Nitrogen-fixing and decay-promoting functional bacterium II471 as well as screening method and application thereof

Abstract

The invention discloses a nitrogen-fixing and decay-promoting functional bacterium II471 and a screening method and application thereof, and the screening method comprises the following steps: collecting a sample to obtain an initial sample; the ammonium nitrogen high-temperature domestication method comprises the following steps: domesticating an obtained initial sample to obtain mother liquor; screening a used culture medium, an MD culture medium and a DSM88 culture medium; carrying out strain screening, separation and purification, sucking mother liquor, carrying out gradient dilution, and culturing to obtain an initial series of functional strains; the nitrogen-fixing and decay-promoting functional bacteria II471 disclosed by the invention are applied to compost, the functional strains are re-screened by a rapid screening method to obtain strains, the strains are identified, then a microbial inoculum is prepared, and the nitrogen-fixing and decay-promoting functional bacteria II471 disclosed by the invention is applied to compost. According to the bacillus flavobacterium thermophilus obtained by screening, the nitrogen loss is reduced, the formation of humus is promoted, and a high-temperature domestication method and a method for rapidly screening a high-temperature ammoxidation functional strain can be used for more rapidly obtaining a target functional strain.

Description

Technical Field

[0001] The invention relates to the technical field of microbial agent preparation, and in particular to a nitrogen-fixing and decay-promoting functional bacterium II 471 and a screening method and application thereof. Background Art

[0002] The so-called kitchen waste refers to organic waste generated in residents' daily life and related activities such as food processing, catering services, and unit catering, including discarded vegetable leaves, leftovers, and leftovers. Its main sources are family kitchens, restaurants, hotels, canteens, markets, and other related industries. With the widespread implementation of garbage classification policies, the separation rate of kitchen waste is getting higher and higher, and its proportion in domestic garbage is increasing year by year. At present, kitchen waste in my country accounts for about 50% to 70% of domestic garbage. In 2004, it surpassed the United States to become the country with the largest amount of kitchen waste in the world. The components of kitchen waste are complex, and are often mainly composed of organic matter such as protein, fat, cellulose, and sugar. Among them, protein accounts for 13% to 27%, and organic components account for 80% to 95%. It is very easy to cause fermentation difficulties, serious nitrogen loss, and difficulty in humification during the composting process. At present, the treatment methods for kitchen waste mainly include incineration, landfill, feed, anaerobic fermentation, and composting. Among them, incineration and landfill technology are prone to cause secondary pollution such as dioxins and leachate, and the risk of environmental pollution is relatively high. Feed and anaerobic fermentation technology require high initial infrastructure and equipment investment, and the technical operation is difficult. Composting technology, as a rapid fermentation technology that integrates the harmlessness of kitchen waste, resource treatment and organic fertilizer production, has a low entry threshold, flexible application scenarios and broad prospects. However, this technology also has problems such as serious nitrogen loss and difficulty in decomposition. At present, the most widely used technologies to reduce nitrogen loss include material regulation, microbial flora function regulation, exogenous improvement material addition, membrane covering, etc., and in order to solve the problem of difficulty in decomposing the pile, the humus content in the pile is mostly improved by initial material regulation, exogenous bacterial agents and exogenous improvement material addition. In view of the serious nitrogen loss and difficulty in decomposition caused by the composting fermentation process, the above problems are mainly solved by technical means such as material regulation, bacterial agents and exogenous improvement material addition. However, the above technologies also have certain shortcomings. For example, through material control technology, that is, by controlling the pH, volatile solid matter content, carbon-nitrogen ratio, moisture content, etc. of the initial material, the nitrogen loss in the composting process can be slowed down. This has clear range requirements for the ratio of raw materials, that is, the pH and carbon-nitrogen ratio of the pile cannot be too low, and the volatile solid matter and moisture content cannot be too high. Otherwise, it will cause difficulties in pile fermentation and difficulty in normal temperature rise, and the composting will fail to mature. The addition of exogenous improvement materials, such as struvite, biochar, phosphogypsum, superphosphate, etc., although the effect is obvious, but the cost is high and it is difficult to apply in a factory. As for the technology of adding microbial agents, the microbial agents currently used generally have a single function and only have the function of nitrogen fixation or promoting decomposition, and the composting effect is not stable, which seriously restricts the resource utilization of kitchen waste during the composting fermentation process.

