New acinetobacter loffithii strain and application thereof in ammonia reduction and deodorization of livestock and poultry farms

Abstract

The invention provides a new strain of acinetobacter loffii, the new strain is acinetobacter loffii N2, and the preservation number of the new strain is CGMCC (China General Microbiological Culture Collection Center) NO.30272. The invention also provides a microbial inoculum prepared from the acinetobacter lophenanthrene N2, and the microbial inoculum is a bacterial liquid cultured to a logarithmic phase. In addition, the invention also provides an application of the acinetobacter lophenanthrene N2 in removing ammonia gas and odor in livestock and poultry farms. The acinetobacter lophenanthrene N2 provided by the invention can effectively degrade odor and ammonia gas in livestock and poultry farms, the degradation rates are respectively 70.07% and 84.55%, and the acinetobacter lophenanthrene N2 has a good industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of environmental microbial technology, and specifically relates to a new strain of Acinetobacter lofivi and its application in ammonia reduction and deodorization in livestock and poultry farms. Background Art

[0002] The scale of livestock and poultry farming is an inevitable trend in the development of modern agriculture. It is of great significance to improve production efficiency and reduce costs. However, with the continuous improvement of the scale of livestock and poultry farming, the environmental problems in the farming process are becoming increasingly serious. In particular, the odor of livestock and poultry farms has not been properly handled due to its complex composition and difficulty in treatment, which has a direct impact on the environment and the quality of life of surrounding residents. The main components of the odor of livestock and poultry farms are: ammonia (NH 3 ), hydrogen sulfide (H 2 S) and volatile organic compounds (VOCs). Ammonia is the main component of odor in livestock and poultry farms. Excessive ammonia emissions not only cause air pollution, but may also cause a series of environmental problems such as soil acidification and water eutrophication. Therefore, ammonia reduction and deodorization solutions are of great significance to the sustainable development and environmental protection of livestock and poultry farms.

[0003] At present, the technical means of reducing ammonia and deodorizing mainly include physical methods, chemical methods, and biological methods. The physical method has a high cost, and the adsorption material needs to be replaced after saturation; although the chemical method can quickly reduce the ammonia concentration, it may cause secondary pollution; the biological method relies on a specific microbial community and there are safety issues with the strains. The odor from livestock and poultry farms is characterized by complex composition, high dust content, and continuous emissions. Considering the construction cost and operating cost of the treatment equipment, the biological method is the most suitable treatment method for the odor from livestock and poultry farms. Therefore, there is an urgent need to screen safe and efficient ammonia reduction and deodorization microorganisms.

[0004] The inventors of the present disclosure screened and obtained a new strain, Acinetobacter lwoffii N2, with a preservation number of CGMCC NO.30272, which can effectively degrade odor and ammonia in livestock and poultry farms. Summary of the invention

[0005] A brief overview of the disclosure is given below in order to provide a basic understanding of certain aspects of the disclosure. It should be understood that this overview is not an exhaustive overview of the disclosure. It is not intended to identify key or important parts of the disclosure, nor is it intended to limit the scope of the disclosure. Its purpose is simply to give certain concepts in a simplified form as a prelude to a more detailed description discussed later.

[0006] In order to solve the above technical problems, the technical solution provided by the present disclosure is:

[0007] In the first aspect, the present application provides a new strain of Acinetobacter lwoffii, which is Acinetobacter lwoffii (Acinetobacter lwoffii) N2, with a preservation number of CGMCC NO.30272. The strain was deposited on April 7, 2024 at the General Microbiology Center of the China Culture Collection Administration at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The Acinetobacter lwoffii N2 of the present application was isolated from a filter element sample of a biological trickling filter device in a pig farm in Chongqing. The sample was gradient diluted, streaked on a plate, and pure bacteria N2 was isolated and purified. Identification showed that N2 was Acinetobacter lwoffii. Experiments found that N2 can effectively degrade ammonia and deodorize livestock and poultry farms.

[0009] In a second aspect, the present application also provides a bacterial agent prepared from the above-mentioned Acinetobacter lofivi N2, wherein the bacterial agent is a bacterial liquid cultured to a logarithmic growth phase or a stable phase.

[0010] Preferably, the bacterial liquid contains at least 1×10 8 CFU / mL. More preferably, the bacterial liquid contains 1×10 8 -1×10 9 CFU / mL.

