A strain of Pseudocanthomonas sp. NJAU-273 and its application in straw composting

Abstract

The present invention discloses a strain of Pseudocanthomonas NJAU‑273, which is classified and named as Pseudocanthomonas Pseudoxanthomonas sp., deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, with a deposit date of March 25, 2024 and a deposit number of CGMCC No.30114. The present invention discloses its application in straw composting. The present invention also discloses the microbial agent prepared by the present invention and the application of the microbial agent in straw composting. Compared with the treatment without inoculation of microbial agent, the treatment with inoculation of NJAU‑273 microbial agent effectively promotes the increase of the temperature of the compost pile, accelerates the composting process, improves the germination index of the compost product, and increases the total nutrients of the compost product.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and in particular to a strain of Pseudocanthomonas NJAU-273 and application thereof in straw composting. Background Art

[0002] Agricultural solid waste refers to various solid wastes generated in the process of agricultural production. If they are not used efficiently, they will cause serious environmental pollution. The resource utilization of agricultural solid waste is an important part of controlling agricultural non-point source pollution, saving biomass resources, saving energy and reducing emissions, protecting the ecological environment, achieving carbon peak and carbon neutrality goals and sustainable social and economic development. Direct return to the field, biogas production, incineration, and production of organic fertilizers are different modes of treating agricultural solid waste. Among them, turning agricultural solid waste into fertilizer is the most promising way to achieve its resource utilization.

[0003] Plant-derived agricultural solid waste (such as straw, rice husk, fruit residue, etc.) contains a large amount of high-molecular organic matter such as cellulose, hemicellulose and lignin. These high-molecular organic matter has a complex structure and is difficult to be directly decomposed by general composting bacteria. It requires the synergistic action of specific enzyme systems and microbial communities to effectively degrade it. Traditional livestock and poultry manure composting initiating strains often lack enzyme systems or related genes that can efficiently degrade these high-molecular organic matter. Therefore, it is difficult to play an efficient role in the composting of plant-derived agricultural solid waste, resulting in a slow composting process and low conversion efficiency. Therefore, the development of initiating strains adapted to the composting process of plant-derived agricultural solid waste is of great significance for promoting the resource utilization of agricultural solid waste and promoting the sustainable development of agriculture. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Pseudoxanthomonas NJAU-273 and its application in straw composting, in view of the lack of efficient detonating strains in composting with plant-based agricultural solid wastes such as straw as the main raw materials, which results in a large amount of high-molecular organic matter such as cellulose contained in the plant-based agricultural solid wastes limiting the rapid conversion of the composting process.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a strain of Pseudocanthomonas sp. NJAU-273, which is classified and named as Pseudocanthomonas sp. Pseudoxanthomonas sp., deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is March 25, 2024, and the deposit number is CGMCC No.30114.

[0007] The second aspect of the present invention provides a bacterial agent prepared by the above-mentioned Pseudocanthomonas sp. NJAU-273.

[0008] Furthermore, the bacterial agent is prepared by the following steps: inoculating Pseudomonas aeruginosa NJAU-273 in a liquid culture medium for fermentation to obtain a fermentation liquid having a bacterial content of 1×10 9 The fermentation liquid and rice bran are mixed at a volume mass ratio of 1:2-4 to obtain the bacterial agent.

[0009] Furthermore, the liquid culture medium includes TSB medium or LB liquid culture medium.

[0010] Furthermore, the rice bran has a water content of no more than 10 wt %.

[0011] The third aspect of the present invention provides the use of the above-mentioned Pseudocanthomonas sp. NJAU-273 in straw composting.

[0012] Furthermore, the straw includes rice straw, and urea or livestock and poultry manure is added to the straw during composting to provide a nitrogen source.

[0013] A fourth aspect of the present invention provides application of the above-mentioned bacterial agent in straw composting.

[0014] Furthermore, the straw includes rice straw, and urea or livestock and poultry manure is added to the straw during composting to provide a nitrogen source.

[0015] Beneficial effects of the present invention:

[0016] The present invention screened a strain of Pseudocanthomonas NJAU-273 from a fertilizer sample of rice straw composting during the high temperature period. Compared with a treatment without inoculation of bacterial agent, the treatment with inoculation of NJAU-273 bacterial agent effectively promoted the increase of the temperature of the compost pile, accelerated the composting process, significantly improved the germination index of the compost product, and increased the total nutrients of the compost product.

[0017] In the round pile composting experiment, the pile treated with NJAU-273 inoculated bacteria reached 58.7℃ on the 3rd day, which was much higher than that of the pile not inoculated with the bacteria. The fast temperature rise brought the high temperature period forward, improved the composting efficiency, and made the pile more thoroughly decomposed. By the end of the 30th day of composting, the germination index of the pile treated with NJAU-273 inoculated bacteria was 121.75%, significantly higher than 70.69% of the pile not inoculated with the bacteria. The total nutrient content of the pile treated with NJAU-273 inoculated bacteria was also higher than that of the pile not inoculated with the bacteria.