[0003] In view of the serious nitrogen loss and difficulty in composting of kitchen waste during composting, the present invention provides a nitrogen-fixing and compost-promoting functional bacterium Ⅱ471 and a screening method and application thereof, so as to realize the screening of efficient and stable bacterial agents with both nitrogen-fixing and compost-promoting functions. Summary of the invention

[0004] The purpose of the present invention is to provide a nitrogen-fixing and decay-promoting functional bacterium II 471 and a screening method and application thereof, so as to fill the gap in the current technology.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A nitrogen-fixing and decay-promoting functional bacterium II 471 and a screening method thereof, wherein the nitrogen-fixing and decay-promoting functional bacterium II 471 is a yellow thermophilic anaerobic bacillus II 471, the yellow thermophilic anaerobic bacillus II 471 is classified and named Anoxybacillus_flavithermus, and the preservation number is CGMCC NO.29861, and the yellow thermophilic anaerobic bacillus II 471 is preserved in the General Microbiological Center of China National Committee for the Preservation of Microorganisms and Bacteria at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing on February 4, 2024;

[0007] The screening method of the nitrogen-fixing and decay-promoting functional bacteria II 471 comprises the following steps:

[0008] (1) Sample collection: Select material samples during the composting process in the reactor, collect them multiple times in a row, and mix them to obtain the initial sample;

[0009] (2) Ammonium nitrogen high temperature acclimation method: The initial sample obtained in step (1) is divided into four parts to obtain a high temperature compost sample, which is transferred to a container and sterile phosphate buffered saline (PBS) and (NH 4 ) 2 SO 4 The solutions are mixed, and the mixed solution is cultured in the dark to obtain a mother solution;

[0010] (3) Screening medium:

[0011] a. MD medium (oligotrophic): nitrilotriacetic acid, NaCl, MgSO 4 7H 2 O, KH 2 PO 4 ,Tryptone,Yeast_extract,Na 2 MoO 4 ·2H 2 O,CuSO 4 , FeCl 3 , MnSO 4 ·H 2 O, ZnSO 4 7H 2 O,H 3 BO3 , CaSO 4 ·H 2 O,CoCl 2 6H 2 O, dissolving the above ingredients in deionized water, adjusting the pH value with NaOH solution, and sterilizing, wherein agar needs to be added to the solid culture medium;

[0012] b. DSM88 medium (nutrient-rich): (NH 4 ) 2 SO 4 , KH 2 PO 4 , MgSO 4 7H 2 O, CaCl 2 ,Tryptone,Yeast_extract,Na 2 MoO 4 ·2H 2 O,FeCl 3 , CuSO 4 , MnSO 4 ·H 2 O,H 3 BO 3 , ZnSO 4 7H 2 O, CoC l2 6H 2 O, dissolving the above ingredients in deionized water, adjusting the pH value with NaOH solution, and sterilizing, wherein agar needs to be added to the solid culture medium;

[0013] (4) Screening and separation and purification of bacterial strains: The mother solution obtained in step (2) is taken for gradient dilution, firstly spread on MD medium and culture, then single colonies are selected on DSM88 medium, streaked and cultured, and preserved to obtain an initial series of functional strains;

[0014] (5) Rapid screening method for functional strain rescreening: Pipette the sterilized liquid culture medium of the initial series of functional strains obtained in step (4) into a 96-well plate, pick up the above-mentioned preserved purified single bacterial liquid, and transfer them to the corresponding numbered 96-well plates in turn, seal the periphery of the well plate with a sealing film to prevent water loss during the culture process, culture the above-mentioned 96-well plate, and then pipette the Griess reagent into the cultured 96-well plate, and wait for the color change to be observed. When the color of the colony changes from light yellow to red, it means that the strain has the ability to transform nitrogen. According to the depth of color, the score is assigned and the strain II471 with the strongest nitrogen transformation function is determined;

[0015] (6) Strain identification:

[0016] a. Colony morphology: Use a sterilized inoculation loop to pick the strain obtained in step (5), streak it on DSM88 medium and LB plate, and culture it;

[0017] b. Molecular identification: Pick a single colony, then mix and stir in Triton_X-100, freeze the EP tube containing Triton_X-100 quickly and then boil it, take the supernatant as a template, add it according to the PCR reaction system of DNA template, Forward_Primer, Reverse_Primer, PCR_Premix, ddH2O, cycle through pre-denaturation, denaturation, annealing, and extension, amplify the target fragment, upload the obtained sequence to NCBI, and search and compare it with BLAST, select the rDNA sequence with high similarity to the sequence, and construct a phylogenetic tree with MEGA7.0. The results show that strain II471 has the highest homology with Anoxybacillus_flavithermus and belongs to the same branch;

[0018] (7) Preparation of bacterial agent: The strain II471 preserved on the slant was selected and placed in a container containing LB liquid culture medium for cultivation to obtain seed solution. The seed solution was inoculated into LB liquid culture medium and the culture was expanded in a shaker to obtain fermentation solution. The OD600 of the fermentation solution was measured by ultraviolet spectrophotometer, and the fermentation solution was diluted to make the final concentration of the bacterial agent 1×10^8 cfu / mL.

[0019] Preferably, in step (1), a high-temperature sample having a material temperature greater than or equal to 60° C. during the reactor composting process is selected.

[0020] Preferably, in step (2), (NH 4 ) 2 SO 4 The solution needs to be sterilized through a sterile filter, and the mixed solution needs to be placed in a shaker for light-proof culture. The shaker parameters are 180-200 rpm, 55-65° C., and the culture is acclimated for 14-28 days. After 14 days of culture, samples are taken for strain screening.

[0021] Further preferably, in step (2), (NH 4 ) 2 SO 4 The solution needs to be sterilized through a sterile filter, and the mixed solution needs to be placed in a shaker for light-proof culture. The shaker parameters are 180-190 rpm, 58-62°C, and the culture is acclimated for 14-24 days. After 14 days of culture, samples are taken for strain screening. More preferably, the shaker parameters are 180 rpm, 60°C.

[0022] Preferably, in step (3), the concentration of the NaOH solution in steps a and b is 4-7 mol / L, the pH of the solution is adjusted to 7.0-9.0, the sterilization treatment conditions are high pressure wet heat sterilization at 120-130° C. for 20-30 min, and the concentration of the agar is 2.5-2.8%.

[0023] Further preferably, in step (3), the concentration of the NaOH solution in steps a and b is 5-6 mol / L, the pH of the solution is adjusted to 7.5-8.5, the sterilization treatment conditions are high pressure wet heat sterilization at 120-125°C for 20-25 min, the concentration of the agar is 2.5-2.7%, more preferably, the concentration of the NaOH solution is 5 mol / L, the pH of the solution is adjusted to 8.0, the sterilization treatment conditions are high pressure wet heat sterilization at 121°C for 20 min, and the concentration of the agar is 2.5%.

[0024] Preferably, the culture conditions for coating using MD culture medium in step (4) are high temperature culture at 55 to 65° C. for 12 to 28 hours.

[0025] Further preferably, the culture condition for coating with MD culture medium in step (4) is high temperature culture at 58-63°C for 12-24h. More preferably, the culture condition for coating with MD culture medium is high temperature culture at 60°C.

[0026] Preferably, in step (5), the 96-well plate is placed at 55-65° C. for incubation, taken out after 18-32 hours, and the Griess reagent is pipetted into the incubated 96-well plate, and the color change is observed after waiting for 8-15 seconds.

[0027] Further preferably, in step (5), the 96-well plate is placed at 57-62°C for incubation, taken out after 20-28 hours, and the Griess reagent is pipetted into the incubated 96-well plate, waiting for 9-12 seconds, and observing the color change. More preferably, the 96-well plate is placed at 60°C for incubation, taken out after 24 hours, and the Griess reagent is pipetted into the incubated 96-well plate, waiting for 10 seconds, and observing the color change.

[0028] Preferably, in step (6) a, streaking is performed on DSM88 medium and LB plate, and cultured at 55-65° C. for 22-30 h.

