Pseudomonas heterodera RSA10 and application thereof

Abstract

The application discloses a bacillus chondroitinus RSA10 and application, and belongs to the field of microorganisms, the strain is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO:68839; the bacillus chondroitinus RSA10 of the application is a strain with high-efficiency degradation of protein and fat under high-temperature conditions, which is isolated and screened from the residue after the treatment of dead pig by rendering.The microbial inoculum prepared from the strain is combined with biochar to be used in high-temperature aerobic composting of the residue after the treatment of dead pig by rendering, so that the composting maturity is significantly improved, nitrogen loss is reduced, and the composting quality of the residue after the treatment of dead pig by rendering is promoted, which provides a strain resource application reference for the resource utilization of the residue after the treatment of dead pig by rendering, helps to promote the harmless treatment and resource utilization of agricultural waste, and has a good popularization and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of phosphate-eating pseudobacterium RSA10 and its applications. Background Technology

[0002] In recent years, with the continuous improvement of people's living standards, the demand for livestock products has increased significantly, and pig farming has become a pillar industry of my country's animal husbandry. However, a large number of dead pigs are generated during the breeding process due to stillbirths, diseases, and other reasons. If these pigs are not disposed of harmlessly and are simply piled up, they will seriously endanger the ecological environment and public health safety. Rendering is currently the main method for harmlessly disposing of dead pigs. The residue after rendering is rich in organic matter such as fat and protein, and has the potential to be used as organic fertilizer. However, due to its long degradation cycle and the risk of secondary pollution, it is not suitable for direct return to the field. High-temperature aerobic composting is an effective way to promote the resource utilization of residue after rendering dead pigs. Traditional composting has drawbacks such as severe nitrogen loss, long composting cycle, and low humification efficiency. Studies have shown that inoculating functional microbial agents can effectively promote the decomposition of macromolecules, rapidly increase the temperature of the compost pile, and shorten the composting process; adding exogenous materials such as biochar can improve the physicochemical environment of the compost pile, reduce nitrogen loss, and promote the humification process.

[0003] Currently, studies have screened thermophilic strains with protein and fat degradation capabilities from the residue of diseased and dead pigs after decomposition, such as Lysinibacillus louembei SCAUH16 (protease 56.9 μmol / mL, lipase 28.6 μmol / mL) and Paenibacillus lactis SCAUF07 (protease 61.2 μmol / mL, lipase 22.9 μmol / mL). However, the upper limit of the growth temperature for these strains is 70 °C. Fictibacillus phosphorivorans, a new species published in 2013, with the type strain Ca7 isolated from bioreactor wastewater, has an optimal growth temperature of 28-30 °C and a growth range of only 15-36 °C. Its known functions include nitrate removal and hydrocarbon degradation. There are no reports of this species producing protease and lipase under high-temperature conditions, nor are there any studies on its use in high-temperature aerobic composting. Furthermore, there are few reports on the combined use of functional microbial agents and biochar for composting residues from the decomposition of diseased and dead pigs. Therefore, screening for high-temperature strains with greater temperature tolerance and efficient protein and fat degradation capabilities, and combining them with biochar, is of great significance for further improving compost quality and promoting the resource utilization of residues from the decomposition of diseased and dead pigs. Summary of the Invention

[0004] The technical problem to be solved by this invention is: for the resource utilization of residues after the decomposition of diseased and dead pigs, to isolate and screen high-temperature strains with efficient protein and fat degradation capabilities, and to use them in combination with biochar in high-temperature aerobic composting of residues after the decomposition of diseased and dead pigs. One objective of this invention is to provide a Bacillus strain with efficient protein and fat degradation capabilities, and another objective is to provide the application of this strain.

[0005] The technical solution of the present invention is: a strain of Fictibacillus phosphorivorans RSA10, abbreviated as RSA10, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 17, 2026, with accession number GDMCC NO: 68839, and the address of the depository is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0006] Microbial inoculants containing Fictibacillus phosphorivorans RSA10.