[0018] In the airflow membrane composting experiment, the pile treated with NJAU-273 inoculated with bacteria increased temperature rapidly during the composting process, with the highest temperature reaching 77.7℃, which was significantly higher than that of the pile not inoculated with bacteria. By the end of the 30th day of composting, the germination index of the pile treated with NJAU-273 inoculated with bacteria was 127.40%, which was significantly higher than the 91.98% of the pile not inoculated with bacteria. The total nutrient content of the pile treated with NJAU-273 inoculated with bacteria was also higher than that of the pile not inoculated with bacteria.

[0019] The pseudoxanthomonas NJAU-273 of the present invention can effectively promote the increase of compost pile temperature under different composting forms and different nitrogen sources, improve composting efficiency, significantly improve the germination index of compost products, increase the total nutrients of compost products, provide technical support for straw composting, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of inoculation of cellulose-degrading bacteria on the temperature of the compost during composting. CK: rice straw + cow dung; J1: rice straw + cow dung + NJAU-190; J2: rice straw + cow dung + NJAU-273; J3: rice straw + cow dung + S48; J4: rice straw + cow dung + G112; J5: rice straw + cow dung + G67; RT: room temperature.

[0021] Figure 2 The effect of inoculation of cellulose-degrading bacteria on the germination index of the compost pile at the end of composting. CK: rice straw + cow dung; J1: rice straw + cow dung + NJAU-190; J2: rice straw + cow dung + NJAU-273; J3: rice straw + cow dung + S48; J4: rice straw + cow dung + G112; J5: rice straw + cow dung + G67.

[0022] Figure 3 The effect of inoculation of cellulose-degrading bacteria on the pH of the compost during the composting process. CK: rice straw + cow dung; J1: rice straw + cow dung + NJAU-190; J2: rice straw + cow dung + NJAU-273.

[0023] Figure 4 The effect of inoculation of cellulose-degrading bacteria on the electrical conductivity (EC) of the compost during composting. CK: rice straw + cow dung; J1: rice straw + cow dung + NJAU-190; J2: rice straw + cow dung + NJAU-273.

[0024] Figure 5 The effect of inoculation of cellulose-degrading bacteria on the organic matter content of the compost during the composting process. CK: rice straw + cow dung; J1: rice straw + cow dung + NJAU-190; J2: rice straw + cow dung + NJAU-273.

[0025] Figure 6 This is a colony photo of strain NJAU-273.

[0026] Figure 7 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of strain NJAU-273.

[0027] Figure 8 To study the effect of inoculation with NJAU-273 bacterial agent on the temperature of straw pile during round composting.

[0028] Fig. 9 To study the effect of inoculation with NJAU-273 bacterial agent on the pH of straw pile during round composting.

[0029] Fig.10 To study the effect of inoculation with NJAU-273 bacterial agent on the electrical conductivity (EC) of straw during round composting.

[0030] Fig.11 To study the effect of inoculation with NJAU-273 bacterial agent on the germination index of straw pile during round composting.

[0031] Fig.12 To study the effect of inoculation with NJAU-273 bacterial agent on the total nitrogen content in the straw round composting process.

[0032] Fig.13 To study the effect of inoculation with NJAU-273 bacterial agent on the total phosphorus content in straw round composting process.

[0033] Fig.14 To study the effect of inoculation with NJAU-273 bacterial agent on the total potassium content in the straw round composting process.

[0034] Fig.15 To study the effect of inoculation with NJAU-273 bacterial agent on the temperature of the pile during straw airflow membrane composting.

[0035] Fig.16 To study the effect of inoculation with NJAU-273 bacterial agent on the pH of the pile during straw airflow membrane composting.

[0036] Fig.17 To study the effect of inoculation with NJAU-273 bacterial agent on the electrical conductivity (EC) of straw during airflow membrane composting.

[0037] Fig.18 To study the effect of inoculation with NJAU-273 bacterial agent on the germination index of the pile during straw airflow membrane composting.

[0038] Fig.19 To study the effect of inoculation with NJAU-273 bacterial agent on the total nitrogen content in the straw pile during airflow membrane composting.

[0039] Fig. 20 To study the effect of inoculation with NJAU-273 bacterial agent on the total phosphorus content in the straw pile during airflow membrane composting.

[0040] Fig.21 To study the effect of inoculation with NJAU-273 bacterial agent on the total potassium content of the pile during straw airflow membrane composting.

[0041] Different letters above the bar graphs indicate significant (P < 0.05) differences defined by one-way ANOVA. * above the bar graphs indicates significant (P < 0.05) differences, and ** indicates extremely significant (P < 0.01) differences.

