Application of bacillus flexus in compost fermentation

Abstract

The invention provides application of bacillus flexus in compost fermentation, and belongs to the technical field of microorganisms. The bacillus flexus can grow at the temperature of 60 DEG C or above, can produce one or more of xylanase, laccase and cellulose hydrolase, obviously improves the composting temperature, prolongs the high-temperature period, accelerates the composting fermentation process, obviously improves the organic carbon content in the bio-organic fertilizer, and improves the yield of the bio-organic fertilizer. The content of heavy metals such as cadmium is reduced, harmful bacteria in the bio-organic fertilizer or compost fermentation are reduced, and the method has important significance and application value in application of compost treatment, preparation of the bio-organic fertilizer and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to application of Bacillus flexus in compost fermentation. Background Art

[0002] At present, pig manure treatment is mainly based on anaerobic and aerobic fermentation. During the anaerobic digestion (AD) process of livestock and poultry manure, most of the cellulose and hemicellulose are converted into methane. The use, sale and storage of combustible gases such as methane and hydrogen have certain safety hazards. Moreover, special equipment is required. Ordinary farmers and small-scale breeding farms do not have the ability to apply and promote them. The wastewater and sludge produced cannot be directly degraded, and there is a risk of secondary environmental pollution. Aerobic fermentation does not rely on special equipment, is relatively simple to operate, and has a high input-output ratio. Therefore, the promotion and application of aerobic fermentation technology on livestock and poultry manure has gradually attracted people's attention.

[0003] Microorganisms play an important role in the transformation of various elements in organic materials during composting, and show obvious changes in community composition. Ecosystem multifunctionality (EMF) targeting the cycle of key nutrients (i.e., carbon, nitrogen, phosphorus, and sulfur) is significantly positively correlated with temperature and significantly negatively correlated with functional gene diversity. Microbial agents include lignocellulose hemicellulose degradation biological agents, thermophilic microbial agents, certain fungal agents, and many antibiotic degradation agents, which can degrade organic matter and enrich nutrients. They also have the ability to degrade pollutants, change microbial communities, enhance the activity of certain enzymes, improve fungal communities, passivate and reduce the biological activity of heavy metals, promote the removal of antibiotic resistance genes (ARGs), and accelerate material transformation. At the same time, different carbon-nitrogen ratios, straw particle sizes, and total solid content (TS%) have significant effects on the composting fermentation process. Therefore, finding a microorganism and its microbial agent is crucial for composting fermentation, but so far there has been no report on high-temperature resistant Bacillus flexus agents. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an application of Bacillus flexus in composting fermentation.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an application of Bacillus flexus in composting fermentation.

[0007] Preferably, the Bacillus flexus is Bacillus flexus FM; the complete genome sequence of the Bacillus flexus FM is shown in SEQ ID NO.1.

[0008] Preferably, the Bacillus flexus increases the composting temperature, prolongs the high temperature period, and accelerates the composting fermentation process;

[0009] The compost comprises one or both of livestock and poultry manure compost and organic waste compost;

[0010] The organic waste includes one or more of straw, fallen leaves, mushroom residue and furfural residue.

[0011] The present invention provides an application of Bacillus flexus in the preparation of S1 and / or S2:

[0012] S1, biological organic fertilizer;

[0013] S2. Lignin and / or cellulose degrading biomass.

[0014] Preferably, when preparing the bio-organic fertilizer, the Bacillus flexus is Bacillus flexus FM; the whole genome sequence of the Bacillus flexus FM is shown in SEQ ID NO.1;

[0015] The bacillus flexus increases the organic carbon content in the biological organic fertilizer and reduces the heavy metal content.

[0016] The present invention provides a microbial agent, wherein the raw materials of the microbial agent include soybean meal and the above-mentioned Bacillus flexus.

[0017] The present invention provides a method for preparing the above-mentioned microbial agent, wherein the Bacillus flexus is fermented in a fermentation medium to obtain a Bacillus flexus liquid fermentation liquid, which is then mixed with soybean meal to obtain a microbial agent.

[0018] Preferably, the mass ratio of the Bacillus flexus liquid fermentation liquid to soybean meal is 1:(0.5-1.5);

[0019] The fermentation medium includes the following components in concentrations: 5.0-7.5 g / L maltose, 10-15 g / L peptone, and 5-10 g / L calcium chloride;

[0020] The pH value of the fermentation medium is 6.8 to 7.2;

[0021] The fermentation temperature is 30-37°C, the fermentation time is 18-24h, and the fermentation speed is 170-190r·min -1 ;

[0022] The inoculation amount of the bacillus flexus is 1.5 to 2.5%.