[0007] Application of Fictibacillus phosphorivorans RSA10 or microbial agents containing this strain in the treatment of residues from the rendering of diseased and dead pigs.

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

[0009] Furthermore, the high-temperature aerobic composting refers to mixing the residue from the decomposition of diseased and dead pigs with sawdust and adding biochar, then inoculating with the aforementioned *Fictibacillus phosphorivorans* RSA10 or microbial agents for high-temperature composting fermentation.

[0010] Furthermore, the high-temperature aerobic composting method is as follows: sawdust is mixed with the residue from the decomposition of diseased and dead pigs as an auxiliary material, and biochar is added at a weight ratio of 5%. The moisture content of the material is adjusted to 50-60%, and the initial carbon-nitrogen ratio is controlled at 25-30. The aforementioned microbial agent is added to the material at a weight ratio of 3% for aerobic composting. During the composting process, the compost is turned over once a week, and aeration is carried out for 3 hours a day. The composting time is 30 days.

[0011] Compared with the prior art, the present invention has the following unexpected technical effects:

[0012] This invention is the first to discover the high-temperature resistance of Fictibacillus phosphorivorans: the upper limit of the growth temperature of the model strain Ca7 is only 36 ℃, and this invention is the first to extend the growth temperature of this strain to 30-75 ℃, doubling the maximum growth temperature, which is a major breakthrough in the temperature adaptability of this strain.

[0013] This invention provides the first quantitative confirmation of the protease and lipase activities of this bacterial strain: although the genome of this strain contains predicted protease and lipase-related genes, specific quantitative data on enzyme activity have never been available. This invention provides, for the first time, quantitative data on protease activity (72.4 μmol / mL) and lipase activity (35.7 μmol / mL).

[0014] This invention is the first to apply this strain to high-temperature aerobic composting: Fictibacillus phosphorivorans was applied for the first time to composting the residue after the decomposition of diseased and dead pigs, achieving effects such as significantly improving the maturity of compost and significantly reducing nitrogen loss.

[0015] The preservation information is as follows:

[0016] Fictibacillus phosphorivorans RSA10, or RSA10 for short, was deposited on August 17, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 68839). The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. Attached Figure Description

[0017] Figure 1 Photograph of the colony morphology of Fictibacillus phosphorivorans RSA10 on tryptic soybean agar (TSA medium);

[0018] Figure 2 Phylogenetic tree of Fictibacillus phosphorivorans RSA10 constructed based on 16S rRNA sequence results;

[0019] Figure 3 For composting temperature changes;

[0020] Figure 4 This refers to changes in the moisture content of the compost.

[0021] Figure 5 pH changes in composting;

[0022] Figure 6 This refers to the change in the electrical conductivity of compost.

[0023] Figure 7 For changes in organic matter in compost;

[0024] Figure 8 This refers to the change in total nitrogen in the compost.

[0025] Figure 9 The change in total phosphorus in compost;

[0026] Figure 10 This refers to the change in total potassium in the compost.

[0027] Figure 11 Changes in the germination index of composted seeds;

[0028] Figure 12 Venn diagram analysis of bacterial communities in samples at different fermentation stages of compost;

[0029] Figure 13 The bacterial community composition of samples at different fermentation stages of composting. Detailed Implementation

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0031] In this application:

[0032] Tryptic soybean agar medium (TSA medium): 15 g tryptic peptone, 5 g soybean peptone, 5 g NaCl, 20 g agar, 1000 mL distilled water;

[0033] Tryptic soybean peptone liquid medium (TSB medium): 17 g tryptic peptone, 3 g soybean peptone, 5 g NaCl, 2.5 g K2HPO4, 2.5 g glucose, 20 g agar, 1000 mL distilled water;

[0034] Protease fermentation medium: 5 g glucose, 10 g yeast powder, 0.5 g KH2PO4, 0.3 g MgSO4·7H2O, 1 g (NH4)2SO4, 1 g CaCl2, 1 g NaCl, 1000 mL distilled water;

[0035] Lipase fermentation medium: 20 g peptone, 20 g beef extract, 5 g sucrose, 1 g KH2PO4, 0.05 g MgSO4·7H2O, 1 g (NH4)2SO4, 20 mL olive oil, 1 g NaCl, 1000 mL distilled water.

