Complex microbial inoculant with cellulose-protein two-component degradation function as well as preparation method and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant with a cellulose-protein two-component degradation function as well as a preparation method and application of the complex microbial inoculant. According to the present invention, the active components of the composite bacterial agent comprise Acinetobacter baumannii (Acinetobacter baumannii) and Providencia alcaligenes (Providencia alcaligenes), and the active components of the composite bacterial agent comprise a bacillus amyloliquefaciens strain, a bacillus amyloliquefaciens strain, a bacillus amyloliquefaciens strain, a bacillus amyloliquefaciens strain, a bacillus amyloliquefaciens strain and a bacillus amyloliquefaciens strain, the number of the acinetobacter baumannii in the China Industrial Microbiological Culture Collection Center is CICC 22933; the providencia alcaligenes is numbered as CICC (China Industrial Microbiological Culture Collection Center) 25067 in the China Industrial Microbiological Culture Collection Center). The strains in the complex microbial inoculant do not have antagonism, and the complex microbial inoculant shows a remarkable synergistic effect when degrading cellulose and protein, so that the waste conversion efficiency and the yield of hermetia illucens can be synchronously improved, the environmental pollution is reduced, and a new technical support is provided for high-value utilization of agricultural wastes.

Description

Technical Field

[0001] This invention relates to the field of microbial agent development and application technology, specifically to a composite microbial agent with cellulose-protein dual-component degradation function, its preparation method and application. Background Technology

[0002] With the rapid development of my country's livestock and poultry farming industry, while meeting the demand for meat, eggs, and dairy products, it has also brought severe environmental pressure. The Second National Pollution Source Census Bulletin shows that livestock and poultry farming accounts for 42% of total nitrogen, 56% of total phosphorus, and 94% of chemical oxygen demand (COD) emissions from agricultural sources. The green, efficient, and circular utilization of livestock manure has become an urgent need. At the same time, rural areas in my country generate over 100 million tons of fruit and vegetable waste, 280 million tons of tofu residue, and 31 million tons of aquatic waste annually. Most of this waste is disposed of by discarding or incinerating, resulting in extremely low resource utilization. Although the government has promoted resource utilization policies such as silage and composting, the high moisture content, easy perishability, and high content of fibrous lignin components that are difficult to completely degrade in these wastes lead to problems such as low nutritional value, significant nutrient loss, and high risk of secondary pollution in the final products. Therefore, developing a new technology capable of efficiently degrading complex components and achieving high-value utilization of agricultural organic waste is imperative.

[0003] Black soldier flies, as a resource insect with high conversion efficiency, can transform various organic wastes into high-quality insect bodies rich in protein (30%–52%) and fat (21%–40%). Their residues can also be used as fertilizer, making it an ideal way to solve the aforementioned problems. However, current production practices using livestock and poultry manure directly for black soldier fly farming suffer from low production efficiency and poor insect quality. Studies have shown that while using composite substrates containing added vegetable waste and slaughterhouse waste can increase yields, the potential environmental emissions are often overlooked, failing to achieve a synergistic effect of "increased production" and "reduced emissions." Therefore, overcoming the efficiency and environmental bottlenecks of the black soldier fly bioconversion system is crucial for supporting the green and efficient utilization of agricultural waste.

[0004] Enhancing the black soldier fly transformation system by adding exogenous probiotics is an effective approach, but existing probiotic agents suffer from limitations such as unstable colonization, limited functionality, and poor synergy with the host. Recent studies have shown that endogenous microorganisms in the black soldier fly gut have better compatibility with the host and can specifically degrade their ingested substrates (such as cellulose and proteins). Therefore, developing a bifunctional endogenous probiotic agent that can efficiently degrade both cellulose and proteins is key to overcoming existing bottlenecks. Currently, the construction of functional probiotic agents targeting the synergistic degradation of cellulose and proteins and their application in driving efficient nutrient utilization and reducing carbon and nitrogen emissions remain areas of research gaps. Summary of the Invention

[0005] The purpose of this invention is to provide a composite microbial agent with dual cellulose-protein degradation function, its preparation method and application. This composite microbial agent can simultaneously improve the waste conversion efficiency and insect yield of black soldier fly larvae and reduce environmental pollution, providing new technology support for the high-value utilization of agricultural waste.

[0006] In a first aspect, the present invention provides a compound bacterial agent, the active ingredient of which is Acinetobacter baumannii (…). Acinetobacter baumannii ) and Alcaligenes providediflora ( Providencia alcalifaciens ); The Acinetobacter baumannii strain mentioned is numbered CICC 22933 by the China Industrial Microbial Culture Collection Center. The Alcaligenes Providencia species is registered at the China Industrial Microbial Culture Collection Center as CICC25067.