[0042] Biomaterial Deposit Information

[0043] strain NJAU-273, taxonomically named Pseudocanthomonas Pseudoxanthomonas sp., deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is March 25, 2024, and the deposit number is CGMCC No.30114. DETAILED DESCRIPTION

[0044] The present invention is further explained below in conjunction with the examples and drawings. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0045] The culture medium formulas involved in the following examples are as follows:

[0046] 1 / 10TSA medium (1L): 1.5g tryptone, 0.5g soytone, 0.5g sodium chloride, 15g agar powder.

[0047] TSA medium (1L): 15g tryptone, 5g soytone, 5g sodium chloride, 15g agar powder.

[0048] TSB medium (1L): 15 g tryptone, 5 g soytone, and 5 g sodium chloride.

[0049] CMC medium (1L): 10g sodium carboxymethyl cellulose, 10g peptone, 5g yeast powder, 5g sodium chloride, 1g potassium dihydrogen phosphate, and 20g agar powder.

[0050] Starch culture medium (1L): 5g beef extract, 10g peptone, 5g sodium chloride, 2g soluble starch, and 20g agar powder.

[0051] Skim milk powder culture medium (1L): 10g skim milk powder, 10g peptone, 5g yeast powder, 5g sodium chloride, 20g agar powder.

[0052] Example 1 Isolation and screening of functional bacteria

[0053] 1. Prepare gradient dilution of fertilizer samples

[0054] The rice straw compost high temperature fertilizer sample from Nanjing Mingzhu Fertilizer Co., Ltd. was collected. 10 g of fertilizer sample was weighed into a sterile conical flask, and 90 mL of sterile water and glass beads (4-5 beads, 6 mm in diameter) were added. After shaking, the sample was shaken at 25 °C and 170 rpm for 30 min. A dilution gradient of 10 -4 , 10 -5 , 10 -6 and 10 -7 of suspension.

[0055] 2. Plate coating and culture

[0056] The dilution gradient was 10 -5 , 10 -6 and 10 -7 Use a vortexer to mix the suspensions of each dilution gradient, take 100 μL of the suspension and add it to a 1 / 10 TSA medium plate and spread it evenly. Repeat three times for each dilution gradient. After spreading, place the plate in an incubator at 30°C and culture it at a constant temperature. Observe and record the growth of the colonies every 24 hours.

[0057] 3. Separation, purification and preservation

[0058] Observe the characteristics of the colonies on the culture medium (color, shape, texture), pick single colonies with different morphological characteristics, and streak them onto new 1 / 10 TSA culture medium plates. After constant temperature culture at 30°C for 48 hours, pick out the single colonies on the plate again and streak them onto new 1 / 10 TSA culture medium plates for purification. The purified strain (bacteria) was inoculated into TSB culture medium, cultured at 30°C and 170rpm until the logarithmic growth phase, and then the bacterial solution was mixed with 40v / v% glycerol in a volume ratio of 1:1, placed in a 2mL sterile culture preservation tube, and stored in a -80°C ultra-low temperature refrigerator for use. A total of 248 culturable bacterial strains belonging to 34 genera were isolated from the high temperature period fertilizer samples of rice straw compost.

[0059] 4. Screening of efficient cellulose-degrading bacteria

[0060] The degradation ability of the culturable bacteria isolated above on cellulose, starch and protein was measured, and a small-scale composting test was conducted to screen out the strain NJAU-273 with strong cellulose degradation ability. The specific operation is as follows:

[0061] (1) Determination of the ability of culturable bacteria to degrade cellulose, starch and protein

[0062] ① Determination of cellulose degradation ability: The strain was spotted on a CMC medium plate and placed in an incubator at 30°C for 48 h. 500 μL of 1 mg mL -1 Congo red solution was added to the plate, and after standing for 1 hour, the dye solution was discarded and 500 μL of 1 mol L -1 After 1 hour of NaCl solution, strains with transparent circles around the colonies have the ability to degrade cellulose. The diameter of the transparent circle (mm) and the diameter of the colonies (mm) were measured, and the Hc value was calculated according to the following formula.

[0063] Hc value = transparent circle diameter / colony diameter

[0064] ② Determination of starch degradation ability: The strain was spotted on a starch culture medium plate, placed in an incubator at 30°C for 48 hours, 500 μL of 1% Lugol's iodine solution was added to the plate, and strains with transparent circles around the colonies showed starch degradation ability. The diameter of the transparent circle (mm) and the diameter of the colony (mm) were measured, and the Hc value was calculated according to the following formula.

[0065] Hc value = transparent circle diameter / colony diameter

[0066] ③Determination of protein degradation ability: The strain was spotted on a skim milk powder medium plate and placed in an incubator at 30°C for 48 hours. The strain with a transparent circle around the colony showed protein degradation ability. The diameter of the transparent circle (mm) and the colony diameter (mm) were measured, and the Hc value was calculated according to the following formula.