[0036] Example 1: Isolation and identification of Fictibacillus phosphorivorans RSA10

[0037] 1.1 Isolation of Fictibacillus phosphorivorans RSA10

[0038] Weigh 10 g of fresh residue from the calcination of diseased and dead pigs into a 250 mL sterile Erlenmeyer flask containing 90 mL of sterile water. Place the flask on a shaker at 50 ℃ and 180 r / min for 30 min. After mixing, let it stand. Take the supernatant and perform serial dilutions, taking 100 μL of each diluted 10⁻⁶ dropper. -3 10 -4 10 -5 The bacterial suspension was spread onto TSA medium and incubated at 50 °C for 48 h. Single colonies were picked and purified on TSA solid medium. The purified strains were placed in 40% (V / V) glycerol cryovials and stored at -80 °C.

[0039] 1.2 Identification of Fictibacillus phosphorivorans RSA10

[0040] 1.2.1 Strain morphology

[0041] Single colonies of strain RSA10 were picked and inoculated onto TSA medium and incubated at 50 °C for 48 h. The colony morphology of the strain was then observed.

[0042] 1.2.2 DNA extraction and PCR amplification of the strain

[0043] (1) Reagents: Chelex 100 (Beijing Solarbio Science & Technology Co., Ltd.), Mix (Sangon Biotech Co., Ltd.).

[0044] (2) Primers

[0045] Forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0046] Reverse primer 1492R: 5'-TACGACTTAACCCCAATCGC-3'.

[0047] (3) Bacterial DNA extraction

[0048] Pick a single colony using a disposable inoculation loop and place it into a 2 mL EP tube. Add 200 μL of 5% Chelex solution, incubate in a metal bath at 100 °C for 15 min, vortex thoroughly to mix, and cool. Centrifuge at 12000 r / min for 5 min, and use the supernatant as a DNA template. Store at -20 °C.

[0049] (4) Bacterial 16S rRNA gene amplification

[0050] The purified bacterial DNA template was amplified using universal primers 27F and 1492R for bacterial 16S rRNA. The 16S rRNA gene amplification conditions were as follows: 1 μL DNA template (50 ng / μL), 12.5 μL 2× Taq PCR mix, 1 μL each of forward and reverse primers (1.0 μmol / L), ddH2O added to a final volume of 25 μL, incubated at 95 ℃ for 5 min, 95 ℃ for 30 s, 55 ℃ for 45 s, 72 ℃ for 1.5 min, for 35 cycles, followed by 72 ℃ for 10 min. After PCR amplification and band detection using 1.5% agarose gel electrophoresis, the products were sent to Sangon Biotech Co., Ltd. for sequencing.

[0051] 1.2.3 16S rRNA gene sequence analysis and phylogenetic tree construction

[0052] The sequence obtained after sequencing (SEQ ID No. 1) was uploaded to the EzBioCloud database for comparison. The 16S rRNA gene sequence of the published standard strain of the same genus with the highest similarity was selected as the reference sequence. The sequence of the Bacillus subtilis (NCIB 3610) standard strain was used as the outgroup. Multiple sequence alignment analysis was performed using Clustal X software, and a phylogenetic tree was constructed using MEGA 11 software based on the Neighbor-joining algorithm to determine the taxonomic position of the strain.