[0007] Based on the above technical solution, the compound bacterial agent is obtained by targeted screening and compounding from the intestinal flora of black soldier fly larvae that can secrete a variety of digestive enzymes to assist in the decomposition of substrates. The antagonism test confirmed that there was no antagonistic effect between the two strains. The bacterial agent showed a significant synergistic effect in degrading cellulose and protein, which can improve the nutrient cycling efficiency of the black soldier fly biotransformation system and reduce environmental gas emissions in practical applications.

[0008] Furthermore, in the compound bacterial agent, the ratio of live bacteria CFU of Acinetobacter baumannii and Providencia alkali-producing bacteria is 1:(2-3).

[0009] Furthermore, the compound microbial agent can be a liquid or solid microbial agent. Correspondingly, the compound microbial agent may also include a carrier. The carrier can be a solid or liquid carrier. For example, the solid carrier can be an organic or inorganic carrier; the organic carrier includes, but is not limited to, at least one of wheat bran, straw powder, peat moss, pine shells, rice straw, peanut shells, corn flour, soybean flour, starch, peat moss, and animal excrement; the inorganic carrier includes, but is not limited to, at least one of zeolite, vermiculite, clay, talc, kaolin, montmorillonite, white carbon, silica, and diatomaceous earth. For example, the liquid carrier includes, but is not limited to, liquid culture medium, physiological saline, or brown sugar water. The inventors compared three organic carriers—wheat bran, straw powder, and peat moss—and two inorganic carriers—zeolite and vermiculite, and found that the number of effective viable bacteria in the compound microbial agent after 7 days of cultivation on wheat bran was significantly higher than that on other carriers. The compound microbial agent using wheat bran as a carrier can improve the activity and preservation capacity of the bacterial strains. If the microbial agent needs to be transported long distances or used after short-term storage, it should be stored on a wheat bran carrier. Furthermore, when wheat bran is used as a carrier, the total effective viable count of the compound microbial agent is 1.55–1.65 × 10⁻⁶. 8 CFU / g (initial viable count at inoculation).

[0010] In a second aspect, the present invention provides a method for preparing the compound microbial agent described in any of the above claims, comprising the following steps: inoculating the seed liquid of Acinetobacter baumannii and Providencia alkali-producing bacteria into a culture medium for cultivation to obtain the compound microbial agent.

[0011] Furthermore, preferably, the volume ratio of the seed culture of Acinetobacter baumannii to the seed culture of Providencia alkaligenes is 1:1. In practice, the inventors measured the enzyme activity values ​​of compound bacterial agents composed of different strains in different proportions. The results showed that when the two strains were compounded in an equal volume ratio (1:1), the best overall effect was achieved, and the activities of the four key enzymes were maintained at a high level and reached a balance with each other.

[0012] Furthermore, the activation of the *Acinetobacter baumannii* and *Providencebrina alkaligenes* strains is carried out at 28–32°C. Unlike the conventional activation temperature of 37°C, this invention, based on the final application scenario of black soldier fly farming (approximately 25–30°C), considers screening and verifying strains that can efficiently produce enzymes at around 30°C from the perspective of application relevance and actual efficacy. This is far more valuable than screening strains that are efficient at 37°C.

[0013] Further, preferably, the carbon source in the culture medium used to prepare the seed culture of Acinetobacter baumannii is sodium carboxymethyl cellulose (CMC-Na). This medium uses CMC-Na as the carbon source, rather than glucose or other carbon sources, because CMC-Na is also a highly efficient cellulase inducer. In the absence of a more readily available carbon source (such as glucose) to compete with, Acinetobacter baumannii is forced to activate its cellulase system to utilize CMC-Na, thus preparing it for enzyme production at the seed stage. Preferably, the nitrogen source in the culture medium used to prepare the seed culture of Acinetobacter baumannii is sodium nitrate. It is understood that the role of the nitrogen source is to support the basic growth of the cells. While the growth rate may be slightly slower than in a medium rich in organic nitrogen sources (such as peptone) using sodium nitrate, the resulting cells are in a "starved" or "stressed" state, which encourages them to more actively express degradative enzyme systems to obtain nutrients. As an example, the specific composition of the culture medium is as follows: 15.0 g of sodium carboxymethyl cellulose, 1.0 g of sodium nitrate, 0.5 g of magnesium sulfate and 1.0 g of potassium dihydrogen phosphate are added to every 1 L of distilled water, with the pH at natural.

