Heterotrophic nitrifying bacteria and bacterial inoculum for reducing nitrogen loss in chicken manure composting and application thereof

Abstract

The application discloses a heterotrophic nitrifying bacterium D31, which is classified as Geobacillus proteiniphilus and has a strain preservation number of CGMCC NO.27788. The strain D31 can perform heterotrophic nitrification on a culture medium containing ammonia nitrogen, can grow on a simple carbon and nitrogen source culture medium and oxidize ammonia. The strain D31 can utilize simple nutrients to rapidly proliferate, is inoculated into compost after aerobic fermentation, can efficiently and rapidly oxidize ammonia nitrogen in livestock and poultry manure into nitrate nitrogen in a high-temperature period, reduces the volatilization of nitrogen elements in the form of ammonia gas in the compost, can not only reduce odor emission and protect the environment, but also can maintain nitrogen nutritional elements in the compost, improves compost quality, and has a wide application prospect. The application further provides application of the heterotrophic nitrifying bacterium D31 in livestock and poultry manure composting.

Description

Technical Field

[0001] This invention belongs to the field of agricultural resources and environment, specifically to the field of solid waste resource utilization. It relates to a highly efficient heterotrophic nitrifying bacterium D31 and its inoculant, as well as its application in livestock and poultry manure composting. In the process of livestock and poultry manure composting, heterotrophic nitrifying bacteria D31 can oxidize ammonia into nitrate nitrogen, reduce ammonia emissions, and realize the resource utilization of agricultural waste. Background Technology

[0002] With the rapid growth of per capita consumption of livestock and poultry products in my country, the annual output of livestock and poultry manure reached 4 billion tons by the end of 2020. If not properly treated, this will cause serious environmental pollution. Resource utilization is one of the important means of sustainable agricultural development and a major method for large-scale digestion of livestock and poultry manure. Composting, in particular, can convert the nutrients in livestock and poultry manure into organic fertilizer, which can both alleviate pollution from livestock and poultry manure and improve soil fertility.

[0003] However, ammonia volatilization is a problem that cannot be ignored in the composting process of livestock and poultry manure. On the one hand, ammonia volatilization leads to a significant loss of nitrogen; on the other hand, ammonia is a major component of the odor emitted by compost. Ammonia volatilization not only degrades the quality of compost products but also contributes to air pollution and the greenhouse effect. Ammonia volatilization during composting is influenced by multiple factors, including the C / N ratio of the raw materials, pH, temperature, and the composting process itself. Methods to control ammonia volatilization mainly include adjusting the C / N ratio, inoculating with microorganisms, and adding chemical additives. Studies have found that a large amount of ammonia volatilization occurs during the high-temperature phase of composting. High temperatures and the high pH resulting from ammonium accumulation cause ammonium ions to convert into ammonia and volatilize. Some nitrifying bacteria can oxidize ammonia into nitrate ions, thereby reducing ammonia volatilization. However, ordinary nitrifying microorganisms cannot survive during the high-temperature phase; only thermophilic heterotrophic ammonia-oxidizing bacteria are effective during this period. Therefore, it is necessary to screen some highly efficient thermophilic heterotrophic nitrifying microorganisms, prepare them into inoculants, and add them to livestock and poultry manure composting. This will reduce the volatilization of ammonia during composting, improve compost quality, reduce environmental pollution, and achieve sustainable development of livestock and poultry farming. Summary of the Invention

[0004] The purpose of this invention is to provide a heterotrophic nitrifying bacterium (Geobacillus proteiniphilus) D31 that can oxidize ammonia into nitrate nitrogen, thereby reducing ammonia emissions. This reduces ammonia volatilization and ensures the smooth development of sustainable agriculture.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A highly efficient heterotrophic nitrifying bacterium, D31, classified as Geobacillus proteiniphilus, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 3, 2023, with accession number CGMCC NO.27788.

[0007] Heterotrophic nitrifying bacteria D31 was isolated from chicken manure high-temperature compost samples from Jintan District, Changzhou City, Jiangsu Province. Specifically, the extract of the chicken manure compost sample was inoculated into DSM88 liquid medium and cultured at high temperature (55℃) for enrichment. This enrichment process was repeated four times. Each time, the enriched solution was diluted and spread on DSM88 solid medium to obtain thermotolerant strains. Single colonies were picked, purified, and inoculated into DSM88 liquid medium for culture. The colorimetric reaction of Griess reagent with nitrite was used to initially screen out strains with nitrifying activity. The initially screened strains were inoculated into heterotrophic nitrification liquid medium and cultured. The total nitrate nitrogen and nitrite nitrogen in the bacterial solution were measured by the sulfanilamide colorimetric method. The strain with the strongest nitrifying ability was screened out. After identification, this strain was identified as *Geobacillus proteiniphilus* and named D31.

