A composite bacterial agent and its application in straw composting

Abstract

The present invention discloses a composite bacterial agent, including Bacillus NJAU‑190 and Pseudoxanthomonas NJAU‑273. The present invention also discloses its application in straw composting. Bacillus NJAU‑190 helps to increase the temperature in the early stage of composting, and Pseudoxanthomonas NJAU‑273 helps to increase the temperature in the later stage of composting. Compounding the two strains can promote the temperature rise of the entire composting cycle, improve the composting efficiency and improve the composting conversion rate. Compared with the treatment without inoculation of bacterial agent, the treatment with inoculation of NJAU‑190+NJAU‑273 composite bacterial agent effectively promotes the increase of the temperature of the compost pile, accelerates the composting process, significantly improves the germination index of the compost product, and significantly increases the total nutrients of the compost product. Compared with the treatment with inoculation of NJAU‑190 bacterial agent or NJAU‑273 bacterial agent, the treatment with inoculation of NJAU‑190+NJAU‑273 composite bacterial agent also significantly 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 composite bacterial agent and application thereof in straw composting. Background Art

[0002] As China's agricultural production scale expands, the output of major crops is increasing, resulting in an increasing output of straw waste. According to statistics, my country's average annual straw output exceeds 900 million tons, accounting for 25% of the world's total straw output. Among them, the straw of crops such as rice, corn and wheat accounts for more than 70%, but the current utilization method of these straws is mainly to return them to the fields directly. Direct return to the fields will not only make it difficult to cultivate the next crop, but will also aggravate the occurrence of pests and diseases, and cause serious pollution to the agricultural ecological environment, hindering the sustainable development of my country's agriculture.

[0003] Compared with direct return to the field, composting straw is a more promising way to achieve its resource utilization. However, straw 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 straw composting, resulting in a slow composting process and low conversion efficiency. Composite microorganisms are communities composed of two or more microorganisms. Composite microbial agents have more advantages than single microbial agents, can adapt to complex environments, and have stronger and more complete effects. Therefore, the development of composite microbial agents adapted to the straw composting process is of great significance to promoting the resource utilization of straw and promoting the sustainable development of agriculture, and is the focus and hot spot of current research. Summary of the invention

[0004] The purpose of the present invention is to provide a composite bacterial agent and application thereof in straw composting to solve the deficiencies of the prior art.

[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 straw composting composite bacterial agent, including Bacillus NJAU-190 and Pseudomonas NJAU-273; the Bacillus NJAU-190 is classified and named as Bacillus Bacillus sp., deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee, the deposit date is March 25, 2024, and the deposit number is CGMCC No.30113; the pseudoxanthomonas NJAU-273 is classified and named as Pseudoxanthomonas Pseudoxanthomonassp., 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] Furthermore, the straw composting composite bacterial agent is prepared by the following steps:

[0008] Bacillus NJAU-190 was inoculated into a liquid culture medium for fermentation to obtain a Bacillus NJAU-190 fermentation liquid, wherein the Bacillus NJAU-190 fermentation liquid had a bacterial content of 1×10 9 More than 100 / ml;

[0009] The pseudoxanthomonas NJAU-273 was inoculated into a liquid culture medium for fermentation to obtain a pseudoxanthomonas NJAU-273 fermentation liquid, wherein the pseudoxanthomonas NJAU-273 fermentation liquid had a bacterial content of 1×10 9 More than 100 / ml;

[0010] The fermentation liquid of Bacillus sp. NJAU-190 and the fermentation liquid of Pseudomonas sp. NJAU-273 were mixed in equal volumes to obtain a mixed fermentation liquid;

[0011] The mixed fermentation liquid and rice bran are mixed in a volume-to-mass ratio of 1:2-4 to obtain the straw composting composite bacterial agent.

[0012] Furthermore, the liquid culture medium includes TSB medium or LB liquid culture medium.

[0013] Furthermore, the rice bran has a water content of no more than 10 wt %.

[0014] The application of the above straw composting composite bacterial agent in straw composting.

[0015] 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.