[0014] Furthermore, in the preparation method of the compound microbial agent, the culture conditions are as follows: culture temperature of 30 ℃, initial pH of the culture medium of pH 7, seed liquid inoculum volume of 8% (based on the total amount of seed liquid of Acinetobacter baumannii and Providencia alkali-producing bacteria), culture time of 3 days, and shaking speed of 120 r / min. The inventors optimized the culture conditions through systematic single-factor experiments, ultimately significantly improving the activities of the four key enzymes (carboxymethyl cellulase, filter paper enzyme, β-glucosidase, and protease) in the compound microbial agent. The specific single-factor experiments are as follows: culture temperature (15–35 ℃), initial pH (5–9), inoculum volume (4%–12%), fermentation time (2–6 days), and shaking speed (100–140 r / min). The optimal combination of enzyme production conditions was determined based on the quantitative enzyme activity results. Similarly, in this invention, the optimized selection of the culture temperature of 15-35℃ is also based on the fact that its final application scenario is the black soldier fly breeding environment (about 25-30℃), and is determined from the perspective of application relevance and actual effectiveness.

[0015] This invention involves screening strains and optimizing enzyme production conditions, all within their practical application temperature range (e.g., 30°C). This ensures that the obtained strains and their produced enzymes can immediately exert their optimal function in the black soldier fly farming environment, thus facilitating their practical application.

[0016] Furthermore, in the preparation method of the compound microbial agent, the culture medium is a compound enzyme-producing culture medium. As an example, the composition of the compound enzyme-producing culture medium is as follows: 5.0 g casein, 5.0 g sodium carboxymethyl cellulose, 5.0 g glucose, 2.0 g potassium dihydrogen phosphate, 1.4 g ammonium sulfate, 0.3 g magnesium sulfate, 0.3 g calcium chloride, 0.005 g ferrous sulfate, 0.0016 g manganese sulfate, 0.0014 g zinc sulfate, 0.002 g cobalt chloride, and 1 L distilled water; adjusted to pH 7.0.

[0017] Thirdly, the present invention provides the use of the compound microbial agent described in any of the above-mentioned claims in at least one of the following: A1) Increase the overall productivity of black soldier fly larvae in aquaculture; A2) Improve the substrate bioconversion rate in black soldier fly farming; A3) Reduce greenhouse gas emissions from black soldier fly farming.

[0018] In the above applications, the greenhouse gas may specifically be environmentally harmful gases such as methane, nitrous oxide, and ammonia.

[0019] Fourthly, the present invention provides a method for cultivating black soldier flies, comprising the following steps: Black soldier fly larvae are inoculated into a culture substrate containing any of the compound microbial agents described above for culture; wherein the culture substrate includes agricultural waste.

[0020] Furthermore, in the black soldier fly farming method, the agricultural waste consists of vegetable waste, slaughterhouse waste, and fresh chicken manure. More specifically, the agricultural waste is composed of vegetable waste, slaughterhouse waste, and fresh chicken manure in a dry weight ratio of 1:1:3. Optionally, the vegetable waste comes from a local vegetable market and mainly consists of leafy vegetables and root vegetables; the slaughterhouse waste is various aquatic and poultry waste collected from market meat stalls; and the fresh chicken manure is obtained from a conveyor belt farming facility without the use of pesticides.

[0021] Furthermore, in the black soldier fly culturing method, the total carbon-to-nitrogen ratio of the culturing substrate is (20-25):1, such as 20:1.

[0022] Furthermore, in the black soldier fly larvae farming method, the moisture content of the farming substrate is 70% to 75%, such as 70%.

[0023] Furthermore, in the black soldier fly larvae farming method, the inoculation density of black soldier fly larvae in the farming substrate is 450-500 larvae / kg fresh material, such as 500 larvae / kg fresh material. The black soldier fly larvae are 4-6 days old.

[0024] Furthermore, in the black soldier fly culturing method, the compound microbial agent is sprayed onto the surface of the culturing substrate in the form of a bacterial solution, and the total number of effective viable bacteria in the bacterial solution is ≥2.75×10⁻⁶. 8 CFU / mL (e.g., 2.75–2.85 × 10⁻⁶) 8 The bacterial solution (CFU / mL) comprises 0.5% to 1.0% of the total dry weight of the substrate, e.g., 0.5%. For ease of spraying and activation, the bacterial solution can be diluted with liquid culture medium or physiological saline. This invention's compound bacterial agent requires a low dosage, achieving a powerful synergistic effect on the black soldier fly biotransformation system, while being cost-effective and easy to operate.

[0025] Furthermore, in the black soldier fly larvae rearing method, the rearing is carried out under constant temperature of 28°C and constant humidity of 60% in a light-protected, open-air environment. The experiment ends when 50% of the larvae reach the prepupa stage, and the black soldier flies are separated from the insect residue. Preferably, gas detection is performed on each treatment every 24 hours, and the pile is then turned evenly to improve the permeability of the pile and the activity of the larvae.

