Cellulose degrading bacterium and application thereof in preparation of bio-organic fertilizer through fermentation of traditional Chinese medicine residues

Abstract

The invention discloses bacillus subtilis for cellulose degradation and application of the bacillus subtilis in preparation of a bio-organic fertilizer through fermentation of mulberry leaf and chrysanthemum cold granule traditional Chinese medicine residues. The bacillus subtilis is collected and separated from decomposed soil under osmanthus fragrans forests in the bead mountain park in the Dalou City; through a sodium carboxymethyl cellulose culture medium, a Congo red dyeing transparent ring and an enzyme activity determination method, an efficient cellulose degradation strain is screened, and the cellulose degradation strain is inoculated with mulberry leaf and chrysanthemum cold granule traditional Chinese medicine residues, rice hull fungus sticks and pig manure as raw materials for solid state fermentation to produce the bio-organic fertilizer. The cellulose degrading bacterium is high in degrading capacity, and the bio-organic fertilizer prepared by fermenting the traditional Chinese medicine residues through the cellulose degrading bacterium is uniform in size and gap, alkalescent, capable of reducing the content of heavy metal, relieving soil acidification and remarkably increasing the number of soil microbial fungi and bacteria. The bio-organic fertilizer prepared by applying the cellulose degrading bacteria to ferment traditional Chinese medicine residues and livestock wastes can significantly improve the yield and quality of citrus.

Description

Technical Field

[0001] The invention belongs to the technical field of biological organic fertilizer production, and particularly relates to the preparation and application of an organic fertilizer fermentation agent Background Art

[0002] Traditional Chinese medicine residues are wastes from the processing and preparation of traditional Chinese medicines, the production of traditional Chinese medicines, and the production and processing of other traditional Chinese medicine-related products. The traditional Chinese medicine residues produced in the production of traditional Chinese medicines are the most, accounting for 70% of the total. According to statistics, in 2017, the planting area of ​​traditional Chinese medicines in my country was nearly 4.67×10 6 hm 2 , the solid waste of Chinese medicine residue generated in the processing of traditional Chinese medicine can reach 5.5×107t per year. Chinese medicine residue contains a variety of nutrients, including crude fiber, crude protein, crude fat, starch, amino acids, vitamins and effective active ingredients of traditional Chinese medicine. The main treatment methods for Chinese medicine residue are stacking, incineration and landfill, which leads to serious waste of resources and environmental pollution.

[0003] The vast majority of Chinese medicine ingredients come from plants, and lignocellulose is one of the main components of plant cell walls. Therefore, cellulose degradation is the key to the utilization of Chinese medicine residues. At present, the use of microorganisms to degrade cellulose is an effective way. Although the cellulase secretion ability of bacteria is generally not as good as that of fungi and actinomycetes, bacteria reproduce quickly. Therefore, screening highly efficient cellulose-degrading bacteria is the key to microbial cellulose degradation. Aerobic composting is one of the effective ways to utilize agricultural waste resources and achieve resource recycling. After the waste of medicinal residues is used as fertilizer, it can be used in the cultivation of Chinese medicinal materials, which can promote the recycling of medicinal residues, which is of great significance to the income generation and environmental protection of Chinese medicine enterprises.

[0004] The number and type of microorganisms play a very important and critical role in the organic fertilizer fermentation process. When commonly used strains are used to ferment antiviral granules of traditional Chinese medicine residues, the activity of the strains is inhibited by the antibacterial components of the residues, and the growth and reproduction are abnormally slow; and the pH value of the residues is too low, which is not conducive to the growth of the strains; and the lack of nutrients in the residues also limits the growth of the strains. In addition, due to the complex composition of traditional Chinese medicine residues, it is necessary to select appropriate strains and / or strain combinations as well as unique fermentation processes and technologies to fully biodegrade the traditional Chinese medicine residues to ensure the safety of the recycled products and fully meet the needs of different organisms for organic fertilizers, so that the biological fermentation products of traditional Chinese medicine residues can be widely used in land improvement, plant fertilization and other fields. Therefore, the selection of fermentation agents is particularly important for the production of biological organic fertilizers. Summary of the invention

[0005] The object of the present invention is to provide a cellulose-degrading bacterium ZG2-3 that can efficiently ferment traditional Chinese medicine residues into organic fertilizer. ZG2-3 is Bacillus subtilis, which has been deposited in the General Microbiological Center of China National Committee for the Preservation and Administration of Microbiological Cultures on November 20, 2023. The address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.29066.