[0023] The present invention provides an application of the above-mentioned microbial agent or preparation method in at least one of the following a to c:

[0024] a. Composting and fermentation;

[0025] b. Preparation of biological organic fertilizer;

[0026] c. Lignin and / or cellulose degrading biological agents.

[0027] Preferably, the microbial agent increases the composting temperature, prolongs the high temperature period, and accelerates the composting fermentation process;

[0028] The microbial agent increases the organic carbon content in the biological organic fertilizer and reduces the heavy metal content.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The invention provides an application of bacillus flexus in compost fermentation. The bacillus flexus of the invention can grow at a temperature above 60°C and can produce one or more of xylanase, laccase and cellulose hydrolase. The bacillus flexus significantly increases the compost temperature, prolongs the high temperature period, accelerates the compost fermentation process, and can significantly increase the organic carbon content in bio-organic fertilizer, reduce the heavy metal content such as cadmium content, and reduce harmful bacteria in bio-organic fertilizer or compost fermentation. The invention has important significance and application value in applications such as compost treatment and preparation of bio-organic fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The bacterial development tree for the 16S rRNA of Bacillus flexus FM;

[0032] Figure 2 The effect of different carbon source liquid fermentation medium on the growth of Bacillus flexus FM;

[0033] Figure 3 The effect of different nitrogen source liquid fermentation medium on the growth of Bacillus flexus FM;

[0034] Figure 4 The effect of different inorganic salt liquid fermentation medium on the growth of Bacillus flexus FM;

[0035] Figure 5 This is the growth curve of Bacillus flexus FM fermented in the optimal liquid fermentation medium;

[0036] Figure 6 The effect of different pH values ​​of liquid fermentation medium on the biomass of fermentation reaction liquid;

[0037] Figure 7 The effect of different fermentation culture temperatures on the biomass of the fermentation reaction liquid;

[0038] Figure 8 The effect of different inoculation amounts on the biomass of the fermentation reaction liquid;

[0039] Fig. 9 The effect of different rotation speeds on the biomass of the fermentation reaction liquid;

[0040] Fig.10 The effect of Bacillus flexus FM on the reaction temperature of compost after 0, 4, 7 and 11 days of fermentation.

[0041] Fig.11 The effect of Bacillus flexus FM on the organic carbon content in compost after 0, 4, 7 and 11 days of fermentation.

[0042] Fig.12 The effect of Bacillus flexus FM on the pH value of compost after 0, 4, 7 and 11 days of fermentation.

[0043] Fig.13 Figure a shows the effect of different treatment groups on the cadmium content in the pile; Figure b shows the effect of different treatment groups on the total amount of microorganisms in the pile. DETAILED DESCRIPTION

[0044] The present invention provides an application of Bacillus flexus in compost fermentation.

[0045] In the present invention, the Bacillus flexus is isolated from fresh pig manure from a farm in Feicheng, Tai'an City, Shandong Province, China, and is capable of producing xylanase, laccase and cellulose hydrolase, has the ability to degrade lignin and / or cellulose, and can grow above 60°C, having the characteristic of high temperature resistance. The Bacillus flexus is preferably Bacillus flexus FM; the whole genome sequence of the Bacillus flexus FM has been disclosed, and the whole genome nucleotide sequence of the Bacillus flexus FM is shown in SEQ ID NO.1.

[0046] The 16S rRNA gene sequence of the Bacillus flexus FM is stored in the NCBI database with accession number TUC8MV23013QUERY_1255113, and the bacterium is preliminarily identified as Bacillus flexus. The applicant promises to release Bacillus flexus FM to the public.

[0047] In the present invention, the bacillus flexus increases the composting temperature, prolongs the high temperature period, and accelerates the composting fermentation process. The compost includes one or both of livestock and poultry manure compost and organic waste compost; the livestock and poultry manure preferably includes one or more of chicken manure, duck manure, goose manure, cow manure, sheep manure, pig manure and horse manure. The organic waste preferably includes one or more of straw, fallen leaves, mushroom residue and furfural residue, and is further preferably mushroom residue. More preferably, the compost is preferably pig manure and mushroom residue, and the mass ratio of pig manure to mushroom residue is 1:1.