[0067] Hc value = transparent circle diameter / colony diameter

[0068] The results are shown in Table 1, indicating that there are 43 bacteria with the ability to degrade cellulose, from which strains with larger Hc values ​​were selected for subsequent experiments. Since S48, NJAU-190, G123, G112 and S95 are all Bacillus bacteria ( Bacillus sp.) (S48, NJAU-190, S95, G123 are Bacillus subtilis, G112 is Bacillus licheniformis), so S48, NJAU-190 and G112 were selected for subsequent experiments. In addition, G67 is a bacterium of the genus Paenibacillus ( Paenibacillus sp.), NJAU-273 is a bacterium of the genus Pseudomonas ( Pseudoxanthommonas sp.), all of which have strong cellulose degradation ability and do not belong to Bacillus, and were also selected for subsequent experiments. Finally, strains S48, NJAU-190, G67, G112 and NJAU-273 (cellulose-degrading bacteria) were selected for subsequent small-scale composting experiments.

[0069]

[0070]

[0071] (2) Small-scale composting test

[0072] Preparation of compost inoculant:

[0073] The strains (S48, NJAU-190, G67, G112 and NJAU-273) stored in -20℃ glycerol tubes were streaked on TSA medium plates for activation and placed in an incubator at 30℃ for 24h. A single colony was picked and placed in 3mL TSB medium. The culture was carried out at 30℃ and 170rpm for 10h to obtain seed solution. The seed solution was then transferred into 250mL TSB medium at an inoculation rate of 1% (seed solution v / TSB medium v). ​​The fermentation was carried out at 30℃ and 170rpm for 72h to obtain a fermentation broth with a bacterial content of ≥1×10 8 The fermentation liquid was directly used as the compost inoculation agent (cellulose degrading agent) for the small-scale composting test, or the fermentation liquid was centrifuged at 4°C to collect the bacteria, which were washed three times with sterile water and resuspended with an equal volume of sterile water and used as the compost inoculation agent (cellulose degrading agent) for the small-scale composting test. For the convenience of carrying, the latter operation was adopted in this embodiment.

[0074] The operation of the small system composting test is as follows:

[0075] Cow dung (fresh) and rice straw (chopped to 2-3 cm) are mixed in a dry weight ratio of 8.5:1.5 (w:w) to make C:N=25:1, and the moisture content is maintained at 65%-70% (65% in this embodiment). After all the compost raw materials are mixed, they are evenly divided into 6 portions, each of which has a dry weight of 7 kg. Five of them are inoculated with S48, NJAU-190, G67, G112 and NJAU-273 cellulose degrading bacteria, respectively, and the inoculation amount is 70 ml, which is 1% (v / w) of the dry weight of the pile raw materials. After fully mixing, each pile is evenly divided into three portions, each of which is placed in a 52×41×32cm 3In the foam box, no need to seal; another portion was inoculated with an equal volume of water as a control (CK) to keep the experimental conditions consistent. Finally, this experiment set up a total of 6 treatments, each with 3 replicates. Turn the compost every 3 days. Measure the temperature at the center of the foam box pile every day (3, 6, 9, 12, and 18 days are before turning the compost). Sampling was performed on the 0th, 1st, 3rd, 6th, 9th, 12th, and 18th days (sampling was performed after turning the compost on the 3rd, 6th, 9th, 12th, and 18th days. Sampling after turning the compost can reduce the error of sample result determination). Sampling was performed according to the five-point method. 40g of compost sample was taken at each of the 5 locations. After sampling, the compost samples taken from the 5 locations were turned and stirred evenly. Each compost sample was divided into two parts, one part was directly or stored in a -20℃ refrigerator for the determination of moisture content and germination index, and the other part was naturally air-dried and crushed through a 20-mesh sieve for the determination of pH, electrical conductivity (EC), organic matter, and total nutrients (total nitrogen, total phosphorus, and total potassium). The physical and chemical properties of the compost samples were tested according to the NY525-2012 standard.

[0076] The temperature change results of the pile body every day for each treatment are as follows Figure 1 As shown in the figure, the temperature of the piles of each treatment began to rise on the first day and reached above 60℃; starting from the second day, the temperature of the piles treated with the addition of cellulose degrading agents was significantly higher than that of the CK treatment, indicating that the addition of cellulose degrading agents can effectively increase the activity of microorganisms, thereby increasing the temperature of the piles. The temperature of the J1 treatment inoculated with the NJAU-190 cellulose degrading agent was significantly higher than that of the other treatments in the early stage of composting, and the highest temperature could reach 70℃. After the piles were turned over on the sixth day, the temperature of the piles rose again, among which the temperature rise rate of the J1 treatment was the fastest. The temperature of the J2 treatment inoculated with the NJAU-273 cellulose degrading agent was lower than that of the other treatments in the early stage of composting, but was significantly higher than that of the other treatments in the late stage of composting.