[0016] Beneficial effects of the present invention:

[0017] The present invention constructs a straw composting composite bacterial agent, including Bacillus NJAU-190 and Pseudoxanthomonas NJAU-273. Bacillus NJAU-190 and Pseudoxanthomonas NJAU-273 are both cellulose-degrading strains screened from fertilizer samples in the high-temperature period of rice straw composting. Bacillus NJAU-190 helps to increase the temperature in the early stage of composting, and Pseudoxanthomonas NJAU-273 helps to increase the temperature in the later stage of composting. Compounding the two strains can promote the temperature rise of the entire composting cycle, improve the composting efficiency and improve the composting conversion rate. Compared with the treatment without inoculation of bacterial agent, the treatment with inoculation of NJAU-190+NJAU-273 composite bacterial agent effectively promotes the increase of the temperature of the compost pile, accelerates the composting process, significantly improves the germination index of the compost product, and significantly increases the total nutrients of the compost product. Compared with the treatments inoculated with NJAU-190 or NJAU-273, the treatment inoculated with NJAU-190+NJAU-273 composite bacteria also significantly increased the total nutrients of the compost products.

[0018] In the round-pile composting experiment, the temperature of the piles of each treatment began to rise from the 3rd day. The temperature of the pile inoculated with NJAU-190+NJAU-273 compound bacteria was significantly higher than that of the LCK treatment, indicating that the addition of NJAU-190+NJAU-273 compound bacteria could significantly increase the temperature of the pile and make the pile enter the high temperature period ahead of time. By the end of the 30th day of composting, the germination index of the pile inoculated with NJAU-190+NJAU-273 compound bacteria was 87.66%, significantly higher than the 70.69% of the LCK treatment. Until the end of composting, the total nitrogen, phosphorus and potassium nutrients of the piles of LCK, L1, L2 and L3 treatments were 5.2%, 5.9%, 5.6% and 6.2%, respectively. The L3 treatment with NJAU-190+NJAU-273 compound bacteria had the best effect.

[0019] In the airflow membrane composting experiment, the temperature of each treatment pile began to rise on the first day. Except for the NCK treatment, the temperature of the piles of the other treatments, that is, the treatments with the addition of cellulose degrading agents, reached above 60°C. The temperature of the pile of the N3 treatment with the addition of NJAU-190+NJAU-273 compound bacteria rose the fastest, reaching about 70°C. On the third day of composting, the temperature of the pile of the N3 treatment with the addition of NJAU-190+NJAU-273 compound bacteria was significantly higher than that of the NCK treatment without the addition of bacteria, indicating that the addition of NJAU-190+NJAU-273 compound bacteria can make the pile enter the high temperature period in advance, making the compost raw materials The compost matured faster. At the end of composting, the temperature of the pile in N3 treated with NJAU-190+NJAU-273 compound bacteria was significantly higher than that in other treatments. At the end of composting on the 30th day, the germination index of the pile in N3 treated with NJAU-190+NJAU-273 compound bacteria was 121.12%, significantly higher than the 91.98% of the LCK treatment. Until the end of composting, the total nitrogen, phosphorus and potassium nutrients in the piles treated with NCK, N1, N2 and N3 were 3.5%, 3.8%, 3.7% and 3.9%, respectively. The N3 treatment with NJAU-190+NJAU-273 compound bacteria had the best effect.

[0020] The straw composting composite bacterial agent constructed by 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, and significantly increase the nutrients of compost products, providing technical support for straw composting and having a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] 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.

[0023] Figure 3The 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.

[0024] 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.

[0025] 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.

[0026] Figure 6 This is a colony photo of strain NJAU-273.

[0027] Figure 7 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of strain NJAU-273.

[0028] Figure 8 This is a colony photo of strain NJAU-190.

[0029] Fig. 9 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of strain NJAU-190.

[0030] Fig.10 The effect of different treatments on the temperature of straw pile during round composting.

[0031] Fig.11 This study shows the effects of different treatments on the pH of the straw pile during round composting.

[0032] Fig.12 Effects of different treatments on the electrical conductivity (EC) of straw pile during round composting.

[0033] Fig.13 This study shows the effects of different treatments on the germination index of straw piles during round composting.