[0048] The present invention provides an application of Bacillus flexus in the preparation of S1 and / or S2:

[0049] S1, biological organic fertilizer;

[0050] S2, lignin and / or cellulose degrading biological agents.

[0051] When preparing biological organic fertilizer, the bacillus flexus is bacillus flexus FM; the whole genome sequence of the bacillus flexus FM is shown in SEQ ID NO.1; the bacillus flexus increases the organic carbon content in the biological organic fertilizer and reduces the heavy metal content. The heavy metal includes cadmium content.

[0052] The present invention provides a microbial agent, wherein the raw materials of the microbial agent include soybean meal and the above-mentioned Bacillus flexus. The present invention has no special limitation on the source of soybean meal, and any product known in the art or commercially available can be used.

[0053] The present invention provides a method for preparing the microbial agent. The Bacillus flexus is fermented in a fermentation medium to obtain a Bacillus flexus liquid fermentation liquid, which is then mixed with soybean meal to obtain the microbial agent.

[0054] In the above preparation method, the Bacillus flexus is fermented in a fermentation medium, and the fermentation medium includes the following concentrations of components: 5.0-7.5 g / L maltose, 10-15 g / L peptone and 5-10 g / L calcium chloride, more preferably 5.0 g / L maltose, 15 g / L peptone and 10 g / L calcium chloride, or 7.5 g / L maltose, 15 g / L peptone and 10 g / L calcium chloride. The pH value of the fermentation medium is preferably 6.8-7.2, more preferably 6.9-7.1, and more preferably 7.0. The present invention uses the above fermentation medium to effectively promote the growth of Bacillus flexus. The fermentation temperature is preferably 30-37°C, more preferably 37°C, the fermentation time is preferably 18-24h, more preferably 18, 19, 20, 21, 22, 23 or 24h, and the fermentation speed is preferably 170-190 r·min -1 , more preferably 175 to 185 r·min -1 , more preferably 180r·min -1 The inoculation amount of the Bacillus flexus is preferably 1.5 to 2.5%, and more preferably 2%. The inoculation amount of the present invention refers to the volume percentage of the Bacillus flexus seed liquid in the fermentation medium. Taking the inoculation amount of 2% as an example, 2 mL of Bacillus flexus FM seed liquid is added to 100 mL of the fermentation medium. The above fermentation conditions of the present invention increase the biomass of Bacillus flexus.

[0055] After obtaining the liquid fermentation liquid of Bacillus flexus, it is mixed with soybean meal to obtain a microbial agent. The mass ratio of the liquid fermentation liquid of Bacillus flexus to soybean meal is preferably 1: (0.5-1.5), and more preferably 1: 1. The present invention has no special limitation on the mixing method, such as stirring, etc., as long as the mixing is uniform.

[0056] The present invention provides an application of the above-mentioned microbial agent or preparation method in at least one of the following a to c:

[0057] a. Composting and fermentation;

[0058] b. Preparation of biological organic fertilizer;

[0059] c. Lignin and / or cellulose degrading biological agents.

[0060] In the present invention, the microbial agent increases the composting temperature, prolongs the high temperature period, and accelerates the composting fermentation process; the microbial agent increases the organic carbon content in the biological organic fertilizer and reduces the heavy metal content, such as cadmium content.

[0061] In the present invention, unless otherwise specified, all raw material components are commercially available products known to those skilled in the art.

[0062] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0063] In the following examples, data statistics were analyzed using IBM SPSS 25 statistical software, and one-way analysis of variance was used to compare the mean values ​​of different data combinations (P<0.05), and Origin2024b software was used for relevant graphics drawing. Shapiro-Wilktest was used to analyze the homogeneity of variance and normality of real-time data, and descriptive (D) tests were used to compare the mean values ​​between different treatments. All data in this example are (mean ± standard error).