[0006] The present invention also aims to provide a method for preparing organic fertilizer, which comprises collecting and separating the decomposed soil under the sweet-scented osmanthus forest in Zhushan Park, Loudi City, Hunan Province, screening an efficient cellulose-degrading strain ZG2-3 through a sodium carboxymethyl cellulose culture medium, Congo red staining transparent circles and enzyme activity determination, and inoculating the cellulose-degrading strain ZG2-3 with Chinese medicine residues, rice husk mushroom sticks and pig manure as raw materials to carry out solid-state fermentation to produce biological organic fertilizer.

[0007] The cellulose degrading bacteria ZG2-3 were subjected to DNA extraction and PCR amplification to obtain a 16S rDNA fragment of about 1000 bp in length. The BLAST sequence comparison and phylogenetic tree construction showed that Figure 3 , strain ZG2-3 was determined to be Bacillus subtilis.

[0008] The microbial agent containing the cellulose degrading bacteria ZG2-3, whose active ingredients are the cellulose degrading bacteria ZG2-3 (Bacillus subtilis) and its extracellular metabolites, is compared with a commercialized decay-promoting agent (manufacturer: Hebi Renyuan Biotechnology Development Co., Ltd.), and 7 strains of Bacillus subtilis CF1, CF2, CF3, CF4, CF5, CF6, and CF7 are separated from the commercially available decay-promoting agent. Experiments verify that the cellulose degradation activity of the cellulose degrading bacteria ZG2-3 of the present invention is better than that of the commercialized decay-promoting agent.

[0009] The preparation method of the microbial agent comprises the following steps:

[0010] (1) Collecting decomposed soil from the natural osmanthus trees growing for many years in Loudi City;

[0011] (2) Weigh 1.00 g of the soil sample and 0.5 g of filter paper into 50 mL of enrichment medium, shake and culture in the enrichment medium at 30°C and 150 r / min until the filter paper strip is completely lysed, and then continuously dilute the enrichment medium with sterile water to obtain a concentration of 10 -4 -10 -6 diluent;

[0012] (3) Take 200 μL of the dilution and spread it evenly on the screening medium containing Congo red, and culture it at a constant temperature of 30°C. After the colonies grow, select single colonies with different culture characteristics and obvious transparent zones and transfer them to the screening medium. Purify them three times and then freeze them for storage;

[0013] (4) The purified strain was transferred to a screening culture plate and cultured at 30°C until colonies grew out, stained with 0.5% Congo red solution for 60 min, and then faded with 1 mol / L NaCl solution for 60 min. The size of the transparent zone after staining was observed, and the cellulose degradation ability was preliminarily determined by the ratio of the transparent zone diameter D to the colony diameter d;

[0014] (5) A single colony of the purified strain with a large D / d ratio (i.e., cellulose-degrading bacteria ZG2-3) was selected and inoculated into a fermentation medium, cultured at 35°C and 180 r / min for 48 h, and centrifuged at 5000 r / min for 5 min to obtain a fermentation broth, which was the microbial agent.

[0015] The enrichment medium is composed of: K 2 HPO 4 1.00g·L -1 、NaCl 0.10g·L -1 MgSO 4 7H 2 O0.30g·L -1 、NaNO 3 2.50g·L -1 、FeCl 3 0.001g·L -1 , CaCl 2 0.10g·L -1 ;

[0016] The screening medium consists of: (NH 4 )2SO 4 2.00g·L -1 MgSO 4 7H 2 O 0.50g·L -1 , K 2 HPO 4 1.00g·L -1 、NaCl 0.50g·L -1 , sodium carboxymethyl cellulose (CMC-Na) 2.00 g·L -1 , agar 20.00 g·L -1 ,

[0017] The screening medium containing Congo red is as follows: 2.00 g·L -1 Congo Red,

[0018] The fermentation medium composition is: peptone 3.00 g·L -1 , Yeast powder 0.50g·L -1 NH 4 ) 2 SO 4 2.00g·L -1 , KH 2 PO 4 4.00g·L -1 , CaCl 2 0.30g·L -1 MgSO 4 7H 2 O 0.30 g·L -1 .