[0053] 1.3 Experimental Results

[0054] Fictibacillus phosphorivorans RSA10 was isolated from the residue of diseased and dead pigs after decomposition. After incubation on TSA medium at 50 °C for 48 h, the colonies were 2-3 mm in size, round and slightly convex, with smooth edges and a rough surface. The colonies were orange-yellow in color, easily picked up, and aerobic. Figure 1The 16S rRNA sequence of strain RSA10 was determined, and BLAST homology comparison was performed in the EzBioCloud database. Phylogenetic analysis was then performed using MEGA 11 software. The 16S rRNA nucleotide sequence homology between this strain and Fictibacillus phosphorivorans was 100%. Based on the combined results of morphological characteristics and 16S rRNA sequence homology analysis, strain RSA10 was identified as Fictibacillus phosphorivorans. Figure 2 ).

[0055] Example 2: Determination of growth temperature and enzyme production function of Fictibacillus phosphorivorans RSA10

[0056] 2.1 Determination of Temperature Range

[0057] Fictibacillus phosphorivorans RSA10 was inoculated onto TSA medium and cultured in incubators at 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, and 80 ℃ for 48 h. Its growth was observed and recorded. Based on the measurement results, the temperature detection range was further narrowed down in 5 ℃ increments until the highest and lowest growth temperatures were determined.

[0058] 2.2 Protein degradation capacity assay

[0059] (1) Drawing the standard curve

[0060] A 100 μg / mL L-tyrosine standard solution was prepared and diluted to obtain L-tyrosine solutions with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. 1 mL of each diluted solution was taken, and 5 mL of 0.4 mol / L sodium carbonate solution and 1 mL of 1 mol / L Folin-phenol reagent solution were added. After shaking well, the solutions were incubated in a 40 ℃ water bath for 20 min for color development. After shaking well, the OD value was measured at 680 nm. An L-tyrosine standard curve was plotted with absorbance as the ordinate and L-tyrosine content as the abscissa.

[0061] (2) Protease activity assay.

[0062] After inoculating the bacterial strain into TSB medium and culturing for 16 h, the bacterial solution was inoculated into protease fermentation medium at a ratio of 3%, and cultured at 50 ℃ and 150 r / min for 48 h. The supernatant was then centrifuged and used for enzyme activity determination. The Folin-phenol method was used to determine the enzyme activity of the crude enzyme solution. A 2% casein solution was placed in a 40 ℃ water bath for 5 min to preheat. 1 mL of crude enzyme solution was added to 1 mL of the preheated casein solution, mixed well, and incubated in a 40 ℃ water bath for 10 min. The reaction was terminated by adding 2 mL of 0.4 mol / L trichloroacetic acid. 1 mL of the supernatant was added to 5 mL of 0.4 mol / L Na₂CO₃, followed by 1 mL of Folin-phenol reagent. The mixture was incubated in a 40 ℃ water bath for 20 min, and the absorbance was measured at 660 nm. The reaction system with added water was used as a blank. Each experiment was repeated in triplicate. The protease activity was calculated using the following formula:

[0063] Protease activity (U / mL) = A × K × 4 ÷ 10 × N

[0064] In the formula: A: OD value of parallel experiments of the fermentation broth;

[0065] K: Absorption constant;

[0066] N: The dilution factor of the protease solution;

[0067] 4: Total volume of the reaction reagents;

[0068] 10: Reaction time 10 min.