[0064] Example 1

[0065] 1 Materials and methods

[0066] 1.1 Isolation of strains

[0067] The sample used for isolating the strain in this example was collected from fresh pig manure from a breeding farm in Feicheng City, Tai'an City, Shandong Province, China. 5 g of fresh pig manure was taken under sterile conditions and dissolved in 45 mL of sterile distilled water. The mixture was shaken at 220 r / min for 30 min. 1 mL of the fresh pig manure suspension was taken and mixed with 9 mL of sterile water to obtain 10 -1 Dilution, take 1mL·10 -1Mix the diluent with 9 mL of sterile water to obtain 10 -2 Dilution, similarly, prepare 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 Use a pipette to draw 100 μL of the dilution solution in the center of each separation medium plate, spread it evenly with a spreader, and culture at 37°C for 1 to 3 days. LB solid medium was used for bacterial isolation, and the selected dilution was 10 -4 , 10 -5 and 10 -6 ; Martin's medium was used for fungal isolation, and the dilution selected was 10 -3 , 10 -4 and 10 -5 ; Gao's medium No. 1 was used for actinomycete isolation, and the dilution was 10 -4 , 10 -5 and 10 -6 .

[0068] 1.2 Screening of strains

[0069] The screening conditions are whether the strains can produce xylanase, laccase or cellulose hydrolase and can tolerate high temperature (60°C). The strains that can produce two or more enzymes are selected as efficient fermentation strains. The method for determining the activity of cellulose hydrolase is in accordance with the Chinese agricultural industry standard "NY / T 1847-2010 General Technical Requirements for Quality Evaluation of Strains for Microbial Fertilizer Production; Laccase activity determination was based on whether a reddish-brown transparent ring could be formed on the guaiacol primary screening plate. Positive strains were inoculated into laccase-producing liquid fermentation medium. The reference method was as follows: Tian Linshuang. Study on standard methods for determination of enzymes related to lignin degradation [J]. Animal Husbandry and Feed Science, 2009, 30(10): 13-15. Xylanase activity was determined by inoculating strains that could form a transparent ring on the xylan primary screening plate into xylanase-producing liquid fermentation medium for screening. The DNS method was used to quantitatively determine the xylanase activity of the functional strains. For xylanase activity determination, see Liu Yali. Study on the conditions for xylanase production by biocontrol Trichoderma strains and the induction of rice disease resistance [D]. Zhejiang University, 2006. After the target strain plates were streaked, they were placed in a constant temperature (60°C) incubator for 18 hours to observe the growth of the strains.

[0070] The bacterial community was isolated and screened from fresh pig manure, which has the ability to decompose xylanase, laccase or cellulose hydrolase and can withstand high temperature (above 60°C). The FM strain can be screened to produce more than two enzymes at the same time.

[0071] Experiments have shown that the FM strain can grow above 60°C and can decompose xylanase, laccase and cellulose hydrolase.

[0072] 1.3 Identification of strain 16S

[0073] The bacterial genomic DNA extraction kit of Aikerui was used to extract the genomic DNA of the strain. The extracted DNA was used as a template, and the 16S rRNA gene of the strain was amplified by PCR using the universal primers 27F / 1492R of bacteria (the amplification of the 16S rDNA gene sequence used the universal primers of bacteria, the forward primer 27F was 5'-AGAGTTTGATCATGGCTCAG-3' (SEQ ID NO.2); the reverse primer 1492R was 5'-GGTACCTTGTTACGACTT-3' (SEQ ID NO.3)). The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared in NCBI blast, and the sequence similarity was analyzed by software MEGA-X, and the phylogenetic tree was constructed.

[0074] Figure 1 The results showed that the 16S rRNA gene sequence of the FM strain was deposited in the NCBI database with the accession number TUC8MV23013 QUERY_1255113, and the bacterium was preliminarily identified as Bacillus flexus, that is, Bacillus flexus FM.

[0075] 1.4 Optimization of strain growth conditions

[0076] 1.4.1 Screening of the best C, N and inorganic salts in fermentation medium

[0077] The standard formula of LB liquid medium (LB liquid medium is 5.0g·L -1 Yeast extract, 10 g L -1 Tryptone and 10.0 g·L -1 Sodium chloride) as a control (T0), the yeast extract (carbon source) in the LB liquid medium was replaced with an equal amount (5.0 g·L -1 ) of glucose (T1), sucrose (T2), mannitol (T3) and maltose (T4), and other substances remained unchanged. Liquid fermentation medium with different carbon sources was prepared. 100 mL of liquid fermentation medium was placed in each 150 mL Erlenmeyer flask. Bacillus flexus FM strain was inoculated into the fermentation medium with different carbon sources according to the inoculation amount of 2% seed liquid (i.e., 2 mL). The culture medium was incubated at 37°C and 180 r·min -1 Oscillate on a shaker for 18 h and measure the OD of the fermentation reaction liquid 600 Values ​​(culture solution with each carbon source was repeated 3 times).