[0019] The cellulose degrading bacteria is used in preparing organic fertilizer by fermenting traditional Chinese medicine residues.

[0020] The process of preparing organic fertilizer by fermenting Chinese medicine residue with cellulose-degrading bacteria comprises the following steps: preparing fermentation matrix with Chinese medicine residue, fungus residue, rice husk and pig manure, then inoculating 5% by mass of microbial agent containing cellulose-degrading bacteria ZG2-3, adjusting the moisture content to 50%-60%, putting the fermentation matrix into a transparent sealed container, fermenting at a constant temperature of 35°C for 60 days, degassing and mixing every three days, and obtaining biological organic fertilizer.

[0021] The Chinese medicine residue is the Chinese medicine residue of Sangju Cold Granules, which comes from the Chinese medicine residue of Sangju Cold Granules produced by Huichuntang Co., Ltd. The main raw materials of Sangju Cold Granules are mulberry leaves, chrysanthemums, forsythia, mint, bitter almonds, platycodon, licorice, and reed roots.

[0022] Application of the biological organic fertilizer in improving the quality and yield of citrus

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention uses the Congo red staining method to screen out a strain with high efficiency in degrading cellulose from the decomposed soil of Zhushan Park in Loudi. The ability of bacteria to degrade cellulose is determined based on the size of the transparent circle formed by staining. Combined with morphological observation, Gram staining and 16S rDNA sequence determination, a phylogenetic tree is constructed to determine that the strain ZG2-3 is Bacillus subtilis. Compared with the strain CF-6 with the highest carboxymethyl cellulase (CMCase) and filter paper enzyme (FPA) activities screened out from commercially available decay promoters (manufacturer: Hebi Renyuan Biotechnology Development Co., Ltd.), the carboxymethyl cellulase (CMCase) activity and filter paper enzyme (FPA) activity of the cellulose-degrading bacteria CF-6 were measured to be 0.026 IU / mL and 0.018 IU / mL, respectively, and the carboxymethyl cellulase (CMCase) activity and filter paper enzyme (FPA) activity of the cellulose-degrading bacteria ZG2-3 were measured to be 0.054 IU / mL and 0.020 IU / mL, respectively, both higher than those of the strain CF-6.

[0025] (2) The present invention uses Chinese medicine residues and livestock feces as main ingredients, and adopts a screened specific strain of cellulose-degrading bacteria ZG-3 that is highly effective for the Chinese medicine residues of Sangju Ganmao Granules. The strain ZG-3 can quickly degrade the plant cellulose in the Chinese medicine residues of Sangju Ganmao Granules, and the various components of the residues are quickly and completely decomposed, thereby shortening the fermentation time and improving the conversion rate of the Chinese medicine residues.

[0026] (3) The present invention does not need to add inorganic salts during the fermentation process, and can reduce the content of heavy metals in organic fertilizers. The microbial agent can adsorb and transform heavy metals, so that the content of heavy metals is lower than the industry standard. The organic fertilizer has a high organic matter content, and the total nutrient content exceeds the minimum limit of 40g / kg of the industry standard, and is better than ordinary commercially available fermentation agents. The moisture content exceeds 30%, the organic fertilizer has uniform gaps, and the number of fungi and bacteria is increased, which can loosen the soil to a certain extent and improve soil fertility.

[0027] (4) The soil in Loudi is mostly acidic. The pH of the organic fertilizer of the present invention is greater than 8.5, which is weakly alkaline. The application of the organic fertilizer increases the soil pH value, alleviates soil acidification, and significantly increases the number of soil microorganisms. The microbial activity accelerates the dissolution of soil trace elements, reduces the loss of nitrogen and phosphorus, and improves soil fertility and fertilizer utilization.