[0069] 2.3 Determination of Fat Degradation Capacity

[0070] After inoculating the bacterial strain into TSB medium and culturing for 16 h, the bacterial solution was inoculated into lipase fermentation medium at a ratio of 3%, and cultured at 50 ℃ and 150 r / min for 48 h. The supernatant was then collected by centrifugation for enzyme activity determination. Lipase activity was determined using an alkaline titration method. Multiple 50 mL Erlenmeyer flasks were prepared, with three designated as control flasks and the remainder as test flasks. Test flasks contained: 4 mL polyvinyl alcohol-ol-ol emulsion and 5 mL phosphate buffer (pH 7.5); control flasks contained: 15 mL 95% ethanol; 40 ℃ water bath for 10 min; test flasks contained: 5 mL crude enzyme solution, 40 ℃ water bath for 20 min; test flasks contained: 15 mL 95% ethanol; test flasks and control flasks contained: 1% phenolphthalein indicator (3 drops), titrated with 0.05 mol / L NaOH solution, and titrated with 0.05 mol / L standard sodium hydroxide solution until a faint pink color appeared. The volume of NaOH solution used in the titration was recorded. Each experiment was repeated in triplicate. Lipase activity was calculated using the following formula:

[0071] Lipase activity (U / g prot) =

[0072] Where: A: Volume of alkali solution consumed by the sample (mL);

[0073] B: Volume of alkali solution consumed by the control group (mL);

[0074] N: The concentration of the alkaline solution, i.e., 0.05 mol / L;

[0075] F: Final dilution factor of crude enzyme solution;

[0076] T: Action time (min).

[0077] 2.4 Experimental Results

[0078] The growth range of *Fictibacillus phosphorivorans* RSA10 was determined to be 30-75 °C, and it possessed the ability to produce protease and lipase, with enzyme activities of 72.4 μmol / mL and 35.7 μmol / mL, respectively. These results indicate that *Fictibacillus phosphorivorans* RSA10 not only exhibits good heat resistance and growth ability but also demonstrates excellent protein and fat degradation capabilities, showing potential for further development and application.

[0079] Example 3 Preparation of microbial inoculants

[0080] This embodiment provides a microbial inoculant, the preparation method of which is as follows:

[0081] Fictibacillus phosphorivorans RSA10 plates were streaked onto TSA medium. Pure strains were picked and placed in sterile physiological saline. The bacterial suspension concentration was adjusted to 10⁻⁶ using the McFarland turbidimetric method. 9 The bacterial suspension was inoculated into TSB medium at a concentration of CFU / mL at a rate of 3%, and cultured at 37 ℃ with shaking at 180 r / min for 16 h to obtain the microbial inoculum, which had an effective viable count of 1.0 × 10⁻⁶. 8 CFU / g.

[0082] Example 4: Application of Fictibacillus phosphorivorans RSA10 in composting residue from the decomposition of diseased and dead pigs.

[0083] 4.1 Compost preparation and sample collection

[0084] 4.1.1 Compost Preparation

[0085] The residue from the decomposition of diseased and dead pigs was provided by Chengdu Kenong Animal Harmless Disposal Co., Ltd., and the biochar was prepared by a certain agricultural technology company in Zhengzhou, Henan Province, by pyrolyzing corn stalks at 500 ℃ for 2 h. High-temperature aerobic composting of the residue from the decomposition of diseased and dead pigs was carried out by stacking. Sawdust was used as an auxiliary material and mixed with the residue from the decomposition of diseased and dead pigs. Biochar was added at a weight ratio of 5%, the moisture content of the material was adjusted to 50-60%, the initial carbon-nitrogen ratio was controlled at 25-30, and the microbial agent described in Example 3 was added to the material at a weight ratio of 3%. The composting process was carried out by turning the pile once a week and aerating for 3 h a day. The composting time was 30 days. Two treatment groups were set up in the experiment: (1) no microbial inoculant was added, only an equal amount of sterile water was added as a blank control group (CK group); (2) microbial agent containing Fictibacillus phosphorivorans RSA10 was added (RSA10 group).

[0086] 4.1.2 Compost Sample Collection

[0087] The daily temperature changes of the compost were monitored and recorded at 10:00 AM and 10:00 PM using an automatic thermometer. Compost samples were taken on days 0, 1, 3, 5, 7, 10, 13, 16, 25, and 30 of the compost. 200 g samples were taken from the top, middle, and bottom parts of the compost pile using the quartering method. After being mixed evenly, the samples were placed in self-sealing bags and brought back to the laboratory for the determination of physicochemical properties and microbial community.