[0008] The biological characteristics of heterotrophic nitrifying bacteria D31 are as follows: when cultured on DSM88 medium plates at 55°C for 3 days, the colonies are white, round, smooth, and have neat edges.

[0009] Heterotrophic nitrifying bacteria D31 can grow in DSM88 liquid medium and convert ammonia in the medium into nitrite or nitrate.

[0010] The formulation of DSM88 liquid culture medium is as follows: (NH4)2SO4 1.30g, MgSO4·7H2O 0.25g, KH2PO4 0.28g, CaCl2 0.01g, peptone 2.00g, yeast extract 2.00g, FeCl3 0.28mg, Na2MoO4·2H2O 0.025mg, MnSO4·H2O 2.2mg, CuSO4 0.016mg, H3BO3 0.5mg, CoCl2·6H2O 0.046mg, ZnSO4·7H2O 0.5mg, and distilled water 1000mL.

[0011] Another object of the present invention is to provide a bacterial agent containing heterotrophic nitrifying bacteria D31, with a bacterial cell content of not less than 10%. 8 per mL.

[0012] Another object of the present invention is to provide a method for preparing the bacterial inoculant, comprising: inoculating strain D31 into LB medium, culturing at 55-60°C and 170-200 rpm for 3-5 days, centrifuging, discarding the supernatant, resuspending the bacterial cells in sterile water to obtain the bacterial inoculant.

[0013] The centrifugation speed was 5000 rpm, and the centrifugation time was 10 min.

[0014] The LB medium consists of 5g yeast extract, 10g trypsin, 10g sodium chloride, and 1000mL distilled water.

[0015] Experiments have shown that ammonia volatilization significantly decreases after livestock and poultry manure compost is inoculated with bacterial agents. Another object of the present invention is to provide the application of the aforementioned heterotrophic nitrifying bacteria D31 in livestock and poultry manure composting.

[0016] Preferably, the application is the use of heterotrophic nitrifying bacteria D31 in reducing ammonia volatilization during the composting of livestock and poultry manure.

[0017] Another object of the present invention is to provide the application of the bacterial agent containing heterotrophic nitrifying bacteria D31 in livestock and poultry manure composting.

[0018] Preferably, the application is the use of bacterial agents to reduce ammonia volatilization in livestock and poultry manure compost.

[0019] Preferably, the application involves inoculating the bacterial agent into chicken manure at an inoculation rate of 5%, mixing it thoroughly, and then composting it.

[0020] The livestock and poultry manure mentioned is chicken manure.

[0021] The beneficial effects of this invention are:

[0022] The strain D31 of this invention can perform heterotrophic nitrification on a medium containing ammonia nitrogen (DMS88), and can grow and oxidize ammonia on a simple carbon and nitrogen source medium (heterotrophic nitrification medium).

[0023] The strain D31 of this invention can rapidly proliferate using simple nutrients. After aerobic fermentation, the bacterial agent is inoculated into the compost. During the high-temperature period (55-60℃), it can efficiently and rapidly oxidize ammonia nitrogen in livestock and poultry manure into nitrate nitrogen, reducing the amount of nitrogen in the compost that is volatilized in the form of ammonia. The ammonia volatilization is significantly reduced, which can not only reduce odor emissions and protect the environment, but also maintain the nitrogen nutrients in the compost and improve the quality of the compost, thus having broad application prospects. Attached Figure Description

[0024] Figure 1This study determined the nitrification capacity of heterotrophic nitrifying bacteria for initial screening; where the same lowercase letter indicates no significant difference between groups, and different lowercase letters indicate significant differences between groups (P<0.05).

[0025] Figure 2 This shows the colony morphology of bacteria D31.

[0026] Figure 3 The image shows the morphology of a single cell after Gram staining of bacteria D31.

[0027] Figure 4 The results of nitrite color development in DSM88 medium for screening heterotrophic nitrifying strains.

[0028] Figure 5 Phylogenetic evolutionary tree of bacteria D31.

[0029] Figure 6 This is a diagram of a fermentation apparatus.

[0030] Figure 7 This shows the ammonia volatilization in compost after inoculation with bacterial agent D31.

