Lysinibacillus loembei SCAUH16 and application thereof

Abstract

The invention discloses a strain of Lysinibacillus loembei SCAUH16 and application thereof, and belongs to the field of microorganisms, the strain is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO: 65794. According to the bacterial strain, residues obtained after treatment of pigs died of illness serve as a separation source, the bacterial strain with the activity of producing protease and lipase under the high-temperature condition is separated and screened out, the bacterial strain can rapidly degrade the residues obtained after treatment of the pigs died of illness, and the degradation rate reaches 61.37%. Furthermore, the residues obtained after the pigs died of illness are converted are used as main raw materials for aerobic composting fermentation, and after the strain is added, the temperature of a compost body is increased, the material conversion is accelerated, the composting time is shortened, the decomposition of the compost is promoted, the excessive loss of nutrients in the composting process is reduced, the nutrients in the compost material are kept, and the quality of the compost is improved; the method has a good application prospect in the aspect of resource utilization of agricultural wastes.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a strain of Lysinibacillus louembei SCAUH16 and an application thereof. Background Art

[0002] With the development of my country's economy and the improvement of residents' living standards, the consumption of livestock products such as pork continues to rise, and the demand for pig farming continues to increase. However, a large number of dead pigs will be produced during the breeding process due to stillbirth, disease, natural disasters and other reasons. According to statistics, my country's pig output in 2022 will be 699.95 million, and the pig inventory will be 452.56 million. The annual mortality rate of pigs caused by various diseases is about 8% to 12%. The bodies of dead pigs pose serious hazards to economic production, ecological environment, food hygiene and human health. Therefore, it is particularly important to harmlessly treat the bodies of dead pigs. The chemical treatment of dead pigs has the advantages of simple operation, good sterilization effect, short treatment cycle, and no smoke. It is one of the main ways to harmlessly treat dead pigs at present, and it is an important way to achieve harmless treatment and resource utilization of dead livestock and poultry. However, the dead pig bodies will produce by-products mainly composed of grease and solid materials after chemical treatment. At present, it is mainly solved by incineration, which will cause serious secondary pollution problems.

[0003] The residues of dead pigs, which are mainly oil and solid materials, contain a large amount of organic matter, nitrogen, phosphorus, calcium, etc., and have the potential to be used as organic fertilizer. However, if the residues of dead pigs are directly used for returning to the field, there is a long decomposition time and the risk of secondary pollution. Therefore, the residues of dead pigs are used as an organic solid waste for high-temperature aerobic composting to make organic fertilizer, so as to achieve the recycling of resources. Since the residues of dead pigs contain a large amount of protein, fat, etc., their composition is complex and it is difficult to achieve rapid decomposition. A large number of studies have shown that inoculating high-temperature and high-efficiency functional microbial agents during the composting process can not only accelerate the decomposition of substances, promote rapid temperature rise of the pile, effectively kill pathogenic microorganisms in the pile, but also control the generation of odor during the composting process, promote the transformation of substances, and shorten the composting process. At present, there have been many reports on the research of adding functional microbial agents in high-temperature aerobic composting, which mainly focus on the development and application of cellulase-producing agents under high temperature conditions, while the screening and application of high-temperature strains with protease and lipase degradation capabilities are still relatively small. Therefore, in order to achieve the harmless disposal and resource utilization of the residues after chemical processing of dead pigs, high-temperature bacterial strains with high efficiency in degrading protein and fat were screened and applied to the high-temperature aerobic composting of the residues after chemical processing of dead pigs to prepare organic fertilizer. This not only improved the quality of the compost, but most importantly, promoted the resource utilization of the residues after chemical processing of dead pigs and reduced the risk of secondary pollution, which is of great significance for achieving sustainable development. Summary of the invention

[0004] The technical problem to be solved by the present invention is: in order to separate and screen high-temperature strains capable of decomposing proteins and fats, and apply them to the decomposition of residues after dead pigs and their high-temperature aerobic composting treatment, one of the purposes of the present invention is to provide a Bacillus strain capable of decomposing proteins and fats, and another purpose is to provide the application of the strain.

[0005] The technical solution of the present invention is: a Lysinibacillus strain Lysinibacillus louembei SCAUH16, referred to as SCAUH16, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 15, 2025, with a deposit number of GDMCC NO: 65794, and the address of the deposit unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0006] A microbial inoculant containing the strain Lysinibacillus louembei SCAUH16.

[0007] The application of the strain Lysinibacillus louembei SCAUH16 or microbial agent described above in the treatment of residues after dead pigs are processed.

[0008] Furthermore, the treatment is high temperature aerobic composting.

[0009] Furthermore, the high-temperature aerobic composting refers to mixing the residues after the dead pigs are processed with sawdust, inoculating the above-mentioned strain Lysinibacillus louembei SCAUH16 or microbial agent, and performing high-temperature composting fermentation.