[0077] Then the germination index of the compost pile was measured at the end of the 18th day of composting for each treatment. Figure 2 As shown in the figure, the germination index of all treatments reached more than 70%, which met the standard of organic fertilizer germination index (more than 70%). In addition, the germination index of the treatments with cellulose-degrading agents was higher than that of the CK treatment. The treatments inoculated with NJAU-190, NJAU-273, S48 and G112 were significantly different from the CK treatment ( P <0.05).

[0078] Since the pile temperatures of the J1 and J2 treatments in the early and late stages of composting were higher than those of the other inoculated treatments, and the germination index of the piles of these two treatments was also significantly higher than that of the CK treatment, the present invention subsequently measured the pile pH, EC, organic matter, and total nutrients (total nitrogen, total phosphorus, and total potassium) of the two treatments and the CK treatment during the composting process.

[0079] Among them, the changes in pH value of the pile during composting are as follows: Figure 3 As shown, a higher pH value in the pile can prevent ammonia volatilization and thus reduce nitrogen loss. The pH value changes of each treatment as the composting progressed were basically the same, and the overall trend was an increase-decrease-increase. On the third day of composting, the pH values ​​of the piles of each treatment were: 7.79 for the CK treatment, 7.92 for the J1 treatment, and 8.05 for the J2 treatment. During the entire composting cycle, the inoculation of cellulose-degrading bacteria significantly increased the pH value of the pile relative to the CK treatment, and the pH value of the J2 treatment was significantly higher than that of the J1 treatment in the early stage of composting. At the end of composting, the pH values ​​of the J1 and J2 treatments were similar, both around 8.34, while the pH value of the CK treatment was 8.25, both less than 8.5, which met the national standard NY525-2012.

[0080] The changes of EC value of the pile during composting are as follows: Figure 4 As shown, the EC values ​​of the piles of each treatment showed an overall trend of first rising and then falling. In the first 12 days of composting, the EC values ​​of all treatments were on an upward trend, and the EC values ​​of the piles of each treatment reached a peak on the 12th day, with the J1 and J2 treatments being 3.37mS / cm and 3.31mS / cm, respectively, and the CK treatment being 3.4mS / cm. After that, the EC value showed a slow downward trend as a whole. On the 18th day, the EC values ​​of the piles of the J1 and J2 treatments were 3.22mS / cm and 3.15mS / cm, respectively, and the EC value of the piles of the CK treatment was 3.27mS / cm, all of which met the EC value standard of fertilizers (lower than 9.0mS / cm).

[0081] Changes in organic matter in the composting process Figure 5 As shown in the figure, the organic matter content of the piles in each treatment showed an overall downward trend. In the early stage of composting, the organic matter content changed greatly. In the later stage of composting, the trend gradually stabilized. Until the end of composting, there was no significant difference in the organic matter content of the piles in each treatment.

[0082] The changes in the total nitrogen, total phosphorus and total potassium contents of the piles of each treatment during composting are shown in Table 2. The total nutrients of the piles of each treatment increased. At the end of composting, the total nutrient contents of the piles of the CK, JI and J2 treatments were 4.4%, 4.6% and 4.5%, respectively; the total nitrogen content of the piles of the CK treatment was 1.5%, the total phosphorus content was 1.0%, and the total potassium content was 1.9%; the total nitrogen content of the piles of the J1 treatment was 1.7%, the total phosphorus content was 1.1%, and the total potassium content was 1.8%; the total nitrogen content of the piles of the J2 treatment was 1.6%, the total phosphorus content was 1.1%, and the total potassium content was 1.8%.

[0083]

[0084] Since the late stage of composting is the main period for humus synthesis, and this process is the key to improving the quality of compost, NJAU-273 was selected to continue the subsequent round pile composting and airflow membrane composting tests.

[0085] Example 2 Identification of functional bacteria

[0086] After strain NJAU-273 was cultured on TSA medium at 30°C for 24 h, Figure 6 As shown, the colony is round and cake-shaped, light yellow in color, the center of the colony is dark brown, and the outer edge of the colony is thicker; the edge of the colony is smooth and neat, the surface is moist, and it is easy to pick up from the culture medium.

[0087] The results of phylogenetic tree comparison analysis based on 16S rRNA gene sequences showed that ( Figure 7 ), strain NJAU-273 and Pseudoxanthomonas suwonensis In the same branch. Combined with the phylogenetic tree analysis results constructed by 16S rRNA gene sequence and colony morphology, strain NJAU-273 was identified as Pseudocanthomonas Pseudoxanthomonas sp. The phylogenetic tree results showed that the genus was harmless to crops and non-pathogenic to humans and animals.

[0088] The strain NJAU-273 has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit date being March 25, 2024 and the deposit number being CGMCC No.30114.