[0034] Fig.14 This is the effect of different treatments on the total nitrogen content in the straw round composting process.

[0035] Fig.15 This is the effect of different treatments on the total phosphorus content in the straw round composting process.

[0036] Fig.16This is the effect of different treatments on the total potassium content in the straw round composting process.

[0037] Fig.17 The effect of different treatments on the temperature of the straw pile during airflow membrane composting.

[0038] Fig.18 Effects of different treatments on the pH of the straw compost during airflow membrane composting.

[0039] Fig.19 Effects of different treatments on the electrical conductivity (EC) of straw during airflow membrane composting.

[0040] Fig. 20 The effect of different treatments on the germination index of the pile during straw airflow membrane composting.

[0041] Fig.21 Effects of different treatments on the total nitrogen content in the straw composting process.

[0042] Fig. 22 Effects of different treatments on the total phosphorus content in the straw composting process.

[0043] Fig.23 Effects of different treatments on the total potassium content in the straw composting process.

[0044] Different letters above the bars indicate significant (P < 0.05) differences as defined by one-way ANOVA.

[0045] Biomaterial Deposit Information

[0046] 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.

[0047] strain NJAU-190, classified as Bacillus Bacillus 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.30113. DETAILED DESCRIPTION

[0048] 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.

[0049] The culture medium formulas involved in the following examples are as follows:

[0050] 1 / 10TSA medium (1L): 1.5g tryptone, 0.5g soytone, 0.5g sodium chloride, 15g agar powder.

[0051] TSA medium (1L): 15g tryptone, 5g soytone, 5g sodium chloride, 15g agar powder.

[0052] TSB medium (1L): 15 g tryptone, 5 g soytone, 5 g sodium chloride.

[0053] CMC medium (1L): 10g sodium carboxymethyl cellulose, 10g peptone, 5g yeast powder, 5g sodium chloride, 1g potassium dihydrogen phosphate, and 20g agar powder.

[0054] Starch culture medium (1L): 5g beef extract, 10g peptone, 5g sodium chloride, 2g soluble starch, and 20g agar powder.

[0055] Skim milk powder culture medium (1L): 10g skim milk powder, 10g peptone, 5g yeast powder, 5g sodium chloride, 20g agar powder.

[0056] Example 1 Isolation and screening of functional bacteria

[0057] 1. Prepare gradient dilution of fertilizer samples

[0058] 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.

[0059] 2. Plate coating and culture

[0060] 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.

[0061] 3. Separation, purification and preservation

[0062] 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.

[0063] 4. Screening of efficient cellulose-degrading bacteria

[0064] The degradation ability of the culturable bacteria isolated above on cellulose, starch and protein was determined, and a small-scale composting test was conducted to screen out two strains NJAU-273 and NJAU-190 with strong cellulose degradation ability. The specific operation is as follows:

[0065] (1) Determination of the ability of culturable bacteria to degrade cellulose, starch and protein

[0066] 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.

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

[0068] 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.

[0069] Hc value = transparent circle diameter / colony diameter

[0070] 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.

[0071] Hc value = transparent circle diameter / colony diameter

[0072] 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.

[0073]

[0074]

[0075] (2) Small-scale composting test

[0076] Preparation of compost inoculant:

[0077] 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 8The 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.

[0078] The operation of the small system composting test is as follows:

[0079] 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 3 In the foam box, no need to seal; another portion of water with the same volume was inoculated 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. Take the temperature at the center of the foam box pile every day (3, 6, 9, 12, and 18 days are before turning the compost). Take samples at 0, 1, 3, 6, 9, 12, and 18 days (take samples after turning the compost on 3, 6, 9, 12, and 18 days. Sampling after turning the compost can reduce the error of sample result determination). Sampling is carried out according to the five-point method. Take 40g of compost sample at each of the 5 locations. After sampling, stir the compost samples taken from the 5 locations 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.

[0080] The temperature change results of the pile body every day for each treatment are as follows Figure 1As 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.

[0081] 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).

[0082] 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.

[0083] 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.

[0084] The changes of EC value of the pile during composting are as follows: Figure 4As 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).