[0088] 4.2 Physicochemical property analysis of compost samples

[0089] Referring to the Chinese agricultural industry standard for organic fertilizers (NY / T 525-2021), the physicochemical properties of compost samples, including moisture content, pH, conductivity, organic matter, total nitrogen, total phosphorus, total potassium, and seed germination index, were analyzed. The moisture content of the compost samples was determined by drying; the pH and conductivity of the compost samples were measured using a pH meter and conductivity meter, respectively; the organic matter content of the compost samples was determined by potassium dichromate titration; the total nitrogen content of the compost samples was determined by the Kjeldahl method; the total phosphorus content of the compost samples was determined by ammonium molybdate spectrophotometry; the total potassium content of the compost samples was determined by flame photometry; and the seed germination index was calculated using radish seeds using the filter paper method.

[0090] 4.3 Microbial community analysis of compost samples

[0091] Weigh 1 g of compost sample and extract total DNA using a DNA extraction kit (MP Biomedicals, CA, USA). DNA purity and concentration were assessed by 1% agarose gel electrophoresis. The V3-V4 region of the DNA was amplified using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The 25 μL amplification reaction mixture consisted of: 5 μL buffer, 2 μL 2.5 mm dNTPs, 1 μL each of primers 338F and 806R, 0.25 μL DNA polymerase, 1 μL DNA template, and 14.75 μL ddH2O. The PCR amplification program was as follows: 94 °C pre-denaturation for 3 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 40 s, for a total of 35 cycles; and a final extension at 72 °C for 10 min. The amplified products were detected using 1% agarose gel electrophoresis. The qualified DNA was then sent to Shanghai Paisenno Biotechnology Co., Ltd. for paired-end sequencing using the Illumina NovaSeq sequencing platform.

[0092] 4.4 Experimental Results

[0093] 4.4.1 Temperature Changes in Compost Samples

[0094] The composting was carried out using a static aerobic composting method, and the temperature changes of each treatment group over time were obtained through 30 days of timed monitoring. Figure 3 Throughout the composting process, each treatment experienced a typical heating phase, a high-temperature phase, and a cooling phase, indicating that the composting process was normal. Among them, the experimental group with added microbial agents (RSA10 group) experienced a more rapid temperature rise in the compost pile, and was able to maintain a longer high-temperature period compared to the CK group, effectively promoting the temperature rise of the compost pile. This is beneficial for the harmlessness of compost and the rapid degradation of organic matter, laying the foundation for subsequent humification.

[0095] 4.4.2 Changes in moisture content of compost samples

[0096] The initial moisture content of the inoculant-added experimental group (RSA10 group) and the CK group was around 70%. As they entered the high-temperature stage, the moisture in the pile began to be continuously consumed, and the moisture content of the pile continued to decrease. Figure 4 As microbial activity in the compost pile slowed, the pile temperature decreased. The condensate from the barrel walls flowed back into the pile, causing a temporary rise in moisture content. Subsequently, as the composting process continued, the moisture content gradually decreased. Throughout the composting process, the moisture content of the RSA10 group was generally slightly lower than that of the CK group, possibly due to the higher fermentation temperature in the RSA10 group. However, at the end of composting, the moisture content of all treatment groups decreased to a suitable range of 40-50%, which is beneficial for subsequent storage and application of the compost.

[0097] 4.4.3 pH changes in compost samples

[0098] like Figure 5 As shown, the initial pH values ​​of both the inoculant-added experimental group (RSA10 group) and the CK group were around 7. On day 1, the pile temperature rose rapidly, and the pH value also showed a significant upward trend. On day 7, the pile temperature decreased, and the pH value also showed a brief downward trend. As the composting process progressed, the pH value of the pile gradually stabilized. At the end of composting on day 30, the pH values ​​of all treatment groups decreased, with the final pH values ​​being 7.84 for the RSA10 group and 7.65 for the CK group. After composting, the pH values ​​of both the RSA10 group and the CK group met the requirements of the Chinese Organic Fertilizer Agricultural Industry Standard (NY / T 525-2021), which stipulates that the pH value of the compost pile should be between 5.5 and 8.5, suitable for crop growth and soil improvement.