[0029] More preferably, in step (6) a, streaking is performed on DSM88 medium and LB plate, and culture is performed at 57-63° C. for 22-28 h, more preferably, culture is performed at 60° C. for 24 h.

[0030] Preferably, the quick freezing temperature in step (6) b is -75 to -85°C, the quick freezing time is 2 to 5 minutes, and after quick freezing, the quick freezing is performed at 95 to 105°C for 2 to 4 minutes.

[0031] Further preferably, the quick freezing temperature in step (6) b is -77 to -82°C, the quick freezing time is 2 to 4 minutes, and after quick freezing, it is boiled at 98 to 103°C for 2 to 3 minutes. More preferably, the quick freezing temperature is -80°C, the quick freezing time is 3 minutes, and after quick freezing, it is boiled at 100°C for 2 minutes.

[0032] Preferably, in step (6) b, the target fragment is amplified according to the reaction conditions of pre-denaturation at 92-95°C for 3-6 min, denaturation at 92-95°C for 20-40 s, annealing at 54-58°C for 20-40 s, extension at 70-75°C for 80-100 s, 30-40 cycles, and 70-75°C for 8-15 min.

[0033] Further preferably, in the step (6) b, the target fragment is amplified according to the reaction conditions of pre-denaturation at 93-94°C for 3-4 min, denaturation at 93-94°C for 25-35 s, annealing at 55-57°C for 25-35 s, extension at 71-73°C for 85-95 s, 32-38 cycles, and 71-73°C for 8-12 min. More preferably, the target fragment is amplified according to the reaction conditions of pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 90 s, 35 cycles, and 72°C for 10 min.

[0034] Preferably, in step (7), the culture conditions for obtaining the seed solution are 55-65° C. and 170-200 rpm for 12-18 h, and the culture conditions for obtaining the fermentation solution are 57-67° C. and 170-200 rpm for 13-19 h.

[0035] Further preferably, in the step (7), the culture conditions for obtaining the seed solution are 56-61°C and 170-190rpm for 13-16h, and the culture conditions for obtaining the fermentation liquid are 57-63°C and 170-190rpm for 13-15h. More preferably, the culture conditions for obtaining the seed solution are 60°C and 180rpm for 14h, and the culture conditions for obtaining the fermentation liquid are 60°C and 180rpm for 14h.

[0036] Among them, the Anoxybacillus_flavithermus is the thermophilic Bacillus flavobacterium, the Triton_X-100 is polyethylene glycol octylphenyl ether, the Forward_Primer is the forward primer, the Reverse_Primer is the rear primer, the PCR_Premix is ​​the reaction premix, the Tryptone is trypsin, the Yeast_extract is yeast powder, the NCBI is the database, and the BLAST and MEGA7.0 are analysis software.

[0037] Due to the application of the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0038] 1. The strain Flavobacterium thermophilic Bacillus II 471 obtained by screening in the present invention is used for the first time as a functional bacterial agent in the composting fermentation process to reduce nitrogen loss and promote humus formation. The bacterial agent used in the present invention is added when the compost fermentation temperature reaches above 55°C, and the nitrogen fixation and composting promotion effect is better.

[0039] 2. The high temperature acclimation method and the method for rapid screening of high temperature ammonia oxidizing functional strains used in the present invention can more quickly obtain the target functional strains.

[0040] 3. The microbial agent of the present invention is simple to prepare, has the advantages of low cost, good application effect, and can be used without compounding, so it has wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, some of the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be made based on these drawings without paying any creative work.

[0042] Figure 1 This is a schematic diagram of the rapid screening method of Example 1 of the present invention;

[0043] Figure 2 This is a practical diagram of the rapid screening method according to Embodiment 1 of the present invention;

[0044] Figure 3 This is a colony morphology diagram of the thermophilic Bacillus II 471DSM88 culture medium of Example 1 of the present invention;

[0045] Figure 4 This is a colony morphology diagram of the thermophilic bacillus II 471 in LB culture medium of Example 1 of the present invention;

[0046] Figure 5This is the phylogenetic tree of the thermophilic Bacillus sp. strain of Example 1 of the present invention. DETAILED DESCRIPTION

[0047] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, a specific implementation scheme is now described in detail.