[0011] Preferably, the preparation method of the bacterial solution includes: using a sterile inoculation loop to pick a single colony of Acinetobacter lofyi N2 and inoculate it into NB liquid culture medium, 30-37°C (such as 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, etc.), 120-180rpm (such as 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, etc.), shaking culture for 10-24h (such as 12h, 15h, 18h, 20h, 22h, etc.); the formula (g / L) of the NB liquid culture medium is as follows: peptone 10.0, beef extract powder 3.0, sodium chloride 5.0, the balance is water, pH 7.2±0.2, 25°C.

[0012] In a third aspect, the present application also provides an application of the above-mentioned Acinetobacter lofivi N2 in removing ammonia and odor from livestock and poultry farms.

[0013] Preferably, the application comprises: preparing the bacterial liquid of Acinetobacter lofivi N2; and adding the bacterial liquid to feces for treatment.

[0014] Preferably, the feces in water are pig manure.

[0015] Preferably, the bacterial liquid contains at least 1×10 8 CFU / mL, more preferably 1×10 8 -1×109 CFU / mL.

[0016] Preferably, the volume-to-weight ratio of the bacterial liquid to the feces is 0.1-0.3 mL / g (such as 0.15 mL / g, 0.20 mL / g, 0.25 mL / g, etc.).

[0017] Preferably, the treatment time is 20-30 h (eg, 22 h, 24 h, 26 h, 28 h, etc.).

[0018] Preferably, the method for preparing the Lofyi Acinetobacter N2 bacterial solution is: using a sterile inoculation loop to pick a single colony of Lofyi Acinetobacter N2 and inoculate it into NB liquid culture medium, 30-37°C (such as 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, etc.), 120-180rpm (such as 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, etc.), shaking culture for 10-24h (such as 12h, 15h, 18h, 20h, 22h, etc.); the formula (g / L) of the NB liquid culture medium is as follows: 10.0 peptone, 3.0 beef extract powder, 5.0 sodium chloride, and the balance is water, and the pH value of the NB liquid culture medium is 7.2±0.2 when prepared at 25°C.

[0019] Preferably, the livestock and poultry farm is a pig farm.

[0020] The beneficial effects of the present invention compared to the prior art include but are not limited to:

[0021] First, the Acinetobacter lofyi N2 provided in the present application is a new strain of bacteria that can degrade the odor and ammonia of livestock and poultry farms. In recent years, the scale of my country's livestock and poultry farming industry has been continuously improved, and the treatment of polluted gases from livestock and poultry farming is particularly important. Among them, the odor of livestock and poultry farms has complex components, high dust content and continuous emissions, making it difficult to treat. Therefore, it is very important to screen out strains that can degrade the odor of livestock and poultry farms. The Acinetobacter lofyi N2 provided in the present disclosure provides a good foundation for degrading the odor and ammonia of livestock and poultry farms.

[0022] Second, Acinetobacter lofivi N2 can effectively degrade odor and ammonia in livestock and poultry farms, with degradation rates of 70.07% and 84.55% respectively, and has good industrial application prospects.

[0023] The preservation date of the Acinetobacter lwoffii N2 disclosed in the present invention is April 7, 2024, the preservation number is CGMCCNO.30272, the classification name is: Acinetobacter lwoffii N2, the name of the preservation unit is: General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short), the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The ammonia removal rate of the strains with an ammonia removal rate exceeding 50% in the strain screening experiment of Example 1 is shown;

[0025] Figure 2 A colony photograph of Acinetobacter loffei N2 in Example 2 is shown;

[0026] Figure 3 The phylogenetic tree of Acinetobacter loffei N2 in Example 2 is shown;

[0027] Figure 4 The growth curve of Acinetobacter loffei N2 in Example 3 is shown. DETAILED DESCRIPTION

[0028] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without violating the spirit and essence of the present invention, the modification or replacement of the method, steps or conditions of the present invention belongs to the scope of the present invention.

[0029] The technical solution of the present disclosure will be described below in conjunction with exemplary embodiments.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Example 1 Isolation and screening of Acinetobacter lofivi N2

[0033] (1) Preparation of strains and culture medium:

[0034] The strain originated from a filter element sample of a biotrickling filter in a pig farm in Chongqing.