[0078] like Figure 2 As shown, in the carbon source screening of Bacillus flexus FM liquid fermentation medium, compared with the liquid fermentation medium with yeast extract as the carbon source, the absorbance of Bacillus flexus FM cultured in the liquid fermentation medium with maltose as the carbon source increased by 19.90%, therefore, the best carbon source is maltose.

[0079] Using the same weight (10g·L -1 ) beef extract (T1), urea (T2), peptone (T3) and yeast extract powder (T4) were used to replace tryptone (nitrogen source) in LB medium, respectively. The fermentation reaction conditions were the same as those for the above carbon source screening, and the best nitrogen source was screened out.

[0080] like Figure 3 As shown, in the nitrogen source screening of Bacillus flexus FM liquid fermentation medium, compared with the liquid fermentation medium with peptone as the nitrogen source, the absorbance of Bacillus flexus FM cultured in the liquid fermentation medium with peptone as the nitrogen source increased by 3.92%, so the best nitrogen source is peptone.

[0081] Using the same weight (10.0g·L -1 ) potassium nitrate (T1), calcium chloride (T2), potassium chloride (T3), magnesium sulfate (T4) and ammonium nitrate (T5) replaced sodium chloride in LB medium, and the fermentation reaction conditions were the same as the above carbon source screening to screen out the best inorganic salt.

[0082] like Figure 4 As shown in the figure, in the inorganic salt screening of Bacillus flexus FM liquid fermentation medium, compared with the liquid fermentation medium with sodium chloride as the nitrogen source, the absorbance of Bacillus flexus FM cultured in the liquid fermentation medium with calcium chloride as the inorganic salt increased by 36.94%, so the best nitrogen source is calcium chloride.

[0083] In summary, the optimal liquid fermentation medium for Bacillus flexus FM is 5.0 g·L -1 Maltose, 10g·L -1 Peptone and 10.0 g·L -1 Calcium chloride.

[0084] Each 150 mL triangular bottle was filled with 100 mL of the best liquid fermentation medium. The Bacillus flexus FM strain was inoculated into the best liquid fermentation medium at a 2% seed liquid inoculation rate and incubated at 37°C and 180 r·min -1 The culture was shaken on a shaker for 16 h, and the OD of each fermentation reaction liquid was measured at 0, 2, 4, 6, 8, 10, 12, 14 and 16 h. 600 Value (the fermentation reaction liquid in each time period was measured 3 times).

[0085] like Figure 5 As shown, the logarithmic growth period of Bacillus flexus FM is 2 to 8 hours, the strain proliferates quickly, is easy to culture, and is suitable for large-scale culture and expansion.

[0086] 1.4.2 Screening and optimization of culture conditions

[0087] Bacillus flexus FM was fermented in the optimal liquid fermentation medium optimized in 1.4.1. The pH value, inoculation amount, culture temperature, rotation speed and culture time in the liquid fermentation medium were screened and optimized in turn, and each treatment was repeated 3 times.

[0088] The pH value of the liquid fermentation medium was set to 5.0, 6.0, 7.0, 8.0 and 9.0. 100 mL of liquid fermentation medium was placed in a 150 mL Erlenmeyer flask. The seed solution was inoculated at 1% and the fermentation temperature was 180 r·min. -1 , 30℃, shake culture for 18h, according to the fermentation reaction liquid OD 600 value, determine the optimal pH value.

[0089] Figure 6 The results showed that when the pH value of the liquid fermentation medium was 7.0, the biomass of the fermentation reaction liquid was the largest.

[0090] After determining the optimal pH value of the fermentation reaction, the fermentation reaction temperature was set to: 37℃, 39℃ or 42℃, 100mL of pH7.0 liquid fermentation medium was placed in a 150mL Erlenmeyer flask, the seed liquid was inoculated at 1%, and the fermentation temperature was set to 180r·min. -1 The culture was shaken for 18 h, and the OD value of the fermentation reaction liquid was calculated. 600 value, screen the optimal reaction temperature.

[0091] Figure 7 The results showed that when Bacillus flexus FM was fermented in liquid fermentation medium, the biomass of the fermentation reaction liquid was the largest at 37°C.