[0048] The present invention is further described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to the different requirements of specific use, and the implementation conditions not indicated are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0049] Example 1

[0050] A nitrogen-fixing and decay-promoting functional bacterium II 471 and a screening method thereof, comprising the following steps:

[0051] (1) Sample collection: 100 g of high-temperature samples with a material temperature of ≥60°C during the reactor composting process were selected, collected multiple times in succession, and mixed to obtain the initial sample;

[0052] (2) Ammonium nitrogen high temperature acclimation method: The initial sample obtained in step (1) was divided into four parts to obtain 25 g of high temperature compost sample, which was transferred to a 1 L conical flask, and 500 ml of sterile 1 mM phosphate buffered saline (PBS buffer) and 25 ml of 0.25 mM (NH 4 ) 2 SO 4 The solution must be sterilized by passing through a 0.22 μm sterile filter. After mixing the above samples, place them in a shaker for dark culture at 180 rpm and 60°C for 14 to 28 days. After 14 days of culture, samples are taken for strain screening.

[0053] (3) Screening medium:

[0054] a. MD medium (oligotrophic): nitrilotriacetic acid 100 mg / L, NaCl 8 mg / L, MgSO 4 7H 2 O100mg / L, KH 2 PO 4 111mg / L, Tryptone (tryptone) 2.00mg / L, Yeast_extract (yeast powder) 2.00mg / L, Na 2 MoO 4 ·2H 2 O10.3μL / L, CuSO 4100.2μL / L, FeCl 3 17.3μL / L, MnSO 4 ·H 2 O 13 μL / L, ZnSO 4 7H 2 O17.4μL / L, H 3 BO 3 80.9 μL / L, CaSO 4 ·H 2 O0.06mg, CoCl 2 6H 2 O19.3μL / L, dissolve the above ingredients in 1L deionized water, adjust the pH to 8.0 with 5mol / L NaOH solution, sterilize at 121℃ high pressure wet heat for 20min, and add 2.5% agar to the solid culture medium;

[0055] b. DSM88 medium (nutrient-rich): (NH 4 ) 2 SO 4 0.50g / L, KH 2 PO 4 0.28g / L, MgSO 4 7H 2 O0.25g / L, CaCl 2 0.01 g / L, Tryptone 2.00 g / L, Yeast_extract 2.00 g / L, Na 2 MoO 4 ·2H 2 O10.3μL / L, FeCl 3 17.3μL / L, CuSO 4 10μL / L, MnSO 4 ·H 2 O 13 μL / L, H 3 BO 3 80.9μL / L, ZnSO 4 7H 2 O17.4μL / L, CoCl 2 6H 2 O19.3μL / L, dissolve the above ingredients in 1L deionized water, adjust the pH to 8.0 with 5mol / L NaOH solution, sterilize at 121℃ high pressure wet heat for 20min, and add 2.5% agar to the solid culture medium;

[0056] (4) Screening and purification of bacterial strains: 100 μl of the acclimated mother solution obtained in step (2) was taken and diluted to 10 -3 , 10-4 and 10 -5 , take 100 μl of the dilution and spread it on MD medium, culture at 60℃ for 16-24h, pick colonies with different morphology from the cultured MD medium, separate and culture them by streaking on DSM88 medium, culture at 60℃ for 16-24h, and wait until they grow into obvious single colonies. Avoid overlapping colonies due to long culture time, and the initial series of functional strains can be obtained;

[0057] (5) Rapid screening method for functional strain rescreening: Use a pipette to draw 200 μl of the sterilized DSM88 liquid culture medium in step (4) into a 96-well plate, pick up 5 single colonies with a sterilized toothpick, and transfer them to the corresponding numbered 96-well plates in turn. Seal the periphery of the well plate with a sealing film to prevent water loss during the culture process. Place the well plate in a 60°C incubator for culture. Take it out after 24 hours, draw 100 μl of Griess reagent into the incubated 96-well plate, wait for about 10 seconds, and observe the color change (in order to avoid possible contamination during transfer and culture, set 3 blank wells, that is, wells without strains added as blank controls). When the colony color changes from light yellow to red, if attached Figure 1 With attached Figure 2 As shown, it means that the strain has nitrogen transformation ability;