[0026] The present invention has the following beneficial effects: (1) Homologous strains and synergistic effect: This invention screens functional strains that have high homology with the host and strong colonization ability, thus avoiding the rejection reaction of exogenous bacterial agents. There is no antagonistic effect between the strains in the constructed compound bacterial agent, and they show a significant synergistic effect in degrading cellulose and protein, which can achieve comprehensive and rapid degradation of complex agricultural waste.

[0027] (2) Process optimization and stable activity: The present invention has comprehensively optimized the fermentation conditions of the microbial agent through systematic single-factor experiments, and innovatively compared a variety of organic and inorganic carriers. Finally, the carrier and process that can maximize the preservation and enhancement of the microbial agent activity were determined, ensuring the high efficiency and stability of the microbial agent product.

[0028] (3) Small dosage, significant benefits: The compound microbial agent of the present invention requires only a low addition of 0.5% to achieve a strong synergistic effect on the black soldier fly biotransformation system. It is low in cost and easy to operate. Verification results show that the microbial agent can simultaneously achieve "increased production" and "reduced emissions". That is, while significantly improving the total productivity of larvae and the biotransformation rate of substrate, it effectively reduces the emissions of harmful environmental gases such as methane, nitrous oxide and ammonia, thus achieving a unity of economic and environmental benefits. Attached Figure Description

[0029] Figure 1 The results are shown in Example 1 of this invention, which demonstrate the degradation of the clear zone by different single strains using cellulose and protein as substrates.

[0030] Figure 2 The results show the quantitative enzyme activities of three cellulase-degrading enzymes and one protein-degrading enzyme from different single strains in Example 1 of the present invention. APase represents alkaline protease, CMCase represents carboxymethyl cellulase, FPase represents filter paper enzyme, and β-Gase represents β-glucosidase.

[0031] Figure 3 The images show the colony morphology, electron micrographology, and antagonistic test results of the two highly efficient cellulose and protein degrading strains screened in Example 2 of this invention.

[0032] Figure 4 The results show the single-factor optimization of cellulose and protein enzyme activities of the high-efficiency compound microbial agent in Example 3 of the present invention under different culture conditions.

[0033] Figure 5 The results show the differences in total larval productivity, greenhouse gas emissions, and bioconversion rate between the black soldier fly conversion system and the system without the addition of the high-efficiency compound bacterial agent in Example 4 of this invention. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0036] The strain A amyloliquefaciens used in the following examples ( Bacillus amyloliquefaciens Acinetobacter baumannii ( ), Acinetobacter johnsonii Acinetobacter baumannii (C.) Acinetobacter baumannii ), D. alkaline-producing Providencia ( Providencia alcalifaciens ) and Escherichia coli (E. freundii) Escherichia fergusonii All strains (A, B, C, D, and E) are strains that have been shown to be widely present in the gut of black soldier flies in previous studies. They were all purchased from the China Industrial Microbial Culture Collection Center (CICC) and their strain numbers are CICC 10035, CICC 24792, CICC 22933, CICC25067, and CICC 24137, respectively.

[0037] Example 1 In this embodiment, the ability of dominant gut strains of black soldier fly to degrade cellulose and protein was detected by means of primary screening with clear zone and secondary screening with quantitative enzyme activity, in order to screen for substrate-degrading strains with homology advantage.

[0038] The culture medium formula used in the clear zone test is as follows: Cellulose transparency test: Sodium carboxymethyl cellulose 15.0 g, sodium nitrate 1.0 g, magnesium sulfate 0.5 g, yeast extract 1.0 g, potassium dihydrogen phosphate 1.0 g, Congo red 0.5 g, agar 20.0 g, distilled water 1 L; pH natural.

[0039] Protein transparency test: Casein 16.0 g, sucrose 30.0 g, dipotassium hydrogen phosphate 1.0 g, sodium chloride 5.0 g, potassium nitrate 2.0 g, magnesium sulfate 0.5 g, agar 15.0 g, distilled water 1 L; adjust pH to 7.0.

[0040] The culture medium formula used for the enzyme activity quantification assay is as follows: Liquid seed culture medium for strain (for cellulase activity assay): 15.0 g sodium carboxymethyl cellulose, 1.0 g sodium nitrate, 0.5 g magnesium sulfate, 1.0 g potassium dihydrogen phosphate, 1 L distilled water; pH: natural.

[0041] Liquid seed culture medium for strains (for determining protein enzyme activity): 10.0 g glucose, 5.0 g peptone, 5.0 g yeast extract, 10.0 g sodium chloride, 0.2 g magnesium sulfate, 1.0 g potassium dihydrogen phosphate, 1 L distilled water; adjust pH to 7.0.