[0085] 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.

[0086] 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%.

[0087]

[0088] Example 2 Identification of functional bacteria

[0089] 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.

[0090] 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.

[0091] 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.

[0092] After strain NJAU-190 was cultured on TSA medium at 30°C for 24 h, Figure 8 As shown, the colony is round or irregular in shape, white in color, smooth and without wrinkles; the colony can secrete mucus similar to sugars, is tightly connected to the culture medium, and is easier to pick up.

[0093] The results of phylogenetic tree comparison analysis based on 16S rRNA gene sequences showed that ( Fig. 9 ), strain NJAU-190 and Bacillus subtilis In the same branch. Combining the phylogenetic tree analysis results constructed by 16S rRNA gene sequence and colony morphology, strain NJAU-190 was identified as Bacillus Bacillus sp. The phylogenetic tree results showed that the genus was harmless to crops and non-pathogenic to humans and animals.

[0094] The strain NJAU-190 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.30113.

[0095] Example 3 Application of composite bacteria in round composting

[0096] Preparation of solid bacterial agent for compost inoculation:

[0097] The strains (NJAU-273 and NJAU-190) 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 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 2LTSB 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 70LTSB 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 agents (NJAU-273 bacterial agent, NJAU-190 bacterial agent, NJAU-190 + NJAU-273 composite bacterial agent). Among them, the NJAU-190 + NJAU-273 composite bacterial agent was obtained by mixing equal volumes of NJAU-273 fermentation liquid and NJAU-190 fermentation liquid and then mixing them with rice bran.

[0098] The round pile composting test was carried out as follows:

[0099] 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). This experiment set up 4 treatments: rice straw urea, rice straw urea + NJAU-190, rice straw urea + NJAU-273, rice straw urea + NJAU-190 + NJAU-273, marked as LCK, L1, L2, L3 respectively, among which, L1 treatment was NJAU-190 inoculation of the pile, and the amount of NJAU-190 added was 4% (w / w) of the dry weight of rice straw, i.e. 4.8kg; L2 treatment was N JAU-273 inoculant, NJAU-273 inoculant addition amount is 4% (w / w) of the dry weight of rice straw, i.e. 4.8kg; L3 treatment is the pile inoculated with NJAU-190+NJAU-273 compound inoculant, NJAU-190+NJAU-273 compound inoculant addition amount is 4% (w / w) of the dry weight of rice straw, i.e. 4.8kg; LCK treatment is the pile without inoculation of inoculant, but adding equal amount of rice bran, other experimental conditions of the four treatments are kept the same. Rice straw, urea and inoculant are mixed by a turning machine, piled into a round pile, covered with a plastic film with a thickness of 0.12mm, and compacted with sandbags around to prevent 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 3d. Measure the temperature at the same time period every day, determine the center point (horizontally, 1.2m), and use three sets of plug-in digital 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 temperatures before turning the pile). Take samples on the 0th, 1st, 3rd, 6, 9, 12, 18, 24th, and 30th days (take samples after turning the pile on the 3rd, 6, 9, 12, 18, 24th, and 30th days, which can reduce the error of sample results). During sampling, random sampling was performed in the upper layer of the pile: 10-30cm (a); the middle layer: 40-60cm (b); the lower layer: 70-90cm (c) (vertical direction), and 3 samples were collected from each layer (numbered a1, a2, a3, b1, b2, b3, c1, c2, c3, 500g 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, and they were stirred evenly. Three mixed samples were obtained for each treatment, that is, 3 replicates. Each mixed sample was divided into 2 parts, one of which was directly or stored in a -20℃ refrigerator for the determination of moisture content and germination index, and the other 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.

[0100] Effects of various treatments on the temperature of the straw composting process Fig.10 As shown, the temperature of the piles in each treatment began to rise from the 3rd day, and the temperatures of the piles in the L1, L2, and L3 treatments were significantly higher than those in the LCK treatment, indicating that the addition of cellulose-degrading bacterial agents could significantly increase the temperature of the piles, causing the piles to enter the high-temperature period ahead of time.