[0058] (6) Strain identification:

[0059] a. Colony morphology: Use a sterilized inoculation loop to pick up a small amount of the above single strain, streak it on DSM88 medium and LB plate, and culture it at 60℃ for 24h. Figure 3 With attached Figure 4 As shown, the colony is off-white with a yellowish tint, round in shape, with a rough and slightly transparent edge, a smooth and translucent surface, and is easy to pick up;

[0060] b. Molecular identification: Pick a single colony with an autoclaved toothpick, then mix and stir in 20 μl 0.1% Triton_X-100, freeze the EP tube containing 20 μl 0.1% Triton_X-100 at -80°C for 3 min, and then boil at 100°C for 2 min, take 1 μl of the supernatant as a template, and prepare the PCR product according to the following steps: DNA template 1 μl, Forward_Primer (27F) 1 μl, Reverse_Primer (1492R) 1 μl, PCR_Premix 12.5 μl, ddH 2O9.5μl PCR reaction system was added, and the target fragment was amplified according to the reaction temperature of pre-denaturation at 94℃ for 4min, denaturation at 94℃ for 30s, annealing at 56℃ for 30s, extension at 72℃ for 90s, 35 cycles, and 72℃ for 10min. The obtained sequence was uploaded to NCBI and searched and compared with BLAST, and rDNA sequences with high similarity to the sequence were selected. The phylogenetic tree was constructed using MEGA7.0, as shown in the attached figure. Figure 5 As shown, the results showed that strain Ⅱ471 had the highest homology with Anoxybacillus_flavithermus and belonged to the same branch;

[0061] (7) Preparation of bacterial agent: A small amount of strain II471 preserved on a slant was picked up with an inoculation loop and placed in a test tube containing 5 mL of LB liquid medium. The culture was carried out at 60°C and 180 rpm for 14 h to obtain a seed solution. 5 mL of the seed solution was inoculated into 500 mL of LB liquid medium and the culture was expanded in a shaker at 60°C and 180 rpm for 14 h to obtain a fermentation solution. The OD600 of the fermentation solution was measured by a UV spectrophotometer and the solution was diluted with ddH 2 O dilute the fermentation broth to a certain concentration so that the final concentration of the bacterial agent is 1×10 8 cfu / mL.

[0062] Comparative Example 1

[0063] CK group: blank control group, without any bacterial agent, natural composting treatment.

[0064] Application of a nitrogen-fixing and decay-promoting functional bacterium Ⅱ471:

[0065] The microbial agent configured in the above embodiment is added at 5‰ of the fresh weight of the initial compost material during the high temperature period of composting, that is, when the temperature of the compost body reaches above 55°C, to clarify the effect of the microbial agent on nitrogen fixation and decomposition promotion in the process of food waste composting.

[0066] Compost raw material composition: kitchen waste and corn stalks are mixed and composted in a ratio of 3:1 to ensure an initial carbon-nitrogen ratio of 25 and a moisture content of 65%.

[0067] Addition of microbial agent: 5‰ of the wet weight of the initial mixed materials in the compost, added during the high temperature period of composting (≥55℃).

[0068] Production process: First, the above compost raw materials are mixed and the uniform materials are transferred to an 8L cylindrical compost reactor for closed fermentation. A temperature sensor is connected to monitor the temperature change in real time. When the temperature of the pile rises to ≥55°C, the diluted bacterial solution is evenly sprayed on the surface of the pile with a spray device (such as a spray pot or a spray gun), and the pile is fully stirred and continued to ferment. This test sets up a CK control group (Comparative Example 1) and a 5‰ inoculation group, which is the bacteria II471 inoculated in Example 1 of the present invention, and the inoculation amount is 5‰. Sampling was performed on days 0, 3, 7, 12, 18, and 28, and the test results are shown in Tables 1-3 below.