[0042] Liquid enzyme-producing culture medium (for cellulase activity assay): 2.0 g potassium dihydrogen phosphate, 1.4 g ammonium sulfate, 0.3 g magnesium sulfate, 0.3 g calcium chloride, 0.005 g ferrous sulfate, 0.0016 g manganese sulfate, 0.0014 g zinc sulfate, 0.002 g cobalt chloride, 5.0 g sodium carboxymethyl cellulose, 5.0 g peptone, 1 L distilled water; adjust pH to 7.0.

[0043] Liquid enzyme-producing medium (for measuring protein enzyme activity): Replace peptone with 10.0 g of casein in the liquid medium.

[0044] The activation steps for each strain are as follows: Each test strain preserved in glycerol tubes at -80℃ was revived and purified under aseptic conditions. The specific steps are as follows: 1. Streak plate revival: Using a sterile inoculating loop, a small amount of bacterial solution from the glycerol tube was taken and streaked on an LB agar plate (10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 15.0 g agar, 1 L distilled water, pH 7.0). The plate was then incubated upside down in a 28℃ incubator for 24-48 hours. 2. Secondary streak activation: Isolated colonies with typical growth and uniform morphology were picked from the revived plate and streaked a second time on a fresh LB agar plate. The plates were then incubated under the same conditions (28℃) for another 24-48 hours to ensure the purity and activity of the strains. After this activation step, the fresh, mature single colonies obtained on the plate can be directly used for subsequent series of tests such as the clear zone. The specific steps for the initial screening of the clear zone and the rescreening of enzyme activity are as follows: (1) Clear zone test: After activating the isolated strain, it was inoculated onto the differential medium by spot inoculation. Each strain was repeated three times and cultured upside down at room temperature for 2-7 days. The cellulose / protein degradation ability of the strain was preliminarily judged by the size of the clear zone Dc value {Dc = clear zone diameter (D, cm) / colony diameter (d, cm)}.

[0045] (2) Enzyme activity quantification: The activated strains were inoculated into the corresponding liquid seed culture media and cultured with shaking at 28℃ and 180 rpm to prepare seed bacterial suspensions. The culture was continued until the strains entered the late logarithmic growth phase (measured by OD). 600 The inoculum value was set at 0.8-1.2 (approximately 12-16 hours). Subsequently, the seed culture was transferred to the corresponding liquid enzyme-producing medium at a 2% inoculum size and cultured with shaking at 28°C and 180 rpm for 48 hours. After culture, the supernatant was collected by centrifugation to obtain the crude enzyme solution. Then, using 1% sodium carboxymethyl cellulose solution, starch-free filter paper strips, and 1% salicin solution as substrates, the crude enzyme solution was reacted at 50°C for 30 minutes. The amount of reducing sugar produced was determined using the DNS method, and the activities of carboxymethyl cellulase, filter paper enzyme, and β-glucosidase were calculated accordingly. For protease activity determination, 1% casein solution was used as substrate, reacted with the crude enzyme solution at 60°C for 10 minutes, and the amount of tyrosine produced was determined using the Folin method, and the protease activity was calculated accordingly. All enzyme activity assays were performed in triplicate. The enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the production of 1 μmol of product from the substrate per minute.

[0046] Experimental results are as follows Figure 1 and Figure 2 As shown in the figure. This invention successfully screened two highly efficient degrading strains from the intestines of black soldier fly larvae using a combination of the clear zone method and enzyme activity quantification method. Strain C exhibited excellent cellulose degradation ability, with carboxymethyl cellulase, filter paper enzyme, and β-glucosidase activities reaching 58.38 U / mL, 42.68 U / mL, and 26.66 U / mL, respectively; strain D showed outstanding protein degradation ability, with a protease activity as high as 61.23 U / mL.

[0047] Example 2 The high cellulase activity strain C obtained in Example 1 was used. Acinetobacter baumannii ) and high protein enzyme activity strain D ( Providencia vermicola Antagonistic experiments were conducted to determine whether the two strains had potential for synergistic effects. Specifically, activated strains were streaked in pairs on PDA medium (4.0 g potato extract, 20.0 g glucose, 15.0 g agar, 1 L distilled water; pH natural) and incubated at 37°C. Once the strains had grown into bands, inhibition zones were observed at the streaks. The results are shown in the table below. Figure 3 It can be seen that both target strain C and strain D grew well at the cross-streaked area without any inhibition zone, indicating that there is no antagonistic effect between the two strains and they can be combined and mixed.