[0031] Figure 8 This shows the total amount of ammonia volatilization in compost after inoculation with bacterial agent D31.

[0032] Biological Preservation Information

[0033] D31, classified as Geobacillus proteiniphilus, was deposited on July 3, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.27788. Detailed Implementation

[0034] Heterotrophic nitrification liquid culture medium (g / L): ammonium acetate 2.8 g / L, NaOH 1.6 g / L, KH2PO4 8.2 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, CuSO4·5H2O 0.5 mg / L, CaSO4·2H2O 0.5 mg / L, ZnSO4·7H2O 0.5 mg / L, FeCl3·6H2O 0.5 mg / L, pH 7.0, 1000 mL distilled water, sterilized by steam at 115℃ for 30 min.

[0035] Heterotrophic nitrification solid medium: Add 20g of agar powder to the above heterotrophic nitrification liquid medium.

[0036] Example 1

[0037] Screening and identification of thermophilic heterotrophic nitrifying strains

[0038] A 1g sample of chicken manure compost taken from Jintan District, Changzhou City, Jiangsu Province was placed in 30mL of sterile water and shaken for 30min. 1mL of the supernatant was added to 100mL of DSM88 liquid medium and incubated at 55℃ and 170rpm for 4 days to complete the first enrichment. 1mL of the enriched solution was then added to 100mL of DSM88 liquid medium and incubated again at 55℃ and 170rpm for 4 days to complete the second enrichment. This enrichment process was repeated four times, with each enriched solution serially diluted with sterile water to prepare 10... -4 10 -5 10 -6 Diluent: Take 100 μL of the above diluent and spread it on a DSM88 medium plate. Incubate at 55°C for 24 hours. Pick a single colony and streak it on a fresh DSM88 medium plate to obtain the purified strain.

[0039] The purified strains were inoculated into shake tubes containing 5 mL of DSM88 liquid medium and cultured at 55 °C and 170 rpm for 3 days. 1 mL of culture medium was centrifuged at 8000 rpm for 5 min, and 200 μL of the supernatant (colorless and transparent) was transferred to a sterile centrifuge tube. Griess reagent A and B were mixed evenly at a volume ratio of 1:1 to obtain Griess reagent (colorless and transparent). 100 μL of Griess reagent was added to the centrifuge tube and mixed evenly with the supernatant. The mixture was incubated for at least half an hour, and the color change was observed. If the color turned red or pink, nitrite nitrogen was produced. A total of 11 heterotrophic nitrifying bacteria strains were screened.

[0040] Eleven heterotrophic nitrifying strains and one strain E41 (a negative control) without heterotrophic nitrification ability were inoculated into 1.5 mL of DSM88 liquid medium and cultured at 55°C and 170 rpm for 1 day. The bacterial concentration was then adjusted to OD using sterile water. 600 =0.5, and inoculated into heterotrophic nitrification liquid medium at an inoculum rate of 1%, and cultured at 55℃ and 170rpm for 2 days. An equal volume of uninoculated heterotrophic nitrification liquid medium was used as a blank control (CK). The cultured bacterial solution was centrifuged, and the supernatant was filtered through a 0.45μm filter membrane. The total nitrate nitrogen and nitrite nitrogen in the bacterial solution were measured by the sulfanilamide colorimetric method. Figure 1 It can be seen that strain D31 produces the most nitrite and nitrate nitrogen, and has the strongest nitrification ability.

[0041] The biological characteristics of strain D31 are as follows: When strain D31 is cultured on DSM88 solid medium at 55℃ for 3 days, the colonies are white, round, smooth, and have regular edges. Figure 2 ).

[0042] The morphology of single cells of strain D31 after Gram staining was observed using a conventional optical microscope, such as... Figure 3 As shown, strain D31 is a Gram-positive bacterium with a bacillus-like shape.

[0043] like Figure 4 Strain D31 can oxidize ammonia to produce nitrite, which makes Griess reagent pink.

[0044] 2. Phylogenetic analysis of thermophilic heterotrophic nitrifying bacteria

[0045] The 16S rRNA gene in the bacterial genome was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3'). The PCR reaction system and procedure are shown in Tables 1 and 2, respectively. The PCR products were sent to a biotechnology company for sequencing. After the sequencing results were returned, they were uploaded to NCBI for gene sequence alignment. Similar sequences were downloaded and analyzed using iqtree software. A phylogenetic tree was constructed using the ML method, and the results were visualized using Figtree software. Figure 5 The species of the strain was preliminarily identified. Sequence analysis showed that strain D31 had 99% similarity to Geobacillus proteiniphilus strain 1017.