[0089] Example 3 Application of functional bacteria in round pile composting

[0090] Preparation of solid bacterial agent for compost inoculation:

[0091] The strain (NJAU-273) stored in a -20℃ glycerol tube was streaked on a TSA medium plate for activation, and then placed in an incubator at 30℃ for 24h. A single colony was picked and placed in 100mL TSB medium, and cultured at 30℃ and 170rpm for 24h to obtain a first-level seed solution. The first-level seed solution was then transferred to 2L TSB medium at an inoculation rate of 5% (first-level seed solution v / TSB medium v), and cultured at 30℃ and 170rpm for 72h to obtain a second-level seed solution. Subsequently, 2L of the second-level seed solution was transferred to 70L TSB medium, and cultured at 30℃ and 210rpm for 72h to obtain a fermentation broth with a bacterial content of 1-3×10 9 / ml, the fermentation liquid was mixed with rice bran (the moisture content of rice bran was not higher than 10wt%) at a ratio of 1:3 (v (mL): w (g)) (rice bran adsorbed fermentation liquid) to obtain compost inoculation solid bacterial agent (NJAU-273 bacterial agent).

[0092] The round pile composting test was carried out as follows:

[0093] The experiment was conducted at Nantong Huinong Bio-Organic Fertilizer Co., Ltd. 300kg wet weight of rice straw (chopped to 2-3cm) and 1.53kg urea were mixed to make C:N=27.5:1, and the moisture content was maintained at 55%-65% (60% in this embodiment). Two treatments were set up in this experiment: rice straw urea, rice straw urea + NJAU-273, marked as LCK and L2 respectively. Among them, the L2 treatment was to inoculate the pile with NJAU-273 inoculant, and the amount of NJAU-273 inoculant added was 4% (w / w) of the dry weight of rice straw, that is, 4.8kg; the LCK treatment was to not inoculate the pile with the inoculant, but add the same amount of rice bran, and the other experimental conditions of the two treatments remained consistent. After mixing rice straw, urea and NJAU-273 bacterial agent using a composting machine, pile them into a round pile, cover them with a plastic film with a thickness of 0.12mm, and press them tightly with sandbags on all sides to prevent air leakage. Each round pile weighs about 300kg, with a diameter of about 2.4m and a height of about 1.2m. Turn the pile every 3 days. Measure the temperature at the same time every day to determine the center point position (horizontally, 1.2m), and use three sets of plug-in digital display thermometers to measure the temperature of the upper layer: 10-30cm, the middle layer: 40-60cm, and the lower layer: 70-90cm (vertically) at this point (3, 6, 9, 12, 18, 24, and 30 days are the temperature measurements before turning the pile). Sampling was conducted at 0, 1, 3, 6, 9, 12, 18, 24, and 30 days (sampling was conducted after turning the pile at 3, 6, 9, 12, 18, 24, and 30 days. Sampling after turning the pile can reduce the error of sample result determination). Random sampling was conducted in the upper layer of the pile: 10-30 cm (a), the middle layer: 40-60 cm (b), and the lower layer: 70-90 cm (c) (vertical direction). Three samples were collected from each layer (numbered a1, a2, a3, b1, b2, b3, c1, c2, and c3, 500 g each). After sampling, the samples of a1, b1, and c1 were mixed, the samples of a2, b2, and c2 were mixed, and the samples of a3, b3, and c3 were mixed. They were turned over and stirred evenly. Three mixed samples were obtained for each treatment, i.e., three replicates. Each mixed sample was divided into two parts, one part was directly or stored in a -20℃ refrigerator for the determination of moisture content and germination index, and the other part was naturally air-dried and crushed through a 20-mesh sieve for the determination of pH, EC, and total nutrients (total nitrogen, total phosphorus, and total potassium). The physical and chemical properties of the compost samples were tested according to the NY525-2012 standard.

[0094] Effect of inoculation of NJAU-273 bacterial agent on the temperature of straw round composting Figure 8As shown, compared with the LCK treatment without adding bacterial agents, the temperature of the pile in the L2 treatment with the addition of NJAU-273 bacterial agent rose faster, reaching 58.7℃ on the 3rd day, which was much higher than that of the pile in the LCK treatment. This showed that the NJAU-273 bacterial agent improved the composting efficiency and made the pile more thoroughly decomposed.

[0095] Effect of inoculation with NJAU-273 bacterial agent on pH value of straw composting process Fig. 9 As shown in the figure, the pH values ​​of the two treatments have roughly the same trend. The pH value of the L2 treatment with NJAU-273 is higher than that of the LCK treatment without adding bacteria. The higher pH of the pile can prevent ammonia volatilization and reduce nitrogen loss. At the end of composting, the pH value of the L2 treatment with NJAU-273 was 8.37, and the pH value of the LCK treatment without adding bacteria was 8.1, both less than 8.5, which meets the national standard NY525-2012.