[0028] (5) The bio-organic fertilizer prepared by cellulose-degrading bacteria ZG2-3 can significantly increase the yield and quality of citrus, promote the upgrading of citrus quality, increase the sales price of citrus and the income of fruit farmers, and solve the problem of poor sales and low prices caused by the decline of citrus quality. It is worthy of large-scale demonstration and promotion. Description of the drawings:

[0029] Figure 1 Congo red staining results (A. Congo red staining results of strain ZG2-3, B. Congo red staining results without transparent zone)

[0030] Figure 2 Colony morphology and Gram staining results of strain ZG2-3 (A. Colony of ZG2-3; B, C. Gram staining of ZG2-3)

[0031] Figure 3 Phylogenetic tree of strain ZG2-3 DETAILED DESCRIPTION

[0032] Example 1

[0033] (1) Screening of cellulose-degrading bacteria

[0034] The decomposed soil under the naturally growing osmanthus tree in Zhushan Park, Loudi City, was collected. 1.00 g of soil sample was weighed and cultured in an enrichment medium at 30°C and 150 r / min until the filter paper strip was completely lysed. The enrichment medium after culture was continuously diluted with sterile water to obtain 10 -4 , 10 -5 , 10 -6 Take 200 μL of the dilution and spread it evenly on the screening medium containing Congo red. Culture it at 30°C. After the colonies grow out, select single colonies with different culture characteristics and obvious transparent circles and transfer them to the screening medium. Purify them three times and then freeze them.

[0035] The purified strain was transferred to the screening culture plate and cultured at 30°C until colonies grew. Stained with 0.5% Congo red solution for 60 minutes, then faded with 1 mol / L NaCl solution for 60 minutes, and the size of the transparent zone after staining was observed by the existing technical method (Liu Tongtong et al., Screening, genetic modification and optimization of biosynthesis conditions of polyhydroxybutyrate in red panda feces, Journal of Tianjin Normal University (Natural Science Edition), 2022, 42(01): 45-50). The cellulose degradation ability was preliminarily determined by the ratio of the transparent zone diameter (D) and the colony diameter (d) (Li Le et al. Screening of a cellulase-producing bacterium and study of enzyme production characteristics, Environmental Science and Technology, 2019, 32(01): 24-29).

[0036] (2) Identification of cellulose-degrading strains

[0037] Morphological identification: The optimal cellulose-degrading bacteria (maximum D / d value) strain ZG2-3 selected in step (1) was inoculated into the screening medium and cultured for 24 h. The colony culture characteristics were observed and Gram staining was performed.

[0038] Molecular biological identification: DNA of the strain was extracted, and the 16S rDNA sequence of the strain was amplified using the bacterial 16S rDNA universal primers 7F (5'-CAGAGTTTGATCCTGGCT-3') and 1540R (5'-AGGAGGTGATCCAGCCGCA-3'). The PCR reaction conditions were: denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 90 s. After 30 cycles, the final extension was performed at 72°C for 10 min to fully amplify the reaction product. The amplified product was sent to Sangon Biotech (Shanghai) Co., Ltd. for identification. The sequencing results were compared with the existing sequences in GenBank using BLAST software, and the phylogenetic tree was constructed using MEGA-7 software. See the attached data. Figure 3 , determine the species of the strain.

[0039] The strain was entrusted to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation, and the strain preservation number is CGMCC NO.29066.

[0040] After morphological identification, strain ZG2-3 had typical bacterial colony characteristics and was a Gram-positive bacillus, see attached Figure 2 The colonies of the strain are off-white, round, with a rough surface and dry. The colonies are white, round, with a wrinkled surface and dry.

[0041] The enrichment medium in this embodiment is composed of: K 2 HPO 4 1.00g·L -1 、NaCl 0.10g·L -1 MgSO 4 7H 2 O 0.30 g·L -1 、NaNO 3 2.50g·L -1 、FeCl 3 0.001g·L -1 , CaCl 2 0.10g·L -1 ;

[0042] The screening medium consists of: (NH 4 )2SO 4 2.00g·L -1 MgSO 4 7H 2 O 0.50g·L -1 , K 2 HPO 4 1.00g·L -1 、NaCl 0.50g·L-1 , sodium carboxymethyl cellulose (CMC-Na) 2.00 g·L -1 , agar 20.00 g·L -1 ,

[0043] The screening medium containing Congo red is as follows: 2.00 g·L -1 Congo red.

[0044] Example 2 Determination of cellulase activity

[0045] (1) Enzyme activity is the amount of reducing sugar equivalent to 1 μmol of glucose produced by hydrolyzing the corresponding substrate per minute in 1 mL of fermentation broth under corresponding conditions. This is defined as 1 enzyme activity unit and expressed as IU / mL.