[0048] Subsequently, seed cultures of strains C and D were prepared separately, with the following specific steps: Strain C was inoculated into a seed culture medium for cellulase activity assay (15.0 g sodium carboxymethyl cellulose, 1.0 g sodium nitrate, 0.5 g magnesium sulfate, 1.0 g potassium dihydrogen phosphate, 1 L distilled water; pH natural); strain D was inoculated into a seed culture medium for protease activity assay (10.0 g glucose, 5.0 g peptone, 5.0 g yeast extract, 10.0 g sodium chloride, 0.2 g magnesium sulfate, 1.0 g potassium dihydrogen phosphate, 1 L distilled water; pH 7.0), and cultured with shaking at 28℃ and 180 rpm for 12-16 hours until the culture medium OD... 600 A pH value of 0.8-1.2 indicates vigorous seed culture. The seed culture medium for strain C does not contain any organic nitrogen sources, aiming to adapt it to a cellulose-based nutrient environment from the seed stage, thereby initiating the cellulase synthesis mechanism early and laying the foundation for efficient enzyme production later. Conversely, the seed culture medium for strain D is rich in readily available carbon and nitrogen sources such as glucose, peptone, and yeast extract, designed to support rapid growth and obtain high-density, high-activity cells, providing a sufficient strain reserve for efficient and rapid protease synthesis in the enzyme-producing medium. The prepared seed cultures of strains C and D are mixed in an equal volume ratio (1:1) to prepare a composite seed culture. Subsequently, at a total inoculum volume of 10%, the culture medium was transferred to a compound liquid enzyme-producing medium (casein 5.0 g, sodium carboxymethyl cellulose 5.0 g, glucose 5.0 g, potassium dihydrogen phosphate 2.0 g, ammonium sulfate 1.4 g, magnesium sulfate 0.3 g, calcium chloride 0.3 g, ferrous sulfate 0.005 g, manganese sulfate 0.0016 g, zinc sulfate 0.0014 g, cobalt chloride 0.002 g, distilled water 1 L; pH adjusted to 7.0). The medium was cultured under the following initial conditions: temperature 25℃, initial pH 7, inoculum volume 10%, fermentation time 3 days, and shaking speed 120 r / min. The initial enzyme activities of the compound microbial agent were measured.

[0049] It should be noted that, before determining the optimal culture conditions, in order to further optimize the enzyme production balance of the compound microbial agent and verify its optimal compounding ratio, this invention investigated the effects of different inoculation ratios (strain C:strain D, with volume ratios of 1:1, 1:2, 1:3, 2:1, and 3:1) on the activities of four key enzymes. All experiments were conducted under the aforementioned initial enzyme production conditions.

[0050] The results are shown in Table 1. The mixing ratio significantly affected the composition and activity of the compound enzyme system. When the proportion of strain C was high (e.g., 3:1 and 2:1), the activities of carboxymethyl cellulase, filter paper enzyme, and β-glucosidase in the compound bacterial agent were relatively high; while when the proportion of strain D was high (e.g., 1:2 and 1:3), the protease activity was more prominent. Notably, when the two strains were mixed in an equal volume ratio (1:1), the best overall effect was achieved. The activities of the four key enzymes were maintained at a high level and were balanced with each other. The activities of carboxymethyl cellulase, filter paper enzyme, β-glucosidase, and protease reached 50.12 U / mL, 30.75 U / mL, 28.18 U / mL, and 53.28 U / mL, respectively, and the overall enzyme activity was significantly better than other non-equal proportion groups. This result proves that the 1:1 mixing ratio is the optimal ratio that has been experimentally verified to achieve synergistic effects. Therefore, a 1:1 ratio was selected for subsequent mixing, and the enzyme production conditions were optimized.

[0051] Table 1. Enzyme activity values ​​(U / mL) of compound bacterial agents composed of different strains in varying proportions.

[0052] Subsequently, the enzyme production conditions of the compound microbial agent were optimized using a single-factor optimization experiment. Specifically, keeping other components of the culture medium constant, single-factor optimization was performed according to the order of culture temperature, pH value, inoculum size, culture time, and culture speed. The changes in cellulase and protease production by the compound microbial community under the influence of single factors were analyzed. For the single-factor experiment on culture temperature, the culture temperature was set as the dependent variable (15, 20, 25, 30, 35℃) based on the initial conditions. For the single-factor experiment on initial pH value, the initial pH value was set as the dependent variable (5, 6, 7, 8, 9) based on the single-factor experiment on culture temperature (taking the optimal culture temperature). This process was repeated to examine the effects of inoculum size, culture time, and culture speed on enzyme production capacity.

[0053] Experimental results are as follows Figure 4 As shown, the optimal enzyme production conditions obtained through optimization are as follows: temperature 30℃, initial pH 7, inoculum size 8%, fermentation time 3 days, and shaking speed 120 r / min. This invention confirms that strain C and strain D have no antagonistic effect and possess good potential for compounding. After compounding the two strains, the culture conditions were optimized through systematic single-factor experiments, ultimately resulting in a significant increase in the activities of four key enzymes in the compound microbial agent, reaching 72.35 U / mL for carboxymethyl cellulase, 54.10 U / mL for filter paper enzyme, 40.51 U / mL for β-glucosidase, and 77.10 U / mL for protease, laying a solid foundation for the application of high-efficiency microbial agents.