[0010] Furthermore, the high-temperature aerobic composting method is: sawdust is used as an auxiliary material and mixed with the residue after the dead pig is processed, the moisture of the material is adjusted to 50-60%, the initial carbon-nitrogen ratio is controlled at 25-30, and the above-mentioned microbial agent is added to the material at a weight ratio of 0.5% for high-temperature aerobic composting for 30 days. When the stacking temperature reaches above 50°C, the first turning of the pile is carried out; when the temperature reaches 60°C, the second turning of the pile is carried out.

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0012] The present invention uses the dead pigs' residue as a separation source, and the Lysinibacilluslouembei SCAUH16 provided by separation and screening can grow under high temperature conditions, has the ability to decompose protein and fat, has the potential for application in high temperature aerobic composting, and belongs to the genus Lysinibacillus. The strain is suitable for growth in a temperature range of 35-70°C, has high protease activity and lipase activity, and the enzyme activities are 56.9 μmol / mL and 28.6 μmol / mL respectively. Inoculation of the strain can significantly reduce the volume of the dead pigs' residue, reduce the protein content by 25.56%, and achieve a degradation rate of 61.37%. The microbial agent prepared by the strain is applied to high-temperature aerobic composting fermentation with dead pig residues as the main material, thereby achieving the effect of promoting the degradation and composting maturity of dead pig residues. The present invention not only shortens the fermentation period of composting of such materials, but also improves the overall quality of the compost, provides strain resources and technical references for the resource utilization of dead pig residues, is beneficial to the harmless treatment and resource utilization of agricultural waste, and has good application prospects.

[0013] The preservation information is as follows:

[0014] The Lysinibacillus strain Lysinibacillus louembei SCAUH16, referred to as SCAUH16, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 15, 2025, with the deposit number GDMCC NO: 65794, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The colony morphology of Lysinibacillus louembei SCAUH16 in tryptic soy agar medium (TSA medium) is shown in the figure.

[0016] Figure 2 Phylogenetic tree of Lysinibacillus louembei SCAUH16 constructed based on 16S rRNA sequence results;

[0017] Figure 3 This is the preliminary screening result of protease produced by Lysinibacillus louembei SCAUH16;

[0018] Figure 4 This is the preliminary screening result of lipase production by Lysinibacillus louembei SCAUH16;

[0019] Figure 5 The pH changes of the culture medium of the residual material after the treatment of dead pigs with Lysinibacillus louembei SCAUH16 and without bacteria (CK) (1-9 days);

[0020] Figure 6 The degradation effect diagram of dead pig residues by Lysinibacillus louembei SCAUH16; a is the blank control without inoculation of strains, b is the experimental group inoculated with Lysinibacillus louembei SCAUH16, and from left to right are the degradation effect diagrams of 1-9 days;

[0021] Figure 7 The degradation rate of the residues after the treatment of dead pigs with Lysinibacillus louembei SCAUH16 and without bacteria (CK) (1-9 days);

[0022] Figure 8 To compare the changes in protein content of the residues after digestion of dead pigs in the treatment group with Lysinibacillus louembei SCAUH16 and the treatment group without bacteria (CK) (1-9 days);

[0023] Fig. 9 The temperature change of the compost pile;

[0024] Fig.10 for compost C / N changes;

[0025] Fig.11 It is the change of germination index (GI) of compost seeds;

[0026] Fig.12 For compost pH changes;

[0027] Fig.13 The change of moisture content of compost;

[0028] Fig.14 for the change in electrical conductivity of the compost;

[0029] Fig.15 For the changes of total phosphorus in compost;

[0030] Fig.16 For the changes of total potassium in compost;

[0031] Fig.17 Venn diagram analysis of bacterial flora in samples of dead pig residue compost at different fermentation stages;

[0032] Fig.18 This is the bacterial community composition of samples from different fermentation stages of composting residues from dead pigs. DETAILED DESCRIPTION

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0034] In this application:

[0035] TSA medium: 15 g tryptone, 5 g soy peptone, 5 g NaCl, 20 g agar, 1000 mL distilled water.

[0036] TSB medium: tryptone 17 g, soy peptone 3 g, NaCl 5 g, K2HPO4 2.5 g, glucose 2.5 g, agar 20 g, distilled water 1000 mL.

[0037] Protease seed medium (g / L): glucose 10 g, yeast powder 20 g, NaCl 5 g, K2HPO4 1 g, MgSO4 0.4 g, distilled water 1000 mL, pH 7.0;

[0038] Protease basic fermentation medium (g / L): glucose 5g, yeast powder 10g, KH2PO4 0.5g, MgSO4.7H2O0.3g, (NH4)2SO4 1g, CaCL2 1g, NaCl 1g, distilled water 1000mL, pH 7.2;

[0039] Dead pig residue culture medium (g / L): 5g glucose, 10g solid residue after harmless treatment of dead pigs, 0.5g KH2PO4, 0.3g MgSO4.7H2O, 1g (NH4)2SO4, 1g CaCl2, 1g NaCl, 1000mL distilled water, pH 7.2. The solid residue after harmless treatment of dead pigs used in the fermentation medium is the dead pig residue after chemical harmless treatment.