[0101] Effects of various treatments on pH value of straw composting Fig.11 As shown in the figure, during the composting period, the pH value of the piles of each treatment showed an increase-decrease-increase trend. The pH value of the piles of each treatment with the addition of cellulose-degrading bacteria was higher than that of the LCK treatment without the addition of bacteria. The higher pH of the piles can prevent ammonia volatilization and reduce nitrogen loss. At the end of composting, the pH value of the piles of each treatment was less than 8.5, which met the national standard NY525-2012.

[0102] Effects of various treatments on the EC value of the straw round composting process Fig.12 As shown in the figure, the EC values ​​of the piles of each treatment mainly showed a trend of first decreasing and then increasing. At the end of composting, the EC values ​​of the piles of each treatment were ranked as follows: L1 (4.78 mS / cm), L3 (4.78 mS / cm) > L2 (4.39 mS / cm) > LCK (4.38 mS / cm). The EC values ​​of each treatment met the standard of fertilizer EC value (less than 9.0 mS / cm).

[0103] Effects of various treatments on the germination index of the straw round composting process Fig.13 As shown in the figure, the germination index of the piles of each treatment was always on an upward trend during the composting period. At the end of composting, the germination index of the piles of L1, L2, and L3 treatments were 85.73%, 121.75%, and 87.66%, respectively, which were significantly higher than that of the LCK treatment (70.69%).

[0104] Effects of various treatments on total nitrogen in straw round composting Fig.14 As shown in the figure, the total nitrogen content of the piles in each treatment showed a trend of first decreasing and then increasing. At the end of composting, the total nitrogen content of the piles in the L1, L2, and L3 treatments was 1.56%, 1.60%, and 1.69%, respectively, which were significantly higher than the total nitrogen content of the pile in the LCK treatment (1.49%). Among them, the total nitrogen content of the pile in the L3 treatment with the addition of the NJAU-190+NJAU-273 compound bacterial agent was the highest.

[0105] Effects of various treatments on total phosphorus during straw round composting Fig.15As 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, L1, L2, and L3 treatments was 0.74%, 0.82%, 0.83%, and 0.97%, respectively. The total phosphorus content of the pile in the L3 treatment with the addition of the NJAU-190+NJAU-273 compound bacterial agent was the highest, significantly higher than that of other treatments.

[0106] Effects of various treatments on total potassium during straw round composting Fig.16 As shown in the figure, consistent with the trend of total phosphorus, the total potassium content of each treatment increased at the end of composting. The total potassium content of the piles in the L1, L2, and L3 treatments was 3.48%, 3.12%, and 3.51%, respectively, which were significantly higher than the total nitrogen content of the pile in the LCK treatment (2.99%). Among them, the total nitrogen content of the pile in the L3 treatment with the addition of NJAU-190+NJAU-273 composite bacteria was the highest.

[0107] By the end of composting, the total nitrogen, phosphorus and potassium nutrients in the piles of LCK, L1, L2 and L3 treatments were 5.2%, 5.9%, 5.6% and 6.2% respectively. The L3 treatment with the addition of NJAU-190+NJAU-273 compound bacteria had the best effect.

[0108] Example 4 Application of composite bacteria in airflow membrane composting

[0109] Preparation of solid bacterial agent for compost inoculation: same as Example 3.

[0110] The airflow membrane composting test operation is as follows:

[0111] 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 at 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 a total of 4 treatments: cow dung rice straw, cow dung rice straw + NJAU-190, cow dung rice straw + NJAU-273, cow dung rice straw + NJAU-190 + NJAU-273, marked as NCK, N1, N2, N3, respectively. Among them, the N1 treatment was the pile inoculated with NJAU-190 bacterial agent, and the amount of NJAU-190 bacterial agent added was 8% (w / w) of the dry weight of cow dung + rice straw, that is, 70kg (2.5t pile); the N2 treatment was the pile inoculated with NJAU-2 73 bacteria agent, NJAU-273 bacteria agent addition amount is 8% (w / w) of the dry weight of cow dung + rice straw, that is, 70kg (2.5t pile); N3 treatment is the pile inoculated with NJAU-190 + NJAU-273 compound bacteria agent, NJAU-190 + NJAU-273 compound bacteria agent addition amount is 8% (w / w) of the dry weight of cow dung + rice straw, that is, 70kg (2.5t pile); NCK treatment is the pile without inoculation of bacteria agent, but adding the same amount of rice bran, the other experimental conditions of the four treatments are consistent. Cow dung, rice straw and bacteria agent were mixed by a turner, transferred to the airflow membrane fermentation site, covered with e-PTFE polymer membrane with selective permeability, and compacted with sandbags on all sides to prevent leakage. Each airflow membrane strip pile weighs about 2.5t, is about 2.5m long, about 1.5m wide and about 1.2m high. According to the dry weight of the pile (0.3L / min / kg), the aeration frequency was set to 31hz. The temperature was measured at the same time every day, and the three central points of the area, namely 70cm, 140cm, and 210cm (horizontally), were determined. At each point, three sets of plug-in digital display thermometers were used to measure the temperature of the upper layer: 5-10cm, the middle layer: 50-60cm, and the lower layer: 100-120cm (vertically). Samples were taken at 0, 1, 3, 6, 9, 12, 18, 24, and 30 days respectively. When sampling, take one sample (numbered a1, a2, a3, b1, b2, b3, c1, c2, c3, 500g each) from the upper layer: 5-10cm (a), middle layer: 50-60cm (b), lower layer: 100-120cm (c) (vertically), and from each layer: 70cm (1), 140cm (2), 210cm (3) (horizontally). Mix the a1, b1, c1 samples, mix the a2, b2, c2 samples, mix the a3, b3, c3 samples, and stir them evenly. Get 3 mixed samples for each treatment, i.e. 3 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.

[0112] Effects of various treatments on the temperature of the straw composting process Fig.17 As shown in the figure, the temperature of each treatment pile showed a trend of first rising and then falling, and all began to rise on the first day. Except for the NCK treatment, the temperature of the piles of other treatments, that is, the treatments with the addition of cellulose degrading agents, reached above 60°C. The temperature of the pile of the N3 treatment with the addition of NJAU-190+NJAU-273 composite bacteria rose the fastest, reaching about 70°C. On the third day of composting, the temperature of the piles of each treatment with the addition of cellulose degrading agents was significantly higher than that of the NCK treatment without the addition of bacteria. This shows that the addition of cellulose degrading agents can make the pile enter the high temperature period in advance, making the compost raw materials mature faster. At the end of composting, the temperature of the pile of the N3 treatment with the addition of NJAU-190+NJAU-273 composite bacteria was significantly higher than that of other treatments.

[0113] Effects of various treatments on pH value of straw composting during airflow membrane composting Fig.18 As shown in the figure, the pH value of the piles in each treatment showed a trend of first rising, then falling, and then rising again. The pH value changed greatly in the early stage of composting, and gradually stabilized as the pile matured. The pH value of the piles in each treatment with the addition of cellulose degrading agents was higher than that of the LCK treatment without the addition of agents. The higher pH of the piles can prevent ammonia volatilization and reduce nitrogen loss. At the end of composting, the pH value of the piles in each treatment was less than 8.5, which met the national standard NY525-2012.

[0114] Effects of various treatments on EC value of straw composting during airflow membrane composting Fig.19 As shown in the figure, the EC values ​​of the piles of each treatment showed a trend of first decreasing and then increasing. At the end of composting, the EC values ​​of the piles of each treatment met the standard of fertilizer EC value (lower than 9.0 mS / cm). The EC value of the pile of N3 treatment was the highest, reaching 3.55 mS / cm, and the EC value of the pile of N2 was the lowest, at 3.19 mS / cm.

[0115] Effects of various treatments on the germination index of the pile during straw airflow membrane composting Fig. 20As shown in the figure, during the composting process, the germination index of the piles of each treatment showed an upward trend. At the end of composting, the germination index of the piles of NCK, N1, N2, and N3 treatments were 91.98%, 99.17%, 127.40%, and 121.12%, respectively. The N1, N2, and N3 treatments with the addition of cellulose-degrading agents were all higher than the NCK treatment without the addition of agents, and the difference between the N2, N3 treatments and the NCK treatment reached a significant level.

