare a composite bacterial agent to further promote the efficient transformation of pitaya branches and chicken manure waste by soldier flies.

[0104] Preparation method of bacterial agent: take 1mL of seed liquid into 100ml LB medium and culture at 37℃, 180r / min shaking for 24h, take out and adjust the bacterial count to 10 8 cfu / mL, adjust the single bacteria to the same concentration, the total volume of the inoculation is constant, and the three strains are compounded into a composite bacterial agent according to different proportions in Table 10 to ensure that the concentration of the inoculated materials is the same (after preparing the mixed bacterial agent according to Table 10, the effective bacterial concentration is 10 8 cfu / mL).

[0105] The experiment used 6-day-old soldier fly larvae, the total weight of the mixed feed was 100g, 100 soldier fly larvae were added, the inoculation amount was 2%, and three replicates were made in each group. The experiment ended when the first prepupa appeared in each barrel, the insect feed was separated, and the weight gain, material reduction rate, bioconversion rate and feed conversion ratio of the soldier fly larvae were measured after sampling and drying, and various conversion indicators were calculated.

[0106] Table 10 Grouping of compound bacterial agent ratio

[0107]

[0108] Table 11 Mass reduction, bioconversion and feed conversion rate of pitaya branches, chicken manure and co-conversion mixture transformed by BSFL

[0109]

[0110]

[0111] Table 12 Improvement of insect weight gain, material reduction rate and bioconversion rate in the group with added microbial agents compared with CK

[0112]

[0113] Tables 11 and 12 show the experimental results of the treatment of pitaya branches and chicken manure mixture by composite bacterial agent combined with black soldier flies. We can see that the material reduction rate and bioconversion rate of group S3 are the best, which are 33.65% and 7.76%, respectively, which are 11.13% and 10.38% higher than CK. Therefore, the optimal compound ratio of the composite bacterial agent C5, BSF-CL, and L3-9 is 4:1:1.

Claims

1. An isolated strain of Bacillus atrophaeus ( Bacillus atrophaeus ) C5, the preservation number of the Bacillus atrophaeus C5 is CCTCC NO: M20242291.

2. The application of Bacillus atrophaeus C5 described in claim 1 in treating pitaya branch waste, wherein the application process is combined with black soldier flies.

3. Application of Bacillus atrophaeus C5 according to claim 1 in preparing a microbial agent for treating pitaya branch waste.

4. A composite microbial agent for treating pitaya branch waste in combination with black soldier flies, comprising: The Bacillus atrophaeus C5, Bacillus subtilis BSF-CL and Lactobacillus buchneri L3-9 described in claim 1; the Bacillus subtilis has a deposit number of CCTCC NO: M2014222, and the Lactobacillus buchneri has a deposit number of CCTCC NO: M2018511.

5. The composite microbial agent according to claim 4, characterized in that: The ratio of effective bacterial concentrations of Bacillus atrophaeus C5, Bacillus subtilis BSF-CL and Lactobacillus buchneri L3-9 is 1-4:1-4:1-4.

6. The composite microbial agent according to claim 7, characterized in that: The ratio of effective bacterial concentrations of Bacillus atrophaeus C5, Bacillus subtilis BSF-CL and Lactobacillus buchneri L3-9 is 4:1:

1.

7. The application according to claim 2, wherein the application process comprises inoculating black soldier fly larvae and microbial agents into a mixture of pitaya branch waste and chicken manure; the mass ratio of the pitaya branch waste to chicken manure is 1-4:1-4, and the microbial agent is the Bacillus atrophaeus C5 according to claim 1 or the composite microbial agent according to claim 4.

8. The use according to claim 7, characterized in that: The inoculation amount of the microorganisms is 1-5%, and the ratio of the soldier flies to the mixture is 5-10 soldier flies: 10g of the mixture.

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