[0040] Skim milk solid culture medium: 2.5 g yeast extract, 2 g peptone, 1.5 g acid hydrolyzed casein, 1.5 g casein, 40 g skim milk, 20 g agar, 1 g glucose, 1000 mL distilled water.

[0041] Protease seed culture medium: glucose 10 g, yeast powder 20 g, NaCl 5 g, K2HPO4 1 g, MgSO4 0.4 g, pH 7.0, distilled water 1000 mL.

[0042] Victoria Blue B solid medium: peptone 10g, beef extract 3g, sodium chloride 5g, agar 20g, olive oil 25mL, distilled water 1000mL, Victoria Blue (4mg / 100mL).

[0043] Example 1 Isolation and Identification of Lysinibacillus louembei SCAUH16

[0044] 1.1 Isolation of Lysinibacillus louembei SCAUH16

[0045] Weigh 10g of fresh dead pig residue after processing and put it into a 250mL sterile conical flask with 90mL of sterile water. Put it into a shaker, set the temperature at 50℃ and the speed at 180r / min, incubate for 30min, mix well and let it stand. Take the supernatant for gradient dilution. The gradient dilution multiple is 10. -1 to 10 -5 , from 10 -3 , 10 -4 , 10 -5 Take 100 μL of each of the liquids, spread evenly on the casein agar medium, and place the plate in a constant temperature incubator at 50°C for 48 hours. Pick the strains with rapid growth and large colonies, and purify them by the dilution plate streak method until the purified strains are obtained. Transfer the purified strains to tryptic soy bean liquid medium (TSB), shake culture at 50°C for 7 days, and then preserve them with 30% glycerol and freeze them at -80°C.

[0046] 1.2 Identification of Lysinibacillus louembei SCAUH16

[0047] 1.2.1 Strain morphology

[0048] A single colony of strain SCAUH16 was picked and inoculated on tryptic soy agar medium (TSA medium), cultured at 50°C for 24 hours, and the colony morphology of the strain was observed.

[0049] 1.2.2 DNA extraction and PCR amplification

[0050] (1) Reagents: Sterile double distilled water, Mix (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0051] (2) Primers

[0052] 27F:5'-AGAGTTTGATCCTGGCTCAG-3

[0053] 1492R:5'-TACGACTTAACCCCAATCGC-3'.

[0054] (3) Bacterial DNA extraction

[0055] Use a disposable inoculation loop to pick a small amount of bacteria into a sterile 1.5mL centrifuge tube, then add 500μL of sterile double distilled water, quickly freeze the centrifuge tube with liquid, then place it in a 99℃ metal bath for 5min, take it out and vortex for 30s, repeat the above operation 1 to 2 times, then centrifuge the obtained mixture of bacteria and sterile double distilled water at 12000rpm for 3min, take the supernatant as a template, and store it at -20℃; detect it by 1% agarose gel electrophoresis.

[0056] (4) Bacterial 16S rRNA gene amplification

[0057] 16S rRNA gene amplification conditions: 95℃ pre-denaturation for 3min, 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 1.5min, 30 cycles, 72℃ total extension for 20min. PCR products were purified by Shanghai Biotech EZ Spin Column PCR Product Purification Kit UNlQ-1 column PCR product purification kit (SK1142-N) according to the operating instructions, and the purified products were sent to Biotech Biotechnology Co., Ltd. for sequencing.

[0058] 1.2.3 16S rRNA gene sequence analysis and phylogenetic tree construction

[0059] The sequence obtained after sequencing (SEQ ID No.1) was searched for similarity in NCBI using BLAST software, and the 16S rRNA gene sequence of the standard strain of the same genus with the highest similarity that had been published was selected as the reference sequence. The standard strain sequence of Bacillus subtilis (AB042061) was used as the outgroup. Multiple sequence alignment analysis was performed using Clustal X software, and a phylogenetic tree was constructed using MEGA7.0 software using the NJ method to determine the taxonomic status of the strain.

[0060] 1.3 Experimental Results

[0061] Lysinibacillus louembei SCAUH16 was isolated from the residue of dead pigs. After culturing on TSA medium at 50℃ for 1 day, the colony size was 4-5mm, round and convex, with irregular edges and smooth surface. The colony color was beige and easy to pick. It was Gram-positive, aerobic, rod-shaped, with spores, and the size of the bacteria was 0.6×5.0μm ( Figure 1 ).

[0062] The 16S rRNA sequence of strain SCAUH16 was determined, and BLAST homology comparison was performed in the NCBI database. The phylogenetic analysis was performed using MEGA7.0 software. The homology between the 16S rRNA nucleotide sequence of the strain and that of Lysinibacillus louembei was 99.93%. Based on the morphological characteristics, physiological and biochemical characteristics, and 16S rRNA sequence homology analysis, strain SCAUH16 was identified as Lysinibacillus louembei ( Figure 2 ).