[0116] Effects of various treatments on total nitrogen in the straw composting process Fig.21 As shown in the figure, the total nitrogen content of the piles in each treatment showed a trend of first decreasing and then increasing. Until the end of composting, the total nitrogen content of the piles in the NCK, N1, N2, and N3 treatments was 1%, 1.12%, 1.03%, and 1.15%, respectively. The total nitrogen content of the piles in the treatments with the addition of cellulose degrading agents was higher than that in the NCK treatment. The total nitrogen content of the piles in the N3 treatment with the addition of NJAU-190+NJAU-273 composite bacteria was the highest, and the difference with the NCK treatment reached a significant level.

[0117] Effects of various treatments on total phosphorus in the straw composting process Fig. 22 As shown in the figure, 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 in the 0th day. At the end of composting, the total phosphorus content of the piles in the NCK, N1, N2, and N3 treatments was 0.64%, 0.71%, 0.72%, and 0.78%, respectively. The total phosphorus content of the piles in each treatment with the addition of cellulose degrading bacteria was higher than that in the NCK treatment, and the difference between the N2, N3 and NCK treatments reached a significant level. The total phosphorus content of the pile in the N3 treatment with the addition of NJAU-190+NJAU-273 composite bacteria was the highest.

[0118] Effects of various treatments on total potassium in the straw composting process Fig.23 As shown, 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 NCK, N1, N2, and N3 treatments on the 30th day was 1.89%, 1.96%, 1.91%, and 1.99%, respectively. The total potassium content of the piles of each treatment with the addition of cellulose degrading bacteria was higher than that of the NCK treatment, and the difference between N1, N3 and NCK treatments reached a significant level. The total potassium content of the pile in the N3 treatment with the addition of NJAU-190+NJAU-273 composite bacteria was the highest.

[0119] By the end of composting, the total nitrogen, phosphorus and potassium nutrients in the piles treated with NCK, N1, N2 and N3 were 3.5%, 3.8%, 3.7% and 3.9% respectively. The N3 treatment with the addition of NJAU-190+NJAU-273 compound bacteria agent had the best effect.

Claims

1. A straw composting composite bacterial agent, characterized in that: Including Bacillus NJAU-190 and Pseudomonas NJAU-273; the Bacillus NJAU-190 is classified and named as Bacillus Bacillus sp., deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee, with a deposit date of March 25, 2024 and a deposit number of CGMCC No.30113; the pseudoxanthomonas NJAU-273 is classified and named as Pseudoxanthomonas Pseudoxanthomonas sp., deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of March 25, 2024 and a deposit number of CGMCC No.30114; The straw composting composite bacterial agent is prepared by the following steps: Bacillus NJAU-190 was inoculated into a liquid culture medium for fermentation to obtain a Bacillus NJAU-190 fermentation liquid, wherein the Bacillus NJAU-190 fermentation liquid had a bacterial content of 1×10 9 More than 100 / ml; wherein the liquid culture medium includes TSB medium or LB liquid culture medium; The pseudoxanthomonas NJAU-273 was inoculated into a liquid culture medium for fermentation to obtain a pseudoxanthomonas NJAU-273 fermentation liquid, wherein the pseudoxanthomonas NJAU-273 fermentation liquid had a bacterial content of 1×10 9 More than 100 / ml; wherein the liquid culture medium includes TSB medium or LB liquid culture medium; The fermentation liquid of Bacillus sp. NJAU-190 and the fermentation liquid of Pseudomonas sp. NJAU-273 were mixed in equal volumes to obtain a mixed fermentation liquid; The mixed fermentation liquid and rice bran are mixed in a volume-to-mass ratio of 1:2-4 to obtain the straw compost composite bacterial agent; wherein the rice bran has a water content of no more than 10wt%.

2. Application of the straw composting composite bacterial agent according to claim 1 in straw composting, wherein the straw comprises rice straw, urea or livestock and poultry manure is added to the straw during composting to provide a nitrogen source, and the straw composting comprises round pile composting and airflow membrane composting.

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