[0096] Effect of inoculation with NJAU-273 bacterial agent on EC value of straw round composting Fig.10 As shown in the figure, the EC values ​​of the two treatments showed a trend of first decreasing and then increasing. At the end of composting, the EC values ​​of the two treatments were ranked as follows: L2 (4.39 mS / cm) > LCK (4.38 mS / cm). The EC values ​​of the piles of each treatment met the standard of fertilizer EC value (less than 9.0 mS / cm).

[0097] Effect of inoculation with NJAU-273 microbial agent on the germination index of straw round composting Fig.11 As shown, the germination index of the L2 treatment with the addition of NJAU-273 inoculant was 121.75% at the end of composting, which was much higher than the germination index of the LCK treatment without the addition of inoculant (70.69%). The L2 treatment with the addition of NJAU-273 inoculant had a better decomposition effect.

[0098] Effect of inoculation with NJAU-273 bacterial agent on total nitrogen in straw round composting Fig.12 As shown in the figure, the total nitrogen content of the piles of each treatment showed a trend of first decreasing and then increasing. At the end of composting, the total nitrogen content of the piles of the L2 treatment with the addition of NJAU-273 inoculant was 1.60%, which was significantly higher than the total nitrogen content of the piles of the LCK treatment without the addition of inoculant (1.49%).

[0099] Effect of inoculation with NJAU-273 bacterial agent on total phosphorus in straw round composting Fig.13As shown in the figure, the total phosphorus content of the piles in each treatment showed an overall upward trend. At the end of composting, the total phosphorus content of the piles in each treatment increased compared with the total phosphorus content of the piles on the 0th day. On the 30th day of composting, the total nitrogen content of the piles in the LCK treatment without adding bacterial agents and the L2 treatment with adding NJAU-273 bacterial agents were 0.74% and 0.83%, respectively. The phosphorus content of the pile in the L2 treatment was significantly higher than that in the LCK treatment.

[0100] Effect of inoculation with NJAU-273 bacterial agent on total potassium in straw round composting Fig.14 As shown in the figure, consistent with the trend of total phosphorus, the total potassium content of the pile increased in each treatment at the end of composting. In addition, the total potassium content of the pile in the L2 treatment with the addition of NJAU-273 inoculant was 3.12%, and the total potassium content of the pile in the LCK treatment without the addition of inoculant was 2.99%. The potassium content of the pile in the L2 treatment was significantly higher than that in the LCK treatment.

[0101] Example 4 Application of functional bacteria in airflow membrane composting

[0102] Preparation of solid bacterial agent for compost inoculation: same as Example 3.

[0103] The airflow membrane composting test operation is as follows:

[0104] The experiment was conducted at Nantong Huinong Bio-Organic Fertilizer Co., Ltd. Cow dung (fresh) and rice straw (chopped to 2-3 cm) were mixed in a ratio of 8.5:1.5 (w:w) according to dry weight, so that C:N=25:1, and the moisture content was maintained at 65%-70% (65% in this embodiment). This experiment set up two treatments: cow dung rice straw, cow dung rice straw + NJAU-273, respectively marked as NCK and N2, among which, N2 treatment was inoculated with NJAU-273 inoculant, and the amount of NJAU-273 inoculant added was 8% (w / w) of the dry weight of cow dung + rice straw, that is, 70kg (2.5t pile); NCK treatment was not inoculated with inoculant, but added with an equal amount of rice bran, and the other experimental conditions of the two treatments remained consistent. After mixing cow dung, rice straw and NJAU-273 inoculant with a turner, transfer them to the airflow membrane fermentation site, cover them with e-PTFE polymer membrane with selective permeability, and press them with sandbags around to prevent leakage. Each airflow membrane strip weighs about 2.5t, is about 2.5m long, 1.5m wide and 1.2m high. According to the dry weight of the pile (0.3L / min / kg), the aeration frequency is set to 31hz. Measure the temperature at the same time every day, determine the center points of the three areas, namely 70cm, 140cm, and 210cm (horizontally), and use three sets of plug-in digital display thermometers to measure the temperature of the upper layer: 5-10cm, the middle layer: 50-60cm, and the lower layer: 100-120cm (vertically) at each point. Sampling is performed at 0, 1, 3, 6, 9, 12, 18, 24, and 30d respectively. When sampling, take one sample (numbered a1, a2, a3, b1, b2, b3, c1, c2, c3, 500g each) from the upper layer of the pile: 5-10cm (a), the middle layer: 50-60cm (b), the lower layer: 100-120cm (c) (vertically), and from each layer 70cm (1), 140cm (2), 210cm (3) (horizontally). Mix the samples of a1, b1, c1, mix the samples of a2, b2, c2, and mix the samples of a3, b3, c3, and stir them evenly. Each treatment will get 3 mixed samples, i.e. 3 replicates. Each mixed sample is divided into 2 parts, one of which is directly or stored in a -20℃ refrigerator for the determination of moisture content and germination index, and the other is naturally air-dried and crushed through a 20-mesh sieve for the determination of pH, EC, and total nutrients (total nitrogen, total phosphorus, and total potassium). The physical and chemical properties of compost samples were tested according to NY525-2012 standard.