[0063] Example 2 Growth temperature and enzyme production capacity determination of Lysinibacillus louembei SCAUH16

[0064] 2.1 Determination of temperature range

[0065] The strain Lysinibacillus louembei SCAUH16 was inoculated on TSA medium and cultured in incubators at 30℃, 40℃, 50℃, 60℃, 70℃ and 80℃ respectively for 48h. The growth was observed and recorded. According to the measurement results, the temperature detection range was further narrowed with 5℃ as the unit until the highest and lowest growth temperatures of the strain were determined.

[0066] 2.2 Protein degradation ability determination

[0067] 2.2.1 Preliminary screening of protein degradation ability

[0068] The strain was evenly spread on tryptic soy peptone agar medium (TSA medium), placed in a 50°C biochemical incubator and cultured until the strain grew evenly on the entire surface of the medium. A bacterial cake was made with a 5mm diameter puncher and inverted on a skim milk medium. After culturing in a 50°C biochemical incubator for 2 to 4 days, the hydrolysis zone diameter (D) and colony diameter (d) were measured respectively, and the ratio of the two (D / d) was calculated. The larger the ratio, the stronger the ability of the strain to degrade protein. Three replicates were set.

[0069] 2.2.2 Determination of protease activity of strains

[0070] (1) Drawing of standard curve

[0071] After preparing 100 μg / mL of L-tyrosine standard liquid, dilute the 100 μg / mL L-tyrosine standard liquid to finally obtain L-tyrosine solutions with concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. Take 1 mL of the diluted solution respectively, add 5 mL of 0.4 mol / L sodium carbonate solution and 1 mL of 1 mol / L Folin-phenol reagent solution respectively, shake evenly, and color develop in a 40±0.2°C water bath for 20 min, then take out, and use a spectrophotometer at a wavelength of 680 nm, a 10 mm cuvette, and use tube 0 without tyrosine as blank to measure the absorbance respectively. With absorbance A as the ordinate and tyrosine concentration c as the abscissa, a standard curve is drawn. Using the regression equation, the amount of tyrosine (μg) when the absorbance is 1 is calculated, which is the absorption constant K value. The K value should be in the range of 95 to 100. A standard curve of L-tyrosine is drawn based on each concentration-absorbance data.

[0072] (2) Preparation of crude enzyme solution.

[0073] Pick out 1 loop of pure culture stored on the slant and inoculate it into the protease seed medium, culture it at 50℃ on a shaker at 150 rpm for 12 h, inoculate it into the protease basal fermentation medium at an inoculum size of 1% (v / v), culture it at 50℃ on a shaker at 150 rpm for 48 h, then centrifuge and take the supernatant for enzyme activity determination.

[0074] (3) Enzyme activity determination

[0075] The crude enzyme solution was assayed for enzyme activity using the Folin-phenol method. Enzyme activity unit definition: At 40°C, 1 μg of tyrosine is produced per minute by hydrolyzing casein substrate, which is defined as one unit of protease activity. Place 2% (mass fraction) casein solution in a 40°C constant temperature water bath and preheat for 5 minutes; take 1 mL of crude enzyme solution and add 1 mL of preheated casein solution, mix well, and place in a 40°C water bath for 10 minutes, then add 2 mL of 0.4 mol / L trichloroacetic acid to terminate the reaction, take 1 mL of supernatant and add 5 mL of 0.4 mol / L Na2CO3, add 1 mL of Folin-phenol reagent to 40±0.2°C water bath for color development for 20 minutes, and then measure the absorbance at 660 nm, with the reaction system with water as the blank.

[0076] (4) Calculation of enzyme activity: Read the enzyme activity of the final dilution from the standard curve in U / mL. The enzyme activity of the original solution is calculated according to the following formula:

[0077] Protease activity (U / mL) = A × K × 4 ÷ 10 × n

[0078] Where: A: OD value of parallel experiment of fermentation stock solution;

[0079] K: Absorption constant;

[0080] n: dilution factor of protease solution;

[0081] 4: Total volume of reaction reagents;

[0082] 10: Reaction time 10 min; the results are expressed to the nearest integer.

[0083] 2.3 Determination of fat degradation ability

[0084] 2.3.1 Preliminary screening of fat degradation ability

[0085] The strain was evenly spread on tryptic soy peptone agar medium (TSA medium), placed in a 50℃ biochemical incubator and cultured until the strain grew evenly on the entire surface of the medium. A bacterial cake was made with a 5mm diameter puncher and inverted on Victoria Blue B solid medium. After culturing in a 50℃ biochemical incubator for 2 to 4 days, the blue color was observed at the inoculation site of the medium. The color change zone diameter (D) and colony diameter (d) were measured respectively, and the ratio of the two (D / d) was calculated. The larger the ratio, the stronger the ability of the strain to degrade fat. Three replicates were set.

[0086] 2.3.2 Determination of lipase activity of strains

[0087] Take several 100mL conical flasks, one as the control flask and the others as the test flasks. The specific method is shown in Table 1.