[0035] Select NB medium, YPD medium, and MRS medium to isolate strains from samples. The components of each medium are as follows:

[0036] NB medium (g / L): peptone 10.0, beef extract powder 3.0, sodium chloride 5.0, the balance is water, the pH value of the prepared medium at 25°C is 7.2±0.2;

[0037] YPD medium (g / L): peptone 20.0, glucose 20.0, yeast extract powder 10.0, the balance is water, the pH value of the medium prepared at 25°C is 6.5±0.2;

[0038] MRS medium (g / L): peptone 10.0, beef extract powder 8.0, yeast extract powder 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 80 1.0, the balance is water, the pH value of the prepared medium at 25°C is 5.7±0.2;

[0039] The solid culture medium corresponding to the above three liquid culture media was prepared by adding 16 g / L agar to the above formula.

[0040] (2) Isolation and purification: Isolate and purify the strains in the sample obtained in step (1). Under a sterile operating environment, take 10g of the sample and add it to 90mL of physiological saline, mix well, and dilute it with sterile distilled water to obtain a bacterial suspension. Take 100μL of the bacterial suspension and spread it on solid culture media (NB culture medium, YPD culture medium, and MRS culture medium respectively), invert it in an incubator, and culture it at a constant temperature of 37°C for 24 hours. In an ultra-clean workbench, use a sterile inoculation loop to pick single colonies of different morphologies and streak them in the culture medium. Repeat the streaking of the plate 3 times or more to obtain the purified strains. Store the strains in -20°C glycerol for later use. A total of 50 strains were isolated and preserved, numbered N1-N50.

[0041] (3) Screening of ammonia removal performance of strains: The above strains were inoculated into the corresponding liquid culture medium described in step (2) respectively, and cultured at 37°C, 150rpm, and shaking for 12 hours to obtain a bacterial solution. Pig feces and pig sewage were mixed in a mass ratio of 1:1 to obtain water-filled feces. 50g of water-filled feces were taken in a 120mm culture dish, and 10mL of bacterial solution was added to the experimental group and 10mL of sterile water was added to the control group. Three parallels were made for each strain. After being placed in a 3L air bag and sealed to exhaust the gas, the air bag was inflated uniformly with a pump for 30s and then sealed. The air bag was cultured at a constant temperature of 30°C for 24 hours, and the ammonia concentration was detected. Calculate the ammonia removal rate after 24 hours, and select strains with an ammonia removal rate of more than 50% for plotting. Figure 1 As shown. Figure 1 It can be seen that strain N2 (isolated and purified from NB medium) had the highest ammonia removal rate after 24 hours, averaging 70.07%, so strain N2 was identified.

[0042] (4) Odor removal performance test of strain N2: strain N2 was inoculated into NB liquid culture medium, and cultured at 37°C, 150rpm, with shaking for 12h to obtain bacterial liquid. Pig feces and pig sewage were mixed in a mass ratio of 1:1 to obtain water-foamed feces. 50g of water-foamed feces was placed in a 120mm culture dish, and 10mL of bacterial liquid was added to the experimental group and 10mL of sterile water was added to the control group. Three parallels were placed in a 3L air bag to seal and exhaust the gas. After the air was uniformly inflated with a pump for 30s, the air bag was sealed and cultured at a constant temperature of 30°C for 24h. The odor concentration was tested. The average odor removal rate after 24h was calculated to be 84.55%.

[0043] Example 2 Identification and preservation of Acinetobacter loffei N2

[0044] (1) Strain identification

[0045] Morphological identification: strain N2 was cultured on NB medium plate at 37℃ for 24h. The colony pattern is as follows: Figure 2 As shown. Figure 2 It can be seen that the colonies of the N2 strain are round or oval, milky white and opaque, with smooth edges and a bulge in the middle. The texture is sticky and easy to pick up.