[0092] After determining the optimal pH value and reaction temperature, under the optimal pH value and temperature conditions, the inoculation amount was set to the following gradient: 1%, 2%, 3%, 5%, 100mL of pH7.0 liquid fermentation medium was placed in a triangular flask, 37℃, 180r·min -1 , cultured for 18 h, and the OD of the fermentation liquid was 600 value, and screen the optimal inoculum size.

[0093] Figure 8 The results showed that the biomass of the fermentation broth of FM strain with 2% inoculation was the largest. Considering the actual situation in industrial production, 2% inoculation was selected as the optimal inoculation amount.

[0094] After determining the optimal pH value, temperature and inoculation amount, the speed is set to: 180r·min -1 、200r·min -1 、220r·min -1 , 100 mL of pH 7.0 liquid fermentation medium was placed in a 150 mL Erlenmeyer flask, the seed solution was inoculated at 2% and cultured at 37°C for 18 h. The OD values ​​of the fermentation solution were calculated according to the different treatments. 600 value, filter the best speed.

[0095] Fig. 9 The results show that with a speed of 200 r·min -1 or 220r·min -1 Comparison, 180r·min -1 The biomass of the fermentation liquid of FM strain was the largest at 180r·min -1 The optimal culture speed.

[0096] 1.4.3 Optimization of liquid fermentation medium component concentrations

[0097] Three concentration gradients were set for the orthogonal test of carbon source, nitrogen source and inorganic salt in the liquid fermentation medium. The concentration of each gradient is shown in Table 1. The absorbance of each combination was measured to determine the optimal combination.

[0098] Table 1 Orthogonal experimental factors and levels of culture medium components

[0099]

[0100] The results in Table 2 show that the absorbance value of the liquid fermentation medium of the combination A1B3C3 is the largest, i.e., 5.0 g / L maltose, 15.0 g / L peptone and 10.0 g / L calcium chloride; and the K value shows that the combination A2B3C3 may be more effective. Considering the cost and the error factors of the detection system, in this embodiment, the combination A1B3C3 with the largest biomass is selected as the optimal combination, and A2B3C3, i.e., 7.5 g / L maltose, 15.0 g / L peptone and 10.0 g / L calcium chloride, can be used as an alternative combination.

[0101] Table 2 Optimization results of concentration of each component in liquid fermentation medium

[0102]

[0103]

[0104] Among them, Ki value: the sum of the experimental project results corresponding to level i under each factor; R is the difference between Kmax and Kmin under each factor.

[0105] Example 2

[0106] Application of Bacillus flexus FM in pig manure composting

[0107] Preparation of Bacillus flexus FM inoculant:

[0108] (1) Preparation of liquid fermentation medium: 5.0 g maltose, 15.0 g peptone, 10.0 g calcium chloride and 1 L water were mixed, the pH was adjusted to 7.0, and sterilized at 121° C. for 30 min to obtain a liquid fermentation medium;

[0109] (2) Each 150 mL Erlenmeyer flask was filled with 100 mL of liquid fermentation medium. The Bacillus flexus FM strain of Example 1 was inoculated into the liquid fermentation medium at a seed liquid inoculation rate of 2% and the fermentation temperature was 37°C and 180 r·min. -1 The culture was shaken on a shaker for 18 hours to obtain the Bacillus flexus FM liquid fermentation liquid.

[0110] (3) The liquid fermentation liquid of Bacillus flexus FM and the soybean meal sterilized at 121° C. were mixed in a mass ratio of 1:1 to obtain the Bacillus flexus FM inoculant.

[0111] In this embodiment, three treatments are set, including A (pig manure + bacterial agent), B (pig manure + mushroom residue), and C (pig manure + mushroom residue + bacterial agent). Among them, A is to add 1kg of Bacillus flexus FM bacterial agent to 1 ton of pig manure for composting fermentation, B is to add 1000kg of mushroom residue to 1 ton of pig manure for composting fermentation (denoted as CK, control group), and C is to add 1000kg of mushroom residue and 1kg of Bacillus flexus FM bacterial agent to 1 ton of pig manure for composting fermentation (denoted as FM). The composts after 0 days, 4 days, 7 days and 11 days of composting fermentation were taken respectively, and the changes in nitrogen, phosphorus, potassium, moisture, organic carbon, heavy metals (cadmium) and total biomass abundance of each compost material were detected.