[0088] Table 1 Lipase activity determination method

[0089]

[0090] Titrate with 0.05M standard sodium hydroxide solution until it turns slightly red, and record the volume of NaOH solution used for titration.

[0091] Calculation: The specific activity of lipase is defined as the amount of enzyme that produces 1 μmol fatty acid per minute when 1 g of lipase hydrolyzes fat at pH 7.5 and 40°C.

[0092]

[0093] Wherein: A is the alkali solution consumed by the sample (ml); B is the alkali solution consumed by the control group (ml); N is the concentration of the alkali solution, i.e. 0.05 μmol; f is the final dilution multiple of the crude enzyme solution; t is the action time (min).

[0094] 2.4 Experimental Results

[0095] It was determined that the growth range of Lysinibacillus louembei SCAUH16 was 35-70°C and it had the ability to produce protease and lipase ( Figure 3 and Figure 4 , Table 2). The relative enzyme activities of protease and lipase were 56.9μmol / mL and 28.6μmol / mL, respectively. (Table 2). The results showed that Lysinibacillus louembei SCAUH16 had good thermostability and ability to produce functional enzymes, and had the potential for further development and application.

[0096] Table 2 Enzyme production capacity of Lysinibacillus louembei SCAUH16

[0097]

[0098] Example 3 Preparation of microbial inoculants

[0099] This embodiment provides a microbial agent, and the preparation method of the microbial agent is as follows:

[0100] Lysinibacillus louembei SCAUH16 was streaked onto a TSA medium, and a single colony was picked and placed into a TSB medium, and cultured at 50-60°C and 160-200rpm for 1 day to obtain a seed solution. The seed solution was inoculated into a fermentation medium at a 5% (v / v) inoculation rate, and cultured at 50-60°C and 160-200rpm for 24-48 hours to obtain a microbial agent with an effective viable count of 1.3×10 8 CFU / g, pH value is 6.0~8.0.

[0101] Fermentation medium formula: peptone 7g, beef extract 1g, sodium chloride 5g, glucose 10g, distilled water 1000mL.

[0102] Example 4 Fermentation culture of strain Lysinibacillus louembei SCAUH16

[0103] 4.1 Experimental materials and process

[0104] Preparation of solid residue after chemical treatment of dead pigs: After the dead pig carcasses are crushed, they are treated in a high temperature and high pressure chemical tank (temperature ≥ 140°C, pressure ≥ 0.5MPa (absolute pressure)) for 4 hours, and then dried in a high temperature and normal pressure drying tank, and finally the dead pig residue is obtained after the liquid is separated by an oil press. The dead pig residue used in the present invention is provided by Chengdu Kenong Animal Harmless Disposal Co., Ltd.

[0105] Experimental setup: a blank control group (CK group) without adding any microbial agents, and a microbial agent containing Lysinibacilluslouembei SCAUH16 (SCAUH16 group, added amount 0.5% (v / v)) were added. Both were cultured at 50°C and 180r / min. Samples were taken continuously for 9 days, and the physical and chemical properties of the samples were analyzed to determine the degradation effect of strain SCAUH16.

[0106] 4.2 Analysis of physical and chemical properties

[0107] 4.2.1 Degradation rate of dead pig residues

[0108] The degradation rate of solid residues after harmless treatment of dead pigs was calculated according to the weight loss method: the residues after harmless treatment of dead pigs by shake flask culture were filtered through filter paper, washed with distilled water for 2 to 3 times, dried at 65℃ for 24h, weighed, and the weight loss and degradation rate were calculated.

[0109]

[0110] Where: A is the initial dry weight of solid residue after harmless treatment of dead pigs (g);

[0111] B is the dry weight of solid residue after harmless treatment of dead pigs after degradation (g).

[0112] 4.2.2 Determination of pH value of dead pig residue culture medium

[0113] Referring to the determination of pH in the Agricultural Industry Standard of the People's Republic of China NY / T 525-2021 (pH meter method), transfer the dead pig residue culture medium from a 250mL Erlenmeyer flask to a 250mL beaker, let it stand for 30 minutes, measure it with a pH acidometer, read the pH value and record it. Before the measurement, calibrate the acidometer with a standard buffer solution (temperature compensation is set to 25°C).

[0114] 4.2.3 Protein content of dead pig residue substrate

[0115] According to the method in the Agricultural Industry Standard of the People's Republic of China NY / T 525-2021 "Determination of Total Nutrient Content in Fertilizers", weigh 0.5g~1.0g (accurate to 0.0001g) of the air-dried sample of the dead pig residue substrate, place it at the bottom of a conical flask of appropriate volume, add 5mL sulfuric acid and 1.5mL hydrogen peroxide, shake carefully, put a small funnel with a curved neck at the mouth of the bottle, and leave it overnight. The next day, slowly heat it until the sulfuric acid smokes, add 15 drops of hydrogen peroxide when it is slightly cool, gently shake the conical flask or digestion tube, heat it for 10 minutes, add 5~10 drops of hydrogen peroxide after it is slightly cool and digest it in batches until the solution is colorless or light yellow clear liquid, continue heating for 15 minutes, and remove the remaining hydrogen peroxide. Remove and cool, carefully add water to 20mL~30mL, gently shake the conical flask or digestion tube, rinse the small funnel with a small amount of water, and collect the washing liquid in the conical flask. Transfer the digestion solution into a 50mL volumetric flask, cool to room temperature, add water to make up the volume, and let it stand to clarify or dry filter it with phosphorus-free filter paper into a stoppered triangular flask for later use. In the blank experiment, the reagent dosage is the same as that of the operation and experimental samples, except that no sample is added. The protein content in the sample is analyzed and calculated using the automatic Kjeldahl nitrogen analyzer method and expressed as mass fraction (%).