[0069] Table 1 Effects of adding inoculant II471 on changes in nitrogen transformation related indicators in the pile:

[0070]

[0071] Table 2 Effects of adding inoculant II471 on changes in humus-related indicators in the pile:

[0072]

[0073] Table 3 Carbon and nitrogen loss and carbon and nitrogen balance changes in the pile after adding inoculant II 471:

[0074]

[0075] From the results in the above table, it can be seen that adding bacterial agents during the high temperature period of kitchen waste composting can significantly reduce the loss of nitrogen during the composting process compared with the blank control, and the emission of NH3 and N2O is significantly reduced, while the conversion rate of ammonium nitrogen to nitrate nitrogen increases, and the organic nitrogen content and total nitrogen content increase, indicating that adding bacterial agents can significantly reduce nitrogen loss. In addition, adding bacterial agents can also promote compost humification, convert fulvic acid into humic acid, increase the humification rate, humification index and polymerization degree, and finally make the seed germination index of the compost exceed 100%, reaching a high level of harmlessness.

[0076] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A nitrogen-fixing and decay-promoting functional bacterium II471 and a screening method thereof, characterized in that: The nitrogen-fixing and decay-promoting functional bacteria II 471 is classified and named Anoxybacillus_flavithermus II 471, with a preservation number of CGMCC NO.29861, and is preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration; The screening method of the nitrogen-fixing and decay-promoting functional bacteria II 471 comprises the following steps: (1) Sample collection: Select material samples during the composting process in the reactor, collect them multiple times in a row, and mix them to obtain the initial sample; (2) Ammonium nitrogen high temperature acclimation method: The initial sample obtained in step (1) is divided into four parts to obtain a high temperature compost sample, which is transferred to a container, and sterile phosphate buffered saline solution and (NH4)2SO4 solution are added to mix, and the mixed solution is cultured in the dark to obtain a mother solution; (3) Screening medium: a. MD medium: nitrilotriacetic acid, NaCl, MgSO4·7H2O, KH2PO4, Tryptone, Yeast_extract, Na2MoO4·2H2O, CuSO4, FeCl3, MnSO4·H2O, ZnSO4·7H2O, H3BO3, CaSO4·H2O, CoCl2·6H2O. Dissolve the above ingredients in deionized water, adjust the pH value with NaOH solution, and sterilize. Agar needs to be added to the solid culture medium. b. DSM88 medium: (NH4)2SO4, KH2PO4, MgSO4·7H2O, CaCl2, Tryptone, Yeast_extract, Na2MoO4·2H2O, FeCl3, CuSO4, MnSO4·H2O, H3BO3, ZnSO4·7H2O, CoC l2 6H2O, dissolve the above ingredients in deionized water, adjust the pH value with NaOH solution, and sterilize. Agar needs to be added to the solid culture medium; (4) Screening and separation and purification of bacterial strains: The mother solution obtained in step (2) is taken for gradient dilution, firstly spread on MD medium and culture, then single colonies are selected on DSM88 medium, streaked and cultured, and preserved to obtain an initial series of functional strains; (5) Rapid screening method for functional strain rescreening: Pipette the sterilized liquid culture medium of the initial series of functional strains obtained in step (4) into a 96-well plate, pick up the above-mentioned preserved purified single bacterial liquid, and transfer them to the corresponding numbered 96-well plates in turn, seal the periphery of the well plate with a sealing film to prevent water loss during the culture process, culture the above-mentioned 96-well plate, and then pipette the Griess reagent into the cultured 96-well plate, and wait for the color change to be observed. When the color of the colony changes from light yellow to red, it means that the strain has the ability to transform nitrogen. According to the depth of color, the score is assigned and the strain II471 with the strongest nitrogen transformation function is determined; (6) Strain identification: a. Colony morphology: Use a sterilized inoculation loop to pick the strain obtained in step (5), streak it on DSM88 medium and LB plate, and culture it; b. Molecular identification: Pick a single colony, then mix and stir in Triton_X-100, freeze the EP tube containing Triton_X-100 quickly and then boil it, take the supernatant as a template, add it according to the PCR reaction system of DNA template, Forward_Primer, Reverse_Primer, PCR_Premix, ddH2O, cycle through pre-denaturation, denaturation, annealing, and extension, amplify the target fragment, upload the obtained sequence to NCBI, and search and compare it with BLAST, select the rDNA sequence with high similarity to the sequence, and construct a phylogenetic tree with MEGA7.