[0046] Identification by molecular biology methods: The genomic DNA of strain N2 was extracted using the TSINGKE kit, and the bacterial universal primers 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTCTTACGAC-3') were used to amplify the 16SrDNA region sequence of the N2 strain. The amplified size was about 1.5 bp, and the 16SrDNA gene was amplified by polymerase chain reaction (PCR). The PCR amplification product was sequenced by the Chongqing Branch of Beijing Qingke Biotechnology Co., Ltd. The sequencing results were spliced ​​using ContigExpress, and the sequencing results were subjected to BLAST similarity analysis with the existing sequences in NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Strain N2 was identified as Acinetobacter lwoffii. The phylogenetic tree was constructed using the Neighbor Joining method of MAGE6 software, as shown in Figure 2. Figure 3 The 16SrDNA sequence of Acinetobacter lofivi N2 is as follows:

[0047] TGCAAGTCGAGCGGGGAAGAGTAGCTTGCTACTTAACCTAGCG

[0048] GCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACA

[0049] ACATCTCGAAAGGGATGCTAATACCGCATACGTCCTACGGGAGAAAG

[0050] CAGGGGACCTTCGGGCCTTGCGCTAATAGATGAGCCTAAGTCGGATT

[0051] AGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCG

[0052] GGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAG

[0053] ACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAA

[0054] CCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTTTGGTTGTA

[0055] AAGCACTTTAAGCGAGGAGGAGGCTACCGAGATTAATACTCTTGGAT

[0056] AGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTCTGTGCCAGC

[0057] AGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTACTGGGC

[0058] GTAAAGCGCGCGTAGGTGGCCAATTAAGTCAAATGTGAAATCCCCG

[0059] AGCTTAACTTGGGAATTGCATTCGATACTGGTTGGCTAGAGTATGGG

[0060] AGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATC

[0061] TGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAATACTGAC

[0062] ACTGAGGTGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGG

[0063] TAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTTTGAGG

[0064] CTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTAC

[0065] GGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAG

[0066] CGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCT

[0067] GGTCTTGACATAGTAAGAACTTTCCAGAGATGGATTGGTGCCTTCGG

[0068] GAACTTACATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGA

[0069] GATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTATTTG

[0070] CCAGCGGGTTAAGCCGGGAACTTTAAGGATACTGCCAGTGACAAAC

[0071] TGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGACC

[0072] AGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACCTC

[0073] GCGAGAGGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGA

[0074] GTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGAT

[0075] CAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG

[0076] TCACACCATGGGAGTTTTGTTGCACCAGAAGTAGGTAGTCTAACCGC

[0077] AAGGAGGAC

[0078] (2) Preservation of strains

[0079] The inventor named strain N2 as Acinetobacter lwoffii N2 and submitted it for preservation. The preservation date is April 7, 2024, the preservation number is CGMCCNO.30272, the classification name is: Acinetobacter lwoffii N2, the name of the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC for short), the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0080] Example 3 Growth curve determination of Acinetobacter lofivi N2

[0081] The growth curve of the strain Acinetobacter lwoffii N2 in Example 2 was measured by photoelectric turbidimetry (reference: Fiester, SE, Arivett, BA, Beckett, AC, et al. (2018). Miltefosine Reduces the Cytolytic Activity and Virulence of Acinetobacter baumannii. Antimicrobial agents and chemotherapy, 63 (1), e01409-18.). The culture solution was taken every 1 hour, and the absorbance of the bacterial solution was measured at a wavelength of 600 nm to obtain the growth curve of the strain Acinetobacter lwoffii N2 as shown in the figure. Figure 4 shown.

[0082] from Figure 4 It can be seen that at 0-3h, Acinetobacter N2 is in the delay period of the growth stage, and its growth rate is slow; at 3-13h, Acinetobacter N2 enters the logarithmic growth phase, and its growth rate reaches the maximum; after 13h, Acinetobacter N2 enters the stable period, and the growth state basically maintains a dynamic equilibrium.

[0083] Example 4 Performance test of Acinetobacter lofivi N2

[0084] (1) Strain non-toxicity test

[0085] Acinetobacter lofivi N2 was inoculated into NB liquid culture medium and cultured at 37°C, 150rpm, with shaking for 12h. The samples were sent to China Inspection and Quarantine Huatongwei International Inspection (Suzhou) Co., Ltd. for oral toxicity median lethal dose LD 50 Detection. Oral toxicity median lethal dose ID 50 The test showed that the acute oral LD50 of the samples in ICR mice 50 >2000mg / kg body weight, according to GB 5085.2-2007 Hazardous Waste Identification Standard Acute Toxicity Screening, oral toxicity median lethal dose LD 50 Test classification criteria for determining LD 50 >500mg / kg body weight, does not have the acute oral toxicity characteristics of hazardous waste.