[0116]

[0117] Where:

[0118] C is the molar concentration of the calibration standard solution, in moles per liter (mol / L);

[0119] V0 is the volume of the calibration standard solution consumed during the blank test, in milliliters (mL);

[0120] V is the volume of the standard solution consumed during sample determination. The unit is milliliter (mL);

[0121] 14 is the molar mass of nitrogen in grams per mole (g / moL);

[0122] M is the value of the air-dried sample mass, in grams (g);

[0123] X is the value of the moisture content of the air-dried sample;

[0124] D is the fractionation multiple, fixed volume / fractionation volume;

[0125] 6.25 is the factor for converting nitrogen to protein.

[0126] 4.3 Experimental Results

[0127] 4.3.1 pH changes of dead pig residues

[0128] The pH values ​​of the samples in the two treatment groups were measured. It was found that the pH values ​​of the samples in the CK group did not change significantly, and the pH value of the fermentation broth was between 5.41 and 5.73. However, the pH value of the samples in the SCAUH16 group showed a significant upward trend from the second day. From the first day to the sixth day, the pH value of the samples in the SCAUH16 group increased from 6.19 to 8.97, and then stabilized at 8.95-9.00 ( Figure 5 ). The results showed that Lysinibacillus louembei SCAUH16 decomposed proteins during metabolism to produce alkaline substances, resulting in changes in pH.

[0129] 4.3.2 Changes in the degradation rate of dead pig residues

[0130] The amount of dead pig residue after adding strain SCAUH16 was significantly reduced compared with the CK group ( Figure 6 ), the degradation rate of dead pig residue samples was calculated by weight loss method and it was found that the dead pig residue samples of the untreated group (CK group) degraded slowly. From the first day to the ninth day, the degradation rate of the dead pig residue samples of the CK group did not change significantly, and the degradation rate was between 31.64% and 36.83%. However, the degradation rate of dead pig residue in the bacteria-added group (SCAUH16 group) increased from 34.41% on the first day to 61.37%, an increase of 26.96%, and the upward trend was obvious. The degradation rate of dead pig residue ( Figure 7 ). The results showed that the addition of strain SCAUH16 could effectively improve the degradation rate of dead pig residues and had the potential to be used as a microbial fermentation agent.

[0131] 4.3.3 Protein changes in dead pig residues

[0132] The protein content of the dead pig residue samples in the CK group did not change significantly, and the protein content was 60.04%-62.00%. However, the protein content of the dead pig residue samples in the SCAUH16 group continued to decrease from 60.30% on the first day of culture to 25.69% on the ninth day, a decrease of 25.56% ( Figure 8 The results showed that the protease produced by Lysinibacillus louembei SCAUH16 can effectively degrade the protein in the dead pig residues, increase the degradation rate of protein, and promote the rapid conversion of organic matter, and has the potential to be used as a microbial fermentation agent.

[0133] Example 5 Application of Lysinibacillus louembei SCAUH16 in Composting of Residues from Dead Pigs

[0134] 5.1 Compost preparation and sample collection

[0135] 5.1.1 Compost preparation

[0136] The dead pig residues were provided by Chengdu Kenong Animal Harmless Disposal Co., Ltd., and the fresh cow dung was from a large state-owned farm in Zhaojue County, Sichuan Province, China. The dead pig residues were aerobic composted in a stacking manner, sawdust was used as an auxiliary material, the moisture content of the material was adjusted to 50-60%, the initial carbon-nitrogen ratio (C / N) was controlled at 25-30, and the microbial inoculant described in claim 2 and Example 3 was added to the material with the moisture content and carbon-nitrogen ratio adjusted at a weight ratio of 0.5%, and the composting was fermented for 30 days. The experiment was divided into two groups, namely, the first group did not add any microbial inoculant as a blank control group (CK), and the second group added a microbial inoculant containing Lysinibacillus louembei SCAUH16 at an addition amount of 0.5% (v / v) (SCAUH16 group), and each treatment was repeated 3 times.

[0137] 5.1.2 Compost sample collection

[0138] An automatic thermometer was used to monitor and record the daily composting temperature (Tm) changes at 8:00 a.m. and 6:00 p.m. Compost samples (3 samples / group) were collected every day for analysis of their physical and chemical properties and microbial flora characteristics. During the composting process, when the pile temperature reached above 50°C, the first turning was performed; when the temperature reached 60°C, the second turning was performed to maintain aerobic conditions and uniformity, promote material degradation and compost maturity.