[0105] Effect of inoculation of NJAU-273 bacterial agent on the temperature of straw composting during airflow membrane composting Fig.15 As shown, compared with the NCK treatment without adding bacterial agents, the temperature of the pile in the N2 treatment with the addition of NJAU-273 bacterial agent rose faster during the composting process, and the maximum temperature could reach 77.7℃, which was significantly higher than that of the NCK treatment.

[0106] Effect of inoculation with NJAU-273 bacterial agent on the pH of the pile during straw airflow membrane composting Fig.16 As shown, the pH change trends of the two treatments are roughly the same. At the end of composting, the pH of the N2 treatment with the addition of NJAU-273 bacterial agent and the pH of the NCK treatment without the addition of bacterial agent are close, both less than 8.5, which is in line with the national standard NY525-2012.

[0107] Effect of inoculation with NJAU-273 bacterial agent on EC value of straw pile during airflow membrane composting Fig.17 As shown in the figure, the EC values ​​of the two treatments showed a trend of first decreasing and then increasing. The EC value of the NCK treatment was 3.25 mS / cm, and the EC value of the N2 treatment was 3.19 mS / cm, both of which met the standard of fertilizer EC value (lower than 9.0 mS / cm).

[0108] Effect of inoculation with NJAU-273 bacterial agent on the germination index of straw pile during airflow membrane composting Fig.18 As shown, the germination index of the pile in the NCK treatment without adding microbial agents was 91.98% at the 30th day, and the germination index of the pile in the N2 treatment with adding NJAU-273 microbial agents was 127.40% at the 30th day, both of which have reached the decomposition requirements; compared with the NCK treatment without adding microbial agents, the N2 treatment with adding NJAU-273 microbial agents had a higher germination index and better decomposition effect.

[0109] Effect of inoculation with NJAU-273 bacterial agent on total nitrogen in straw composting process Fig.19 As shown in the figure, the total nitrogen content of the two treatments showed a trend of first decreasing and then increasing. At the end of composting, the total nitrogen content of the NCK treatment without adding bacterial agents and the N2 treatment with adding NJAU-273 bacterial agents were 1% and 1.03% respectively.

[0110] Effect of inoculation with NJAU-273 bacterial agent on total phosphorus in straw composting process Fig. 20 As shown, the total phosphorus content of the piles in each treatment showed an upward trend, and the total phosphorus content of the piles in each treatment on the 30th day was higher than that on the 0th day. At the end of composting, the total phosphorus content of the piles in the NCK treatment without adding bacterial agents and the N2 treatment with adding NJAU-273 bacterial agents were 0.64% and 0.72%, respectively, and the total phosphorus content of the piles in the N2 treatment was significantly higher than that in the NCK treatment.

[0111] Effect of inoculation with NJAU-273 bacterial agent on total potassium in the straw composting process Fig.21As shown in the figure, it is consistent with the changing trend of total phosphorus during the composting process. Until the end of composting, the total potassium content of the piles of each treatment increased. The total potassium content of the piles of the NCK treatment without adding bacterial agents and the N2 treatment with adding NJAU-273 bacterial agents on the 30th day was 1.89% and 1.91%, respectively. The total potassium content of the piles of the N2 treatment was significantly higher than that of the NCK treatment.

Claims

1. A strain of Pseudocanthomonas NJAU-273, which is classified as Pseudocanthomonas Pseudoxanthomonas sp., deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is March 25, 2024, and the deposit number is CGMCC No.30114.

2. The bacterial agent prepared by the Pseudocanthomonas sp. NJAU-273 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is prepared by the following steps: inoculating Pseudomonas aeruginosa NJAU-273 in a liquid culture medium for fermentation to obtain a fermentation liquid having a bacterial content of 1×10 9 The fermentation liquid and rice bran are mixed at a volume mass ratio of 1:2-4 to obtain the bacterial agent.

4. The bacterial agent according to claim 3, characterized in that The liquid culture medium includes TSB medium or LB liquid culture medium.

5. The bacterial agent according to claim 3, characterized in that The rice bran has a water content of no more than 10 wt %.

6. Use of the Pseudoxanthomonas sp. NJAU-273 according to claim 1 in straw composting, wherein the straw composting includes round pile composting and airflow membrane composting.

7. The use according to claim 6, characterized in that: The straw includes rice straw, and urea or livestock and poultry excrement is added to the straw during composting to provide a nitrogen source.

8. Use of the microbial agent according to any one of claims 2 to 5 in straw composting, wherein the straw composting includes round pile composting and airflow membrane composting.

9. The use according to claim 8, characterized in that: The straw includes rice straw, and urea or livestock and poultry excrement is added to the straw during composting to provide a nitrogen source.

Images