[0112] Fig.10 The results showed that during the composting process, the addition of Bacillus flexus FM greatly increased the reaction temperature of organic materials and prolonged the high temperature period. Therefore, Bacillus flexus FM accelerated the composting process.

[0113] Fig.11 The results showed that the addition of Bacillus flexus FM significantly increased the organic carbon content during composting.

[0114] Fig.12 The results showed that the pH value changed steadily during the composting fermentation process when Bacillus flexus FM was added.

[0115] Fig.13The results showed that the addition of Bacillus flexus FM had a significant effect on the cadmium (Cd) content in each treatment. After 11 days of composting, the cadmium content in treatment C was lower than that in treatment B (see Fig.13 a) in the figure.

[0116] Fig.13 The results of b in the figure show that the changes in the total amount of microorganisms in each treatment are significantly different. Only the addition of mushroom residues reduces the abundance of microorganisms in the compost material. However, after the addition of Bacillus flexus FM inoculant, the total amount of microorganisms increases. After compost fermentation, Fig.10 It can be seen that after 11 days of fermentation, the C treatment was still in the high temperature period, but the biomass of the C treatment samples increased compared with the B treatment samples. Therefore, Bacillus flexus FM can still grow at about 60°C and promote compost fermentation.

[0117] The Bacillus flexus FM of the present invention is a strain that can tolerate high temperatures and has the ability to decompose lignin and cellulose. It can grow and reproduce at high temperatures above 60°C, and has good application value for composting organic wastes such as straw, fallen leaves, mushroom residues, furfural residues, etc. generated in industrial and agricultural production, especially livestock and poultry manure. The addition of the Bacillus flexus FM inoculant increases the temperature and time of the high temperature period of organic materials, promotes the conversion of related substances and energy, accelerates the fermentation and decomposition process, and degrades heavy metal ions. The solid fermentation materials used for industrial cultivation are cheap and easy to obtain, which is conducive to large-scale promotion and application.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Bacillus flexus in composting fermentation.

2. The use according to claim 1, characterized in that: The Bacillus flexus is Bacillus flexus FM; the complete genome sequence of the Bacillus flexus FM is shown in SEQ ID NO.

1.

3. The use according to claim 1 or 2, characterized in that: The bacillus flexus increases the composting temperature, prolongs the high temperature period, and accelerates the composting fermentation process; The compost comprises one or both of livestock and poultry manure compost and organic waste compost; The organic waste includes one or more of straw, fallen leaves, mushroom residue and furfural residue.

4. Application of Bacillus flexus in the preparation of S1 and / or S2: S1, biological organic fertilizer; S2. Lignin and / or cellulose degrading biomass.

5. The use according to claim 4, characterized in that: The Bacillus flexus is Bacillus flexus FM; the complete genome sequence of the Bacillus flexus FM is shown in SEQ ID NO.1; The bacillus flexus increases the organic carbon content in the biological organic fertilizer and reduces the heavy metal content.

6. A microbial agent, characterized in that: The raw materials of the microbial agent include soybean meal and the Bacillus flexus of claim 2.

7. A method for preparing the microbial agent according to claim 6, characterized in that: The bacillus flexus is fermented in a fermentation medium to obtain a bacillus flexus liquid fermentation liquid, which is then mixed with soybean meal to obtain a microbial agent.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the Bacillus flexus liquid fermentation liquid to soybean meal is 1:(0.5N1.5); The fermentation medium includes the following components in concentrations: 5.0-7.5 g / L maltose, 10-15 g / L peptone, and 5-10 g / L calcium chloride; The pH value of the fermentation medium is 6.8 to 7.2; The fermentation temperature is 30-37°C, the fermentation time is 18-24h, and the fermentation speed is 170-190r·min -1 ; The inoculation amount of the bacillus flexus is 1.5-2.5%.

9. Use of the microbial agent according to claim 6 or the preparation method according to claim 7 or 8 in at least one of the following a to c: a. Composting and fermentation; b. Preparation of biological organic fertilizer; c. Lignin and / or cellulose degrading biological agents.

10. The use according to claim 9, characterized in that: The microbial agent increases the composting temperature, prolongs the high temperature period, and accelerates the composting fermentation process; The microbial agent increases the organic carbon content in the biological organic fertilizer and reduces the heavy metal content.

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