[0099] 4.4.4 Changes in electrical conductivity of compost samples

[0100] The change in electrical conductivity during composting is the result of the combined effects of ion generation due to the release of soluble salts from the decomposition of organic matter, greenhouse gas production, and ion loss through leaching. The changes in electrical conductivity during composting in the inoculant-added experimental group (RSA10 group) and the control group are shown below. Figure 6 As shown, the addition of RSA10 microbial inoculant significantly increased EC (P<0.05), which may be due to the increased microbial metabolic activity during the high-temperature period, promoting the ionization and release of organic matter and reducing salt loss. After composting, the conductivity values ​​of all treatment groups were less than 2000 µs / cm, which is lower than the organic fertilizer salt damage threshold (<3000 µs / cm), thus ruling out the inhibitory effect of salt on crop growth.

[0101] 4.4.5 Changes in organic matter in compost samples

[0102] The changes in organic matter content between the experimental group with added inoculant (RSA10 group) and the control group are as follows: Figure 7 As shown, the organic matter content of the RSA10 group generally showed a fluctuating downward trend throughout the entire composting fermentation stage. At the end of 30 days of composting, the organic matter content of the RSA10 group was significantly higher than that of the CK group (P<0.05), indicating that the addition of exogenous microbial agents reduced the loss of organic matter in the compost samples.

[0103] 4.4.6 Changes in total nitrogen in compost samples

[0104] The composting process involves nitrogen fixation and release, and some degree of nitrogen loss usually occurs after the compost is fully decomposed. The changes in total nitrogen in the experimental group with added microbial agents (RSA10 group) and the control group are shown below. Figure 8As shown, the total nitrogen content of the initial compost samples was around 2%. The total nitrogen content of both the RSA10 group and the CK group showed a fluctuating decrease and then gradually stabilized during the composting process. Throughout the entire composting fermentation stage, the total nitrogen content of the RSA10 group was significantly higher than that of the CK group (P<0.05), indicating that the addition of exogenous microbial agents increased the total nitrogen content in the compost samples and reduced nitrogen loss during fermentation.

[0105] 4.4.7 Changes in total phosphorus and total potassium in compost samples

[0106] The changes in total phosphorus content between the experimental group with added inoculant (RSA10 group) and the control group are as follows: Figure 9 and Figure 10 As shown, in the initial stage of composting, the total phosphorus and total potassium contents of the RSA10 group were significantly higher than those of the CK group (P<0.05), which may be due to the addition of exogenous microbial agents. Furthermore, throughout the composting process, the total phosphorus and total potassium contents of each treatment group showed a fluctuating upward trend. There was no gaseous loss of phosphorus during composting; the increase in its content was mainly attributed to the "concentration effect," meaning that the total mass of the compost decreased due to water evaporation, organic matter degradation, and gas emissions, leading to an increase in the relative concentration of total phosphorus and total potassium.

[0107] 4.4.9 Changes in seed germination index of compost samples

[0108] Seed germination index (GI) is a key indicator reflecting compost maturity. The changes in GI values ​​between the inoculant-added experimental group (RSA10 group) and the control group are shown below. Figure 11 As shown, the GI values ​​of each treatment group showed a continuous increasing trend throughout the composting process, indicating that as organic matter in the compost is continuously decomposed and transformed, substances harmful to plant growth are gradually eliminated after high-temperature composting fermentation. Referring to the Chinese organic fertilizer agricultural industry standard (NY / T 525-2021), a GI value ≥70% indicates that the compost has reached maturity. The RSA10 group reached maturity with a GI value of 84.3% on day 13, while the CK group's GI value was only 63.7%, indicating that it had not reached maturity. This demonstrates that adding RSA10 microbial inoculant can effectively accelerate the composting process and ensure the safety of subsequent use.