[0046] Glucose standard curve drawing: add 2.50, 2.45, 2.40, 2.35, 2.30, 2.25mL of phosphate buffer solution to the washed and dried test tubes, then add 0, 0.05, 0.10, 0.15, 0.20, 0.25mL of glucose standard solution in sequence, and finally add 2mL of DNS solution to each test tube, mix well, heat in a boiling water bath for 5min, take out the test tube and cool it to room temperature with running water, distilled water to 25mL, mix well and measure the absorbance at 540nm, repeat 3 times. With absorbance as ordinate and glucose mass as abscissa, the glucose standard curve y=0.5931x+0.0388 (R2=0.9972) is obtained.

[0047] (2) Take a single colony of the cellulose-degrading bacteria strain ZG2-3 screened in Example 1, and eight strains of Bacillus subtilis CF1, CF2, CF3, CF4, CF5, CF6, and CF7 isolated from a commercially available probiotic (manufacturer: Hebi Renyuan Biotechnology Development Co., Ltd.) and inoculate them into a fermentation medium. Cultivate them at 35°C and 180 r / min for 48 h, and centrifuge them at 5000 r / min for 5 min to obtain a fermentation broth. Repeat three times for each strain. The fermentation medium consists of: 3.00 g·L peptone; -1 , Yeast powder 0.50g·L -1 NH 4 ) 2 SO 4 2.00g·L -1 , KH 2 PO 4 4.00g·L -1 , CaCl 2 0.30g·L -1 MgSO 4 7H 2 O0.30g·L-1 .

[0048] (3) CMCase activity assay: 0.5 mL of fermentation broth and 2 mL of 0.5% CMC-Na solution were placed in a 25 mL stoppered test tube and incubated in a 50°C water bath for 30 min. Then 2 mL of DNS solution was added and heated in a boiling water bath for 5 min. The mixture was cooled to room temperature with running water, fixed to volume, and the OD540 value was measured. The fermentation broth heated in a boiling water bath for 10 min was used as a blank sample.

[0049] (4) FPA activity assay: Fold Whatman No. 1 (1 cm × 6 cm) into an "M" shape, immerse in 2 mL of phosphate buffer solution, and add 0.5 mL of fermentation broth. Incubate in a 50°C water bath for 30 min, then add 2 mL of DNS solution, heat in a boiling water bath for 5 min, cool to room temperature with running water, make up to volume, and measure the OD540 value. The fermentation broth heated in a boiling water bath for 10 min was used as a blank sample.

[0050] Calculation formula: enzyme activity (IU / mL) = (Aa-Ab-c) / K×V×T×M)×1000.

[0051] Wherein, Aa is the OD540 value of the fermentation broth; Ab is the OD540 value of the blank control; K is the slope of the glucose standard curve; c is the intercept of the glucose standard curve; V is the volume of the fermentation broth (mL); T is the reaction time (min); and M is the molecular weight of glucose.

[0052] The reducing sugar content generated by the reaction of the fermentation broth with CMC-Na and NEWSTAR filter paper was determined by the DNS method, and the CMCase and FPA activities of strain ZG2-3 and Bacillus subtilis isolated from commercially available decay promoters were determined (CF-1, CF-4, and CF-5 had poor effects and were not listed in Table 1).

[0053] Table 1 FPA and CMCase activity results of isolated strains

[0054]

[0055] As can be seen from Table 1, the cellulose degrading bacteria ZG2-3 screened by the present invention are compared with the strain CF-6 with the maximum carboxymethyl cellulase (CMCase) activity and filter paper enzyme (FPA) activity screened from the commercially available decay promoter (manufacturer: Hebi Renyuan Biotechnology Development Co., Ltd.). The carboxymethyl cellulase (CMCase) activity and filter paper enzyme (FPA) activity of the cellulose degrading bacteria CF-6 are measured to be 0.026 IU / mL and 0.018 IU / mL, respectively, and the carboxymethyl cellulase (CMCase) activity and filter paper enzyme (FPA) activity of the cellulose degrading bacteria ZG2-3 are measured to be 0.054 IU / mL and 0.020 IU / mL, respectively, which are higher than the commercially available decay promoter strain CF-6.