[0086] (2) Testing of the strain’s ability to degrade ammonia and odor,

[0087] The method for testing the ammonia and odor degradation performance of the strain was the same as in Example 1. The ammonia and odor removal rates of Acinetobacter loffei N2 were 70.07% and 84.55%, respectively (see Table 1).

[0088] (3) Testing of strain degradation performance of VOCs

[0089] Acinetobacter lofivi N2 was inoculated into NB liquid culture medium, and cultured at 37°C, 150rpm, and shaken for 12h to obtain bacterial liquid. Pig feces and pig sewage were mixed in a mass ratio of 1:1 to obtain water-filled feces. 50g of water-filled feces was placed in a 120mm culture dish, and 10mL of bacterial liquid was added to the experimental group and 10mL of sterile water was added to the control group. Three parallels were placed in a 3L air bag and sealed to discharge the gas. After the air was uniformly inflated for 30s with a pump, the air bag was sealed and cultured at a constant temperature of 30°C for 24h, and the VOCs concentration was detected. The VOCs removal rate after 24h was calculated, see Table 1.

[0090] (4) Bacteria degrade H 2 S performance test

[0091] Acinetobacter lofivi N2 was inoculated into NB liquid culture medium and cultured at 37°C, 150rpm, with shaking for 12h to obtain bacterial liquid. Pig feces and pig sewage were mixed at a mass ratio of 1:1 to obtain water feces. 50g of water feces was taken into a 120mm culture dish. 10mL of bacterial liquid was added to the experimental group and 10mL of sterile water was added to the control group. Three parallel plates were placed in a 3L air bag and sealed to discharge the gas. After the air was uniformly inflated with a pump for 30s, the air bag was sealed and cultured at a constant temperature of 30°C for 24h to detect H 2 S concentration. Calculate H after 24 hours 2 For S removal rate, see Table 1.

[0092] Table 1 Effects of Acinetobacter lofibrinosum N2 on VOCs, odor, and NH 3With H 2 S removal rate

[0093]

[0094] Finally, it should be noted that, in the present disclosure, if any, relational terms such as first and second, etc., are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprises", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the statement "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device including the element.

[0095] Although the present disclosure has been disclosed above through the description of the specific embodiments of the present disclosure, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present disclosure within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed by the present disclosure.

Claims

1. A new strain of Acinetobacter lwoffii, Acinetobacter lwoffii N2, with a deposit number of CGMCC NO.30272.

2. A bacterial agent prepared from the Acinetobacter lofivi N2 according to claim 1, wherein the bacterial agent is a bacterial liquid cultured to a logarithmic growth phase or a stable phase.

3. The bacterial agent according to claim 2, It is characterized in that The bacterial liquid contains at least 1×10 8 CFU / mL.

4. The bacterial agent according to claim 3, It is characterized in that The bacterial content of the bacterial solution is 1×10 8 -1×10 9 CFU / mL.

5. The bacterial agent according to any one of claims 2 to 4, It is characterized in that The preparation method of the bacterial liquid comprises: picking a single colony of Acinetobacter lofivi N2 with a sterile inoculation loop and inoculating it into a NB liquid culture medium, 30-37°C, 120-180rpm, and shaking culture for 10-24h; the formula (g / L) of the NB liquid culture medium is as follows: 10.0g / L of peptone, 3.0g / L of beef extract powder, 5.0g / L of sodium chloride, and the balance is water, and the pH value is 7.2±0.2 when the preparation temperature is 25°C.

6. Use of the Acinetobacter lofivi N2 described in claim 1 in removing ammonia and odor from livestock and poultry farms.

7. The use according to claim 6, It is characterized in that The application comprises: preparing the bacterial agent of Acinetobacter lofivi N2 as described in any one of claims 2 to 5; and adding the bacterial agent to water-soaked feces for treatment.

8. The use according to claim 7, It is characterized in that The blistered feces are pig feces.

9. The use according to claim 8, It is characterized in that The volume-to-weight ratio of the bacterial liquid to the feces is 0.10-0.30 mL / g.

10. The use according to any one of claims 7 to 9, It is characterized in that The processing time is 20-30h.

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