New strain of Acinetobacter lofi and its application in ammonia reduction and deodorization in livestock and poultry farms

Abstract

The present disclosure provides a new strain of Acinetobacter lwoffii, which is Acinetobacter lwoffii N2, with a deposit number of CGMCC NO.30272. A bacterial agent prepared from the above-mentioned Acinetobacter lwoffii N2 is also provided, and the bacterial agent is a bacterial liquid cultured to the logarithmic growth phase. In addition, a use of the above-mentioned Acinetobacter lwoffii N2 in removing ammonia and odor from livestock and poultry farms is also provided. The Acinetobacter lwoffii N2 provided by the present disclosure can effectively degrade odor and ammonia from livestock and poultry farms, with degradation rates of 70.07% and 84.55%, respectively, and has good industrial application prospects.

Description

Technical Field

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

[0002] The scale-up of livestock and poultry farming is an inevitable trend in the development of modern agriculture, and it is of great significance for improving production efficiency and reducing costs. However, as the scale of livestock and poultry farming continues to increase, environmental issues in the farming process are becoming increasingly serious. In particular, odor from livestock and poultry farms, due to its complex composition and difficulty in treatment, is not properly managed, which has a direct impact on the environment and the quality of life of surrounding residents. The main components of odor from livestock and poultry farms are ammonia (NH3), hydrogen sulfide (H2S), and volatile organic compounds (VOCs). Ammonia is the main component of odor from livestock and poultry farms. Excessive ammonia emissions not only cause air pollution but can also cause a series of environmental problems such as soil acidification and water eutrophication. Therefore, solutions for ammonia reduction and deodorization are of great significance for the sustainable development and environmental protection of livestock and poultry farms.

[0003] Currently, the main technical approaches for ammonia reduction and deodorization include physical, chemical, and biological methods. Physical methods are costly, and the adsorption material needs to be replaced once saturated. While chemical methods can quickly reduce ammonia concentrations, they may cause secondary pollution. Biological methods rely on specific microbial communities and pose safety concerns. Odors from livestock and poultry farms are characterized by complex composition, high dust content, and continuous emissions. Considering the construction and operating costs of treatment equipment, biological methods are the most suitable treatment method for livestock and poultry farm odors. Therefore, there is an urgent need to identify safe and efficient microorganisms for ammonia reduction and deodorization.

[0004] The inventors of the present disclosure screened and obtained a new strain, Acinetobacter lwoffii N2, with a deposit 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 present disclosure is provided below to provide a basic understanding of certain aspects of the present disclosure. It should be understood that this overview is not an exhaustive overview of the present disclosure. It is not intended to identify key or important aspects of the present disclosure, nor is it intended to limit the scope of the present disclosure. Its purpose is simply to present certain concepts in a simplified form as a prelude to the more detailed description discussed later.

[0006] To solve the above technical problems, the technical solutions provided by the present disclosure are:

[0007] In the first aspect, the present application provides a new strain of Acinetobacter lwoffii, which is 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, 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 biotrickling 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, which is a bacterial liquid cultured to the logarithmic growth phase or the 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 liquid includes: picking a single colony of Acinetobacter lofivi N2 with a sterile inoculating loop and inoculating 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 lofyi 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 into feces for treatment.

[0014] Preferably, the blistered feces are pig manure.

[0015] Preferably, the bacterial liquid contains at least 1×10 8 CFU / mL, more preferably 1×10 8 -1×10 9CFU / mL.

[0016] Preferably, the volume-to-weight ratio of the bacterial liquid to the water-soaked 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 hours (such as 22 hours, 24 hours, 26 hours, 28 hours, etc.).

[0018] Preferably, the method for preparing the Acinetobacter lofyi N2 bacterial solution is: 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: 10.0% peptone, 3.0% beef extract powder, 5.0% sodium chloride, and the balance is water. 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 disclosure compared to the prior art include but are not limited to:

[0021] First, the Acinetobacter loffei N2 provided in this 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 continued to improve, 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 crucial to screen out strains that can degrade the odor of livestock and poultry farms. The Acinetobacter loffei N2 provided in this 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 deposit date of the Acinetobacter lwoffii N2 disclosed in the present invention is April 7, 2024, the deposit number is CGMCCNO.30272, the classification name is: Acinetobacter lwoffii N2, the name of the depository is: General Microbiology Center of China Culture Collection Administration of Microorganisms (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 rates of the strains with an ammonia removal rate exceeding 50% in the strain screening experiment of Example 1 are shown;

[0025] Figure 2 A colony photograph of Acinetobacter lofivi 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 lofivi N2 in Example 3 is shown. DETAILED DESCRIPTION

[0028] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are within the scope of the present invention.

[0029] The technical solutions 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] NB medium, YPD medium, and MRS medium were used to isolate the strains from the sample. 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%, balance water. The pH value of the prepared medium at 25°C is 7.2±0.2.