[0046] Table 1. PCR reaction system

[0047]

[0048]

[0049] Table 2. PCR reaction procedure

[0050]

[0051] Note: Steps 2-4 constitute one loop, requiring 30-35 loops.

[0052] Strain D31, classified as Geobacillus proteiniphilus, was deposited on July 3, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.27788.

[0053] Example 2

[0054] Chicken manure inoculation with D31 high-temperature fermentation experiment

[0055] Strain D31 was inoculated into LB medium and cultured at 55°C and 170 rpm for 3 days. After centrifugation at 5000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were resuspended in sterile water and the OD of the bacterial culture was adjusted. 600 Up to 0.5 (hanging drop count is 1.2 × 10⁻⁵) 8 (number / mL) to obtain bacterial inoculum.

[0056] like Figure 6 As shown, a high-temperature fermentation experiment of chicken manure was conducted using a fermentation apparatus, which included a fermentation bottle (9cm inner diameter, 12cm height) 1, equipped with a lid. Bacterial inoculum was inoculated into 100g of fresh chicken manure at a 5% inoculation rate and mixed thoroughly. The mixed chicken manure was then spread evenly on a surface as shown in the image. Figure 6 At the bottom of the fermentation bottle shown, two layers of sponges (each layer 12cm in diameter and 2cm thick) were placed on top of the mixed chicken manure; 20mL of ammonia absorption solution was injected into each layer of sponges beforehand using a syringe; and 0.8mol·L⁻¹ phosphoric acid was prepared using deionized water. -1 And the concentration of glycerol is 0.7 mol·L -1 (Ammonia absorption liquid), leave a gap of about 1cm between the two layers of sponge. The upper layer of sponge is used to isolate the influence of ammonia in the air, and the lower layer of sponge is used to collect the ammonia produced during fermentation. Tighten the lid to seal, and place the fermentation device in a constant temperature and humidity incubator at 55℃ and 70% humidity for 13 days.

[0057] During sampling, remove the lower layer of sponge and replace it with a new sponge filled with 20 mL of ammonia absorption solution. Place the removed sponge into a 500 mL Erlenmeyer flask and add 300 mL of a 1.0 mol·L⁻¹ solution. -1 KCl solution, shake for 1 hour, collect an appropriate amount of extract and retain it. Determine the ammonia content using the Nessler colorimetric method. Daily ammonia volatilization amount is shown in [reference needed]. Figure 7 The total ammonia volatilization is obtained by adding up the daily ammonia volatilization amounts, such as... Figure 8 As shown, compared with CK (no bacterial inoculation, only an equal amount of sterile water inoculated), ammonia volatilization decreased by 315.8 mg after inoculation with D31, and the relative reduction in ammonia volatilization was 50.74%.

Claims

1. A heterotrophic nitrifying bacterium D31, which is classified and named as Geobacillus proteophilus (G. proteophilus). Geobacillus proteiniphilus and deposited at the China General Microbiological Culture Collection Center on July 3, 2023, and has an accession number of CGMCC NO. 27788.

2. The application of heterotrophic nitrifying bacteria D31 in livestock and poultry manure composting according to claim 1.

3. Use according to claim 2, characterized in that: The application is the application of heterotrophic nitrifying bacteria D31 in reducing ammonia volatilization in livestock and poultry manure composting.

4. A bacterial inoculant characterized in that: The bacterial agent contains the heterotrophic nitrifying bacteria D31 of claim 1, and the content of the bacterial body is not less than 10 8 individuals / mL.

5. A method for preparing the bacterial inoculant of claim 4, characterized by: Comprise: Inoculate strain D31 into LB medium, cultivate at 55-60 ℃, 170-200 rpm for 3-5 days, centrifuge, discard supernatant, resuspend bacterial body with sterile water, and obtain bacterial inoculum.

6. The application of bacterial inoculum in livestock and poultry manure composting according to claim 4.

7. Use according to claim 6, characterized in that: The application is the application of bacterial inoculum in reducing ammonia volatilization in livestock and poultry manure composting.

8. Use according to claim 6, characterized in that: The application is to inoculate bacterial inoculum into chicken manure according to 5% inoculation amount, mix well, and compost.

9. Use according to claim 2 or 6, characterized in that: The livestock and poultry manure is chicken manure.

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