[0139] 5.2 Analysis of compost physical and chemical properties

[0140] According to the Chinese organic fertilizer agricultural industry standard (NY / T 525-2021), the physicochemical properties of the compost samples, such as moisture content, pH, conductivity, organic matter (OM), total nitrogen (N), total phosphorus (P2O5), total potassium (K2O), and seed germination index (GI), were analyzed. The moisture content of the compost samples was determined by drying. The pH value of the 1:5 aqueous solution was detected by a pH meter (INESAPHSJ-3F, China). The conductivity of the 1:10 aqueous solution was detected by a conductivity meter (DDS-307A, China). The organic matter content in the compost samples was determined by potassium dichromate titration. 0.5 g of air-dried manure was digested in concentrated H2SO4 and H2O2 to determine total N, P2O5, and K2O. The total nitrogen was determined by the Kjeldahl method, the total P2O5 was determined by ammonium molybdate spectrophotometry, and the total K2O was determined by flame photometry.

[0141] 5.3 Analysis of microbial flora in compost

[0142] Take 0.5g of compost sample and use Total DNA was extracted using the SPIN Kit for Soil (MP BIO Laboratories, California, USA), and the DNA purity and concentration were detected using an ultra-micro UV spectrophotometer. Qualified DNA samples were tested and the V4-V5 region of bacterial 16S was amplified using primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGAGTTT-3'). After amplification, subsequent library construction, Miseq sequencing, and sequence splicing were all performed on the Penosun Illumina MiSeq sequencing platform. Bioinformatics analysis of high-throughput sequencing data was performed on the Penosun Gene Cloud (https: / / www.genescloud.cn / home). According to sequence similarity, valid sequences were clustered into OTUs (97% similarity), and species annotation of OTUs was performed to obtain taxonomic information of each sample, bacterial diversity was calculated, and relevant indices were obtained, and the species composition of bacteria in the samples was analyzed.

[0143] 5.4 Experimental Results

[0144] 5.4.1 Temperature changes of compost samples

[0145] The temperature of the piles in the groups without bacteria and with bacteria changed after the start of composting. The temperature of the pile in the experimental group with bacteria (SCAUH16 group) rose more rapidly and quickly entered the thermophilic stage on the second day after the start of composting. It reached the thermophilic stage faster than the CK (sterile group) and was able to maintain a longer high temperature period ( Fig. 9 ). The results showed that in this study, the addition of SCAUH16 bacterial preparation in aerobic composting could accelerate the composting of dead pig residues into the thermophilic stage.

[0146] 5.4.2 Changes in C / N of compost samples

[0147] like Fig.10 As shown in the data, the C / N ratio in the CK group decreased from 25.21 to 24.69, while the C / N ratio in the SCAUH16 group decreased from 25.28 to 20.16. This may be because the addition of exogenous microbial agents promoted the decomposition of protein in the dead pig residues, resulting in a decrease in the C / N ratio of the compost and promoting the analysis of organic matter in the compost.

[0148] 5.4.3 Changes in seed germination rate (GI) of compost samples

[0149] GI is an indicator of compost maturity. Fig.11As shown in the figure, the GI of the CK group and the SCAUH16 group showed a trend of first decreasing and then increasing during the entire composting process, indicating that with the decomposition and transformation of organic matter in the compost, harmful substances to plant growth in the compost were gradually removed in the compost. At the end of composting, the GI value of the CK group was 52.26%, while the GI value of the SCAUH16 group was 80.36%. According to the national standard "NY / T525-2021", a GI value of ≥70 means that the compost is mature, indicating that the compost of the group without the addition of microbial agents has not reached the maturity requirements and cannot be further used for field production. The addition of SCAUH16 microbial agents can accelerate the maturity process of the compost of the residue source after the dead pigs are processed, ensuring the safety of subsequent use.

[0150] 5.4.4 pH changes of compost samples

[0151] pH is one of the important factors affecting the growth of microorganisms. Generally, the suitable pH for microorganisms is neutral and slightly alkaline. Too high or too low pH will affect the smooth progress of composting. According to the China Organic Fertilizer Agriculture Industry Standard (NY / T 525-2021), the pH value of the compost pile should be between 5.5 and 8.5. Fig.12 As shown, the pH value of the CK group was between 7.8 and 8.02, and the pH value of the SCAUH16 group was between 7.88 and 8.24, both of which were in line with the national standards.

[0152] 5.4.5 Changes in moisture content of compost samples

[0153] The moisture content of compost is an important parameter that affects the composting process. If the moisture content is too high, the ventilation of the entire compost will be poor, which may cause local anaerobicity in the compost and produce acid and odor. It is also not conducive to the exudation of nutrients, resulting in nutrient loss. If the moisture content is too low, it is easy to increase the mass transfer resistance of nutrients and reduce the metabolism of microorganisms. Fig.13 As shown in the figure, affected by the temperature of the pile, the moisture content of the compost in the CK group and the SCAUH16 group was slowly decreasing, and there was no significant change between the two groups.