0. The results show that strain II471 has the highest homology with Anoxybacillus_flavithermus and belongs to the same branch; (7) Preparation of bacterial agent: The strain II471 preserved on the slant was selected and placed in a container containing LB liquid culture medium for cultivation to obtain seed solution. The seed solution was inoculated into LB liquid culture medium and the culture was expanded in a shaker to obtain fermentation solution. The OD600 of the fermentation solution was measured by ultraviolet spectrophotometer, and the fermentation solution was diluted to make the final concentration of the bacterial agent 1×10^8 cfu / mL.

2. The nitrogen-fixing and decay-promoting functional bacteria II471 and the screening method thereof according to claim 1, characterized in that: In the step (1), a high-temperature sample is selected in which the material temperature during the reactor composting process is greater than or equal to 60°C.

3. The nitrogen-fixing and decay-promoting functional bacteria II471 and the screening method thereof according to claim 1, characterized in that: The (NH4)2SO4 solution in step (2) needs to be sterilized through a sterile filter, and the mixed solution needs to be cultured in a shaker in the dark. The shaker parameters are 180-200 rpm, 55-65°C, and the culture is acclimated for 14-28 days. After 14 days of culture, samples are taken for strain screening.

4. The nitrogen-fixing and decay-promoting functional bacteria II471 and the screening method thereof according to claim 1, characterized in that: In step (3), the concentration of the NaOH solution in steps a and b is 4-7 mol / L, the pH of the solution is adjusted to 7.0-9.0, the sterilization treatment conditions are high pressure wet heat sterilization at 120-130° C. for 20-30 min, and the concentration of the agar is 2.5-2.8%.

5. The nitrogen-fixing and decay-promoting functional bacteria II471 and the screening method thereof according to claim 1, characterized in that: The culture conditions for coating using MD culture medium in step (4) are high temperature culture at 55 to 65° C. for 12 to 28 hours.

6. The nitrogen-fixing and decay-promoting functional bacteria II471 and the screening method thereof according to claim 1, characterized in that: In the step (5), the 96-well plate is placed at 55-65° C. for incubation, taken out after 18-32 hours, and the Griess reagent is pipetted into the incubated 96-well plate, and the color change is observed after waiting for 8-15 seconds.

7. The nitrogen-fixing and decay-promoting functional bacteria II471 and the screening method thereof according to claim 1, characterized in that: In step (6) a, streaking is performed on DSM88 medium and LB plate, and culture is performed at 55-65° C. for 22-30 hours.

8. The nitrogen-fixing and decay-promoting functional bacteria II471 and the screening method thereof as claimed in claim 1, characterized in that: The quick freezing temperature in step (6) b is -75 to -85°C, the quick freezing time is 2 to 5 minutes, and after quick freezing, the quick freezing is performed at 95 to 105°C for 2 to 4 minutes.

9. The nitrogen-fixing and decay-promoting functional bacteria II471 and the screening method thereof according to claim 1, characterized in that: In the step (6) b, the target fragment is amplified according to the reaction conditions of pre-denaturation at 92-95°C for 3-6 min, denaturation at 92-95°C for 20-40 s, annealing at 54-58°C for 20-40 s, extension at 70-75°C for 80-100 s, 30-40 cycles, and 70-75°C for 8-15 min.

10. The nitrogen-fixing and decay-promoting functional bacteria II471 and the screening method thereof according to claim 1, characterized in that: In the step (7), the culture conditions for obtaining the seed solution are 55-65° C. and 170-200 rpm for 12-18 hours, and the culture conditions for obtaining the fermentation solution are 57-67° C. and 170-200 rpm for 13-19 hours.

11. Use of the nitrogen-fixing and decay-promoting functional bacteria II471 as claimed in any one of claims 1 to 10 or the nitrogen-fixing and decay-promoting functional bacteria II471 obtained by the screening method of the nitrogen-fixing and decay-promoting functional bacteria II471 as claimed in any one of claims 1 to 10 in composting.

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