[0056] Example 3 Preparation of biological organic fertilizer

[0057] Based on 100 parts by mass of Chinese medicine residue, fungus residue and rice husk (dry matter ratio of 1:1) are supplemented to make the total weight of medicine residue, fungus residue and rice husk 500 parts by mass, and then 1000 parts by mass of pig manure are added and fully mixed to prepare a fermentation matrix. The fermentation liquid (inoculation amount of 5% by mass) obtained by cellulose degrading bacteria ZG2-3 in Example 2 is inoculated, and the moisture content is adjusted to 50% to 60%, and the control standard is "holding a mass of material by hand, no liquid flows out, and the material does not disperse after letting go", and the fermentation matrix is ​​loaded into a transparent sealed container, and fermented at a constant temperature of 35°C for 60 days, and degassing and mixing are performed every three days, which is recorded as biological organic fertilizer A1.

[0058] In Example 4, the amount of medicinal residue used is 150 parts by mass, and the remaining process is the same as in Example 3 to obtain bio-organic fertilizer A2.

[0059] In Example 5, the amount of medicinal residue used is 200 parts by mass, and the remaining process is the same as in Example 3 to obtain biological organic fertilizer A3.

[0060] In Example 6, the amount of medicinal residue used is 250 parts by mass, and the remaining process is the same as in Example 3 to obtain bio-organic fertilizer A4.

[0061] The blank comparative example CK was not inoculated with a microbial agent, and the rest was the same as Example 3.

[0062] The positive control PC is a blank control inoculated with a commercially available rot-promoting agent (added at 5% by weight), and the manufacturer is Hebi Renyuan Biotechnology Development Co., Ltd.

[0063] The bio-organic fertilizers prepared in Examples 3-6 were analyzed and tested, and the results are as follows

[0064] Table 2 Determination of heavy metal content in bio-organic fertilizer prepared from traditional Chinese medicine residues

[0065]

[0066] After 60 days of solid-state fermentation, the heavy metal content in the bio-organic fertilizer is shown in Table 2. There are significant differences between each treatment and the control. Except for the arsenic in the control group exceeding the limit index, the heavy metal content in other treatment groups is lower than the limit index of the bio-fertilizer. Moreover, the addition of cellulose-degrading bacteria ZG2-3 can significantly reduce the heavy metal content in the bio-organic fertilizer, indicating that the microbial agent of the present invention can adsorb and transform heavy metals, so that the heavy metal content in the bio-organic fertilizer is lower than that in the control treatment.

[0067] Table 3-1 Nutrient elements and total nutrients of organic fertilizer

[0068]

[0069] Table 3-2 Nutrient elements and organic matter content of organic fertilizer

[0070]

[0071] As can be seen from Table 3, there are significant differences in each embodiment. The total nitrogen content of A4 treatment is the highest, and the total nitrogen content of the positive control is the lowest. The total phosphorus content of A2 treatment is the highest, followed by A4 treatment, and the total phosphorus content of the positive control is the lowest. The total potassium content of A4 treatment is the highest, and the total potassium content is the lowest in the positive control. The total nutrient content of A4 and A2 treatments is 41.05g / kg and 40.62g / kg respectively, both exceeding the minimum limit of 40g / kg of the industry standard, which meets the industry standard. The application of biological organic fertilizer can supply the energy and nutrients required for the growth of soil microorganisms, provide favorable conditions for microbial activities, thereby affecting the diversity and function of the microbial community structure.

[0072] Table 4 Determination of physical and chemical properties of organic fertilizer

[0073]

[0074] There was no significant difference in the bulk density of each experimental example, with the highest being CK 0.43g / cm3 and the lowest being the positive control 0.35g / cm3. There was no significant difference in total porosity between the various treatments, with the highest being CK 69.43% and the lowest being A1 62.95%. There was no significant difference in the size pore ratio between the various treatments, and the size of the biological organic fertilizer of the present invention was uniform, and there was a significant difference between the various treatments of pH, with pH greater than 8.5, being weakly alkaline, with the highest being A4 and A2, and the lowest being A1. pH value is an important factor affecting the number of soil microbial populations, and the application of the organic fertilizer of the present invention improves soil pH value, alleviates soil acidification, and significantly increases the number of soil microorganisms, and microbial activity accelerates the dissolution of soil trace elements.

[0075] Table 5. Determination of fungi and bacteria counts in organic fertilizers

[0076]

[0077] There were significant differences in the number of bacteria among the treatments, and the number of bacteria in the PC group was significantly higher than that in the other treatments. There were no significant differences in the number of fungi among the treatments. The method of the present invention increases the number of fungi and bacteria in the biological organic fertilizer.