[0054] Example 3 This embodiment aims to screen the optimal preservation carrier that can effectively maintain the activity of the compound microbial agent (composed of strain C and strain D in a certain proportion) and determine its preparation process.

[0055] Three organic carriers—wheat bran, straw powder, and peat—and two inorganic carriers—zeolite and vermiculite—were selected as candidates. All carriers were pulverized through a 40-mesh sieve, sterilized at 121°C for 30 minutes, and then dried for later use. Under aseptic conditions, the composite bacterial solution prepared under the optimal enzyme-producing conditions of Example 2 (with a volume ratio of strain C to strain D seed culture of 1:1 and a total viable count of 2.75 × 10⁻⁶) was used. 8 CFU / mL (the ratio of the two bacteria after culturing in the bacterial solution is approximately 1:2 to 1:3) was uniformly mixed with various carriers at a mass-to-volume ratio of 5g carrier: 15mL bacterial solution. The inoculated carriers were then incubated at 30℃. Samples were taken on day 7 after incubation, and the viable cell count in each carrier was determined using the plate count method to evaluate the ability of different carriers to maintain bacterial activity during short-term preservation. Each treatment was performed in triplicate.

[0056] Table 2 shows the effective viable cell counts of the high-efficiency compound microbial agent after 7 days of cultivation on different carriers. Wheat bran was the optimal preservation carrier. This invention addresses the short-term preservation and application requirements of microbial agents, selecting wheat bran as the best carrier. Solid microbial agents prepared on this carrier can achieve an effective viable cell count of up to 3.24 × 10⁻⁶. 8 The CFU / g ratio is significantly higher than other carriers, effectively ensuring the activity and stability of the bacterial agent during storage and transportation.

[0057] Table 2. Number of viable bacteria after 7 days of culture of compound microbial agent on different carriers

[0058] Example 4 This embodiment aims to verify the actual effects of the cellulose-protein composite degrading microbial agent provided by the present invention on system productivity and environmental gas emissions in actual black soldier fly farming. Two treatment groups were set up: a control group (CK) without microbial agent and a group treated with the self-made composite microbial agent (OMA), with each treatment group being replicated three times.

[0059] The substrate for aquaculture consists of vegetable waste, slaughterhouse waste, and fresh chicken manure in a dry weight ratio of 1:1:3. The vegetable waste and slaughterhouse waste are mechanically crushed to approximately 2 cm and then mixed evenly with the fresh chicken manure. The total carbon-to-nitrogen ratio of the mixture is adjusted to 20:1 using sawdust, and the moisture content is controlled at around 70%. The mixture is then spread evenly indoors.

[0060] Black soldier fly eggs were hatched in an artificial climate chamber at a constant temperature of 28°C and a relative humidity of 30%. After they showed obvious mobility, they were transferred to a wheat bran roughage diet (coarse wheat bran and distilled water) for 4-6 days of culture. Subsequently, they were inoculated into the aforementioned culture substrate at a density of 500 flies / kg. The inoculation rate was 0.5% of the total dry weight of the substrate. w / w Weigh out the liquid bacterial suspension under optimal single-factor culture conditions, dilute and activate it with physiological saline, and then spray it evenly onto the substrate surface. Before application, use the plate count method to control the total effective viable count to be between 2.75 and 2.85 × 10⁻⁶. 8 CFU / mL (where the ratio of strain C to strain D is approximately 1:2 to 1:3). The treatment without added bacterial agent serves as the control group (CK). Open culture was carried out in the dark under constant temperature of 28°C and constant humidity of 60%.

[0061] After the start of cultivation, greenhouse gas collection of the substrate was conducted every 24 hours using a dynamic chamber method, and subsequent measurements were performed using gas chromatography. The substrate was then evenly turned, and a small amount of moisture was added to maintain larval activity. The experiment was stopped when 50% of the larvae reached the black prepupal stage. The larvae and substrate were sieved and weighed to calculate the bioconversion efficiency and larval yield. Bioconversion efficiency (%) = Total dry weight of black soldier fly larvae after cultivation / (Initial dry weight of added substrate - Dry weight of remaining substrate after cultivation) × 100%. Total productivity (kg / day) -1 m -3 = Total fresh body yield of black soldier fly larvae after cultivation (kg) / [conversion time (d) × conversion container volume (m³)] 3 Greenhouse gas emission equivalent (g CO2-eq kg) -1 DM) = E CH4 ×34×10 -3 + E N2O ×298×10 -3 + E NH3 ×298×1%×10 -3 in, E CH4 Represents the cumulative methane emissions (mg / kg) throughout the entire conversion process. -1 ), E N2O Represents the cumulative emissions of nitrous oxide during the entire conversion process (mg / kg). -1 ), E NH3 Represents the cumulative ammonia emissions (mg / kg) throughout the entire conversion process. -1The 1% figure refers to the indirect nitrous oxide emission standard factor for ammonia. 34 and 298 represent the global warming potential of methane and nitrous oxide, respectively.