[0154] 5.4.6 Changes in conductivity of compost samples

[0155] The conductivity reflects the nutrient decomposition effect and product quality during the composting process. Fig.14 As shown in the figure, the conductivity of the CK group and the SCAUH16 group was between 2.77 and 5.39, with no significant changes.

[0156] 5.4.7 Changes in total phosphorus and total potassium in compost samples

[0157] like Figures 15-16As shown in the figure, total nutrients (total phosphorus and total potassium) gradually increased during the composting process, and the nutrient content of the compost sample treated with SCAUH16 inoculant was richer. The total phosphorus and total potassium of the SCAUH16 group were significantly higher than those of the CK group on the 25th day, and the addition of bacteria was beneficial to the maintenance of nutrients in the compost material.

[0158] 5.4.8 Analysis of compost microbial flora diversity

[0159] The representative sequences of bacterial OTUs in samples of dead pig residues from different aerobic composting stages were specially annotated and analyzed. Fig.17 As shown in the figure, there were 29 bacterial OTUs in different fermentation periods of composting, and the unique OTUs in the SCAUH16 inoculation treatment and the non-inoculation treatment at different periods were 3689, 1852, 1843, 910, 797 and 3592, 1639, 1347, 559, 791, respectively, indicating that there were complex and diverse original bacterial flora in the samples of dead pig residues after composting, and the microbial communities in the compost changed significantly with the advancement of composting fermentation. Compared with the treatment without adding bacterial agents, the number of OTUs in the treatment with SCAUH16 was always higher than that in the group without adding bacterial agents, indicating that the SCAUH16 group with the addition of high-temperature degradation bacterial agents could quickly adapt to the rapid temperature rise of the pile, as well as the high-protein and high-fat dead pig residue materials, and its proliferation increased the abundance of bacterial flora in the compost. SCAUH16 could quickly adapt to the environment and quickly become the dominant bacterial flora. Therefore, the addition of SCAUH16 bacterial agents could drive the transformation of substances and promote the maturity of composting.

[0160] The analysis results of the bacterial community composition in samples of dead pig residues at different aerobic composting stages showed that at the phylum level, the dominant bacterial communities were mainly Firmicutes, Proteobacteria, Actinobacteria, and Bacteridetes, but their relative abundance varied in different periods of composting. Firmicutes, Proteobacteria, Actinobacteria, and Bacteridetes were the main dominant phyla in the compost at the beginning of fermentation. With the increase of composting temperature over time, the relative abundance of Firmicutes increased significantly in the treatment of SCAUH16 addition on day 1, while Proteobacteria increased significantly in the group without adding the agent. However, Bacteridetes, an important microbial component in the early stage of fermentation, was almost undetectable at the end of composting. Firmicutes was the absolute dominant phylum in the compost ( Fig.18The increase in composting temperature also led to significant changes in the composition of the microbial community at the genus level in the compost at different fermentation stages. The addition of the inoculant SCAUH16 also led to significant differences in the composition of the microbial community at the genus level in the compost compared to the compost without the addition of the inoculant ( Fig.18 The results show that as the composting progresses, the decomposition and transformation of materials by microorganisms and the increase in temperature have a significant impact on the composition of the microbial flora in the compost, and as the temperature of the pile changes, there are obvious differences in the microbial flora at each stage of composting fermentation. The addition of SCAUH16 inoculant reshapes the microbial composition in the compost, affects the transformation of materials in the compost, increases the temperature of the pile, and is conducive to the maturity of the compost.

Claims

1. A Lysinibacillus strain Lysinibacillus louembeiSCAUH16, deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCC NO: 65794.

2. A microbial agent containing the strain Lysinibacillus louembei SCAUH16 according to claim 1.

3. Use of the strain Lysinibacillus louembei SCAUH16 according to claim 1 or the microbial agent according to claim 2 in treating residues after dead pigs are processed.

4. The use according to claim 3, characterized in that: The treatment is high temperature aerobic composting.

5. The use according to claim 4, characterized in that: The high temperature aerobic composting refers to mixing the residues after the dead pigs are processed with sawdust, inoculating the strain Lysinibacillus louembei SCAUH16 described in claim 1 or the microbial agent described in claim 2, and performing high temperature composting fermentation.

6. The use according to claim 4, characterized in that: The high-temperature aerobic composting method comprises: using sawdust as an auxiliary material and mixing it with the residue after the dead pigs are processed, adjusting the moisture content of the material to 50-60%, controlling the initial carbon-nitrogen ratio to 25-30, adding the microbial agent according to claim 2 to the material at a weight ratio of 0.5% to carry out high-temperature aerobic composting for 30 days, and turning the pile for the first time when the pile temperature reaches above 50°C; and turning the pile for the second time when the temperature reaches 60°C.

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