[0078] Example 7: Bio-organic fertilizer planting experiment

[0079] Example 4 (A2) for preparing bio-organic fertilizer and the positive control comparative example (PC) were selected for field test.

[0080] The experiment set up three kinds of furrowing treatments, namely, Example 4 (A2), positive control comparative example (PC), and chemical fertilizer (CF), to determine the effects of different treatments on citrus yield and quality. The specific steps were as follows:

[0081] A total of three treatments, A2, PC, and CF, were tested at the citrus base of Jiu'er Fruit and Vegetable Planting Professional Cooperative in Loudi City, Hunan Province. The biological organic fertilizer treatments A2 and PC were applied at 1000 kg / mu each, and the chemical fertilizer group CF was applied with 185 kg of ammonium bicarbonate + 92.5 kg of superphosphate per mu. All fertilizers were applied once in March. Each treatment area had 15 citrus trees, arranged in random blocks. The citrus yield and quality were measured after harvest.

[0082] The test results are as follows:

[0083] Table 6. Yield composition and yield of different treatments

[0084]

[0085] The experiment showed that the citrus yield of the two treatments of bio-organic fertilizer A2 and PC was significantly higher than that of the chemical fertilizer treatment. The best one was the bio-organic fertilizer with Chinese medicinal residue, with a yield of 3105kg / mu. The yield of commercial fruit per plant in the treatment with only chemical fertilizer was lower than that of bio-organic fertilizer. The possible reason was that the chemical fertilizer was applied in March, and the soil fertilizer efficiency was reduced in the later citrus fruiting period, affecting the yield.

[0086] Table 7 Nutritional quality of citrus fruits under different treatments

[0087]

[0088] The experiment showed that the application of bio-organic fertilizer A2 and PC had a significantly higher effect on the quality of citrus than the chemical fertilizer treatment (CF), and the effect of the microbial agent of the present invention was better than the commercially available decay-promoting agent, indicating that the bio-organic fertilizer of the present invention can improve the quality of citrus fruit.

[0089] The determination of bio-organic fertilizer indicators is carried out in accordance with the requirements and standards of organic fertilizer (NYT525-2021). The nutrient content and heavy metal content are sent to Nanjing Jisi Huiyuan Biotechnology Co., Ltd. for determination, and other indicators are completed in the laboratory of the College of Agriculture and Biotechnology, Hunan University of Humanities Science and Technology.

[0090] The physical and chemical properties of the bio-organic fertilizer matrix were determined by referring to the experimental method introduced in the literature (He Manqiao. Biotransformation of mushroom cultivation waste and its application in vegetable seedling matrix [D]. Hangzhou: Zhejiang University, 2012).

[0091] Microbial fungi in bio-organic fertilizers are determined according to GB / T 14643.4-2009, Determination of algae and bacteria in industrial circulating cooling water Part 4: Determination of soil fungi. Plate counting method

[0092] The microbial bacteria in biological organic fertilizer were determined according to the plate counting method recorded in the literature (Zhu Xiuxiu. Effects of culture medium treatment and culture time on the culturability of soil bacteria [D]. Nanjing Agricultural University, 2019).

[0093] The water content in bio-organic fertilizer is determined according to the vacuum oven method in GB / T 8576-2010 Determination of free water content in compound fertilizers.

[0094] The soluble solids in citrus quality were determined by a handheld saccharimeter, the total sugar was determined by 3,5-dinitrosalicylic acid colorimetry, the titratable acid content was determined by acid-base neutralization titration, and the vitamin C content was determined by molybdenum blue colorimetry.

[0095] The above experiments show that the present invention starts with the soil decomposed under the sweet-scented osmanthus forest in Zhushan Park, Loudi City, Hunan Province, and develops a bacterial agent with high degradation ability for cellulose, and ferments the Chinese medicine residue to make biological organic fertilizer, and the application of biological organic fertilizer is conducive to the accumulation of soil organic carbon, improves soil structure, increases soil porosity and water and fertilizer retention capacity, enhances soil enzyme activity, alleviates soil acidification, reduces nitrogen loss, improves soil fertility and fertilizer utilization rate, realizes soil fertilization, and promotes crop yield increase. The present invention provides efficient strains for biological organic fertilizer fermentation by screening cellulose-degrading bacteria, explores the recycling of wastes such as Chinese medicine residues, livestock and poultry manure, and rice husk fungus residues in Loudi area, uses organic fertilizer to replace part of chemical fertilizers, reduces the application of chemical fertilizers, thereby reducing the adverse effects of nitrogen and phosphorus loss on the environment, maintaining the sustainable production capacity of the soil, and providing technical references for the rational optimization of fertilization mode for crops.