[0062] Experimental results are as follows Figure 5 As shown, the present invention applies the developed compound microbial agent to the black soldier fly farming system. Compared with the control group without the addition of microbial agent, it can significantly improve the system performance, increase the total productivity of larvae and the substrate bioconversion rate by 18.96% and 18.20% respectively, and achieve a synergistic effect of reducing greenhouse gas emissions by 9.60%, demonstrating excellent promotion and application value and economic and environmental benefits.

[0063] The above embodiments demonstrate that the present invention has successfully developed a composite microbial agent with synergistic degradation function of cellulose and protein. Through efficient strain screening, compound optimization and carrier enhancement, its application efficiency in the black soldier fly bioconversion system has been significantly improved, providing reliable technical support for achieving synergistic effects of efficient resource utilization of agricultural organic waste and environmental emission reduction in the breeding process.

[0064] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A compound microbial agent, characterized in that, Its active ingredient is Acinetobacter baumannii ( Acinetobacter baumannii ) and Alcaligenes providediflora ( Providencia alcalifaciens ); The Acinetobacter baumannii strain mentioned is numbered CICC 22933 by the China Industrial Microbial Culture Collection Center. The Alcaligenes Providencia species is registered at the China Industrial Microbial Culture Collection Center as CICC 25067.

2. The compound microbial agent according to claim 1, characterized in that: The ratio of live bacteria (CFU) of Acinetobacter baumannii and Providencia alkaligenes is 1:(2-3).

3. The compound microbial agent according to any one of claims 1-2, characterized in that: The compound microbial agent also includes a carrier.

4. The compound microbial agent according to claim 3, characterized in that: The carrier is wheat bran.

5. The method for preparing the compound microbial agent according to any one of claims 1-4, characterized in that, The process includes the following steps: inoculating the seed cultures of Acinetobacter baumannii and Providencia alkali-producing bacteria into a culture medium for cultivation to obtain the compound bacterial agent.

6. The method for preparing the compound microbial agent according to claim 5, characterized in that: The volume ratio of the seed culture of Acinetobacter baumannii to the seed culture of Alcaligenes providediflora is 1:

1. And / or, the activation of the Acinetobacter baumannii and the Alcaligenes providentem strain is carried out at 28–32°C; And / or, the culture conditions are as follows: culture temperature of 15-35℃, initial pH of culture medium of 5-9, seed liquid inoculation amount of 4%-12%, culture time of 2-6 days, and rotation speed of 100-140 r / min; preferably, the culture conditions are as follows: culture temperature of 30℃, initial pH of culture medium of pH 7, seed liquid inoculation amount of 8%, culture time of 3 days, and rotation speed of 120 r / min.

7. The use of the compound microbial agent according to any one of claims 1-4 or the compound microbial agent prepared by the method according to any one of claims 5-6 in at least one of the following: A1) Increase the overall productivity of black soldier fly larvae in aquaculture; A2) Improve the substrate bioconversion rate in black soldier fly farming; A3) Reduce greenhouse gas emissions from black soldier fly farming.

8. A method for cultivating black soldier fly larvae, characterized in that, Includes the following steps: Black soldier fly larvae are inoculated into an agricultural waste aquaculture substrate containing the compound microbial agent according to any one of claims 1-4 or the compound microbial agent prepared by the method according to any one of claims 5-6, and then cultured; wherein the culture substrate includes agricultural waste.

9. The method for cultivating black soldier flies according to claim 8, characterized in that: The agricultural waste consists of vegetable waste, slaughter waste and fresh chicken manure, and more specifically, the agricultural waste consists of vegetable waste, slaughter waste and fresh chicken manure in a dry weight ratio of 1:1:

3. And / or, the total carbon-to-nitrogen ratio of the culture substrate is (20-25):1; And / or, the moisture content of the culture substrate is 70% to 75%.

10. The black soldier fly culturing method according to any one of claims 8-9, characterized in that: The inoculation density of black soldier fly larvae in the culture substrate is 450-500 larvae / kg of fresh material; And / or, the black soldier fly larvae are 4 to 6 days old; And / or, the compound microbial agent is sprayed onto the surface of the aquaculture substrate in the form of a bacterial solution, wherein the total number of effective viable bacteria in the bacterial solution is ≥2.75×10⁻⁶. 8 The bacterial culture is CFU / mL, and its mass is 0.5% to 1.0% of the total dry weight of the substrate.

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