[0109] 4.4.10 Analysis of Microbial Diversity in Compost Samples

[0110] The representative sequences of bacterial OTUs in samples from the experimental group with added inoculant (RSA10 group) and the control group at different aerobic composting stages (initial stage 0 days, high-temperature stage 3 days, cooling stage 13 days, and maturity stage 30 days) were annotated and analyzed. The results are as follows: Figure 12As shown, there were a total of 38 OTUs in each treatment group at different fermentation stages of composting. The unique OTUs in the RSA10 inoculation treatment and the uninoculated treatment at different stages were 1624, 1515, 1455, 1551 and 1700, 1448, 1478, 1550, respectively. This indicates that the residue of diseased and dead pigs after decomposition carries a complex and diverse bacterial microbial community, which changes significantly as composting fermentation progresses.

[0111] 4.4.11 Analysis of Microbial Community Composition of Compost Samples

[0112] Analysis of bacterial community composition in compost samples from the experimental group (RSA10 group) and the control group at different aerobic composting stages (initial stage 0 days, high-temperature stage 3 days, cooling stage 13 days, and maturity stage 30 days) with added microbial agents revealed that at the phylum level... Figure 13 In the phylum A, Bacillota and Pseudomonadota remained the dominant phyla throughout the composting process, with their relative abundance changing continuously as fermentation progressed. During the 3-day high-temperature phase, Bacillota became the absolutely dominant phylum. This is likely because the excessively high temperature during this phase inhibited the growth of most microorganisms, while most Bacillota microorganisms, due to the presence of spores, were able to tolerate the high temperatures, leading to an increase in their relative abundance. Further in-depth analysis of the bacterial community composition at the genus level was conducted. Figure 13 In the initial stage (B group), the main taxa in both the RSA10 and CK groups consisted of Bacillus, Escherichia-Shigella, Corynebacterium, and Pusillibacter. The relative abundance of Escherichia-Shigella in the CK group was significantly higher than that in the RSA10 group, indicating that the addition of exogenous microbial agents could effectively inhibit the growth of potential pathogenic microorganisms. During the 3-day high-temperature stage, Bacillus became the dominant genus, which is related to its strong heat resistance. During the composting cooling and maturation stages, the abundance of Pusillibacter and Tepidimicrobium increased significantly, suggesting their potential involvement in the degradation of complex organic matter during composting.

Claims

1. A strain of Fictibacillus phosphorivorans RSA10 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 68839.

2. A microbial inoculant containing Fictibacillus phosphorivorans RSA10 as described in claim 1.

3. The application of the Fictibacillus phosphorivorans RSA10 as described in claim 1 or the microbial agent as described in claim 2 in the treatment of residues after the rendering of diseased and dead pigs.

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

5. The application according to claim 4, characterized in that, The high-temperature aerobic composting refers to mixing the residue from the decomposition of diseased and dead pigs with sawdust and adding biochar, then inoculating with Fictibacillus phosphorivorans RSA10 as described in claim 1 or the microbial agent as described in claim 2, and carrying out high-temperature composting fermentation.

6. The application according to claim 5, characterized in that, The specific method of high-temperature aerobic composting is as follows: sawdust is mixed with the residue after the decomposition of diseased and dead pigs as an auxiliary material, and biochar is added at a weight ratio of 5%. The moisture content of the material is adjusted to 50-60%, and the initial carbon-nitrogen ratio is controlled at 25-30. The microbial agent described in claim 2 is added to the material at a weight ratio of 3% for high-temperature aerobic composting. The compost is turned over once a week during the composting process, and aeration is carried out for 3 hours a day. The composting time is 30 days.

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