[0037] YPD medium (g / L): 20.0g peptone, 20.0g glucose, 10.0g yeast extract powder, and the balance 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, balance water. The pH of the medium at 25°C was 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: The strains in the sample obtained in step (1) were isolated and purified. Under a sterile operating environment, 10 g of sample was added to 90 mL of physiological saline and mixed, and gradient dilution was performed with sterile distilled water to obtain a bacterial suspension. 100 μL of bacterial suspension was spread on solid culture medium (NB culture medium, YPD culture medium, and MRS culture medium respectively), inverted in an incubator, and cultured at a constant temperature of 37°C for 24 hours. In an ultra-clean workbench, a sterile inoculation loop was used to pick single colonies of different morphologies and streak them on the culture medium. After repeating the streak for 3 or more times, the purified strains were obtained and stored in glycerol at -20°C for future 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 shaken for 12 hours to obtain bacterial solution. Pig feces and piggery sewage were mixed at a mass ratio of 1:1 to obtain water-soaked feces. 50g of water-soaked feces were taken into a 120mm culture dish. 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 discharge the gas, the air bag was uniformly inflated with a pump for 30s and then sealed. The bag was cultured at a constant temperature of 30°C for 24 hours, and the ammonia concentration was detected. The ammonia removal rate after 24 hours was calculated, and strains with an ammonia removal rate exceeding 50% were selected for plotting, such as Figure 1 As shown. Figure 1 It can be seen that strain N2 (isolated and purified from NB culture medium) has the highest ammonia removal rate after 24 hours, with an average of 70.07%. Therefore, 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, 150 rpm, with shaking for 12 h to obtain a bacterial solution. Pig feces and piggery wastewater were mixed at a mass ratio of 1:1 to obtain water-soaked feces. 50 g of water-soaked feces was placed in a 120 mm culture dish. 10 mL of bacterial solution was added to the experimental group and 10 mL of sterile water was added to the control group. Three replicates were placed in a 3 L air bag, sealed to expel the gas, and then inflated with a pump for 30 seconds, sealed, and cultured at a constant temperature of 30°C for 24 h. The odor concentration was tested. The average odor removal rate after 24 h was calculated to be 84.55%.

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

[0044] (1) Strain identification

[0045] Morphological identification: strain N2 was cultured on NB medium plate at 37℃ for 24h. The colony was as shown in the figure below. 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 biological 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 the following figure: Figure 3 The 16S rDNA 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 Acinetobacter lwoffii N2 and submitted it for preservation. The preservation date was April 7, 2024, the preservation number was CGMCCNO.30272, the classification name was: Acinetobacter lwoffii N2, the name of the preservation unit was: General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC for short), the address was: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0080] Example 3 Acinetobacter lofivi N2 growth curve determination

[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 balance.

[0083] Example 4 Performance detection 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, 150 rpm, and shaken for 12 h. The culture was 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 sample to 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 standard 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 lofophilum 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 with shaking at 37°C, 150 rpm, for 12 hours to obtain a bacterial suspension. Pig feces and piggery wastewater were mixed in a 1:1 mass ratio to obtain a slurry of feces. 50 g of the slurry was placed in a 120 mm Petri dish. 10 mL of bacterial suspension was added to the experimental group, while 10 mL of sterile water was added to the control group. Three replicates were placed in a 3 L air bag, sealed to remove air, and then uniformly inflated with a pump for 30 seconds. The bag was then sealed and incubated at a constant temperature of 30°C for 24 hours. VOC concentrations were then measured. VOC removal rates after 24 hours were calculated (see Table 1).

[0090] (4) Detection of H2S degradation performance of strains

[0091] Acinetobacter lofivi N2 was inoculated into NB liquid culture medium and cultured with shaking at 37°C, 150 rpm, for 12 hours to obtain a bacterial suspension. Pig feces and piggery wastewater were mixed in a 1:1 mass ratio to obtain a slurry of feces. 50 g of the slurry was placed in a 120 mm Petri dish. 10 mL of bacterial suspension was added to the experimental group, while 10 mL of sterile water was added to the control group. Three replicates were placed in a 3 L air bag, sealed to remove air, and then uniformly inflated with a pump for 30 seconds. The bag was then sealed and incubated at a constant temperature of 30°C for 24 hours. H2S concentration was then measured. The H2S removal rate after 24 hours was calculated, as shown in Table 1.

[0092] Table 1 Removal rate of VOCs, odor, NH3 and H2S by Acinetobacter lofibrinosum N2

[0093]

[0094] Finally, it should be noted that in this disclosure, if any, relational terms such as first and second, etc., are used only 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 variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus comprising 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 may design various modifications, improvements or equivalents of the present disclosure within the spirit and scope of the attached solutions. Such modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed by the present disclosure.

Claims

1. A strain of Acinetobacter lofi Acinetobacter lwoffii )N2, the deposit number is CGMCC NO.30272.

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

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

4. The microbial agent according to claim 3, 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, characterized in that The preparation method of the bacterial liquid includes: using a sterile inoculation loop to pick a single colony of Acinetobacter lofivi N2 and inoculate it into NB liquid culture medium, and shaking culture at 30-37°C, 120-180 rpm, for 10-24 hours; the formula (g / L) of the NB liquid culture medium is as follows: 10.0 g / L peptone, 3.0 g / L beef extract powder, 5.0 g / L 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 according to claim 1 in removing odor from livestock and poultry farms.

7. The use according to claim 6, characterized in that The odor includes ammonia, hydrogen sulfide and volatile organic compounds.

8. The use according to claim 6 or 7, characterized in that The application comprises: preparing the bacterial agent according to any one of claims 2 to 5; and adding the bacterial agent to water-soaked feces for treatment.

9. The use according to claim 8, characterized in that The water-soaked feces is pig feces.

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

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

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