Claims

1. A cellulose-degrading bacterium ZG2-3, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.29066.

2. The cellulose degrading bacteria according to claim 1, characterized in that: The cellulose degrading bacteria ZG2-3 is Bacillus subtilis.

3. A microbial agent using the cellulose degrading bacteria ZG2-3 according to claim 2, characterized in that: Its active ingredients are cellulose-degrading bacteria ZG2-3 (Bacillus subtilis) and its extracellular metabolites; The microbial agent is prepared by the following method: (1) Collecting decomposed soil from the natural osmanthus trees growing for many years in Loudi City; (2) Weigh 1.00 g of the soil sample and 0.5 g of filter paper into 50 mL of enrichment medium, shake and culture in the enrichment medium at 30°C and 150 r / min until the filter paper strip is completely lysed, and then continuously dilute the enrichment medium with sterile water to obtain a concentration of 10 -4 -10 -6 diluent; (3) Take 200 μL of the dilution and spread it evenly on the screening medium containing Congo red, and culture it at a constant temperature of 30°C. After the colonies grow, select single colonies with different culture characteristics and obvious transparent zones and transfer them to the screening medium. Purify them three times and then freeze them for storage; (4) The purified strain was transferred to a screening culture plate and cultured at 30°C until colonies grew out, stained with 0.5% Congo red solution for 60 min, and then faded with 1 mol / L NaCl solution for 60 min. The size of the transparent zone after staining was observed, and the cellulose degradation ability was determined by the ratio of the transparent zone diameter D to the colony diameter d; (5) Pick a single colony of the strain with a large D / d value and inoculate it into a fermentation medium, culture it at 35°C and 180 rpm for 48 h, and centrifuge it at 5000 rpm for 5 min to obtain a fermentation broth; that is, a microbial agent; The enrichment medium composition is: K2HPO4 1.00g·L -1 、NaCl 0.10g·L -1 、MgSO4·7H2O0.30g·L -1 、NaNO3 2.50g·L -1 、FeCl3 0.001g·L -1 、CaCl2 0.10g·L -1 ; The screening medium composition is: (NH4)2SO4 2.00g·L -1 MgSO4·7H2O 0.50g·L -1 、K2HPO41.00g·L -1 、NaCl 0.50g·L -1 , sodium carboxymethyl cellulose (CMC-Na) 2.00 g·L -1 , agar 20.00 g·L -1 , The screening medium containing Congo red is as follows: 2.00 g·L -1 Congo Red, The fermentation medium composition is: peptone 3.00 g·L -1 , Yeast powder 0.50g·L -1 、(NH4)2SO4 2.00g·L -1 、KH2PO4 4.00g·L -1 、CaCl2 0.30g·L -1 MgSO4·7H2O 0.30g·L -1 .

4. The application of the microbial agent in preparing organic fertilizer by fermenting Chinese medicinal residues according to claim 3.

5. The use according to claim 4, characterized in that: The fermentation process of the Chinese medicine residue is as follows: the Chinese medicine residue, fungus residue, rice husk and pig manure are made into a fermentation matrix, and then 5% by mass of the microbial agent described in claim 3 is inoculated, the moisture content is adjusted to 50%-60%, the fermentation matrix is ​​placed in a transparent sealing device, and fermented at a constant temperature of 35°C for 60 days, and gas is released and mixed every three days to obtain a biological organic fertilizer.

6. The use according to claim 5, characterized in that: The Chinese medicine residue is the Chinese medicine residue of Sangju Cold Granules, which comes from the Chinese medicine residue of Sangju Cold Granules produced by Huichuntang Co., Ltd. The main raw materials of Sangju Cold Granules are mulberry leaves, chrysanthemums, forsythia, mint, bitter almonds, platycodon, licorice, and reed roots.

7. Use of the bio-organic fertilizer according to claim 6 in improving the quality and yield of citrus.

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