Novel klebsiella aerogenes strain and application thereof in degradation of VOCs in livestock and poultry farms

Abstract

The invention provides a novel Klebsiella aerogenes strain, which is Klebsiella aerogenes N1, and the preservation number of the novel Klebsiella aerogenes strain is CGMCC (China General Microbiological Culture Collection Center) NO.30271. The invention further provides a preparation method of the novel Klebsiella aerogenes strain. The invention also provides a microbial inoculum prepared from the klebsiella aerogenes N1, and the microbial inoculum is a bacterial liquid cultured to a logarithmic phase. In addition, the invention also provides an application of the klebsiella aerogenes N1 in degradation of VOCs in livestock and poultry farms. The klebsiella aerogenes N1 provided by the invention is a new strain capable of degrading VOCs in livestock and poultry farms. According to the present invention, the bacterial liquid prepared from the klebsiella aerogenes N1 is added into the blistered manure to treat, such that the VOCs in the pig farm can be effectively degraded, the degradation rate is up to 85.37%, and the bacterial liquid has different degrees of degradation effects on odor, ammonia gas and hydrogen sulfide in the livestock and poultry farm so as to provide good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental microorganisms, and in particular relates to a new strain of Klebsiella aeruginosa and its application in degrading VOCs in livestock and poultry farms. Background Art

[0002] Intensive and large-scale livestock and poultry farming is conducive to reducing breeding costs and improving production efficiency, which is the only way for livestock and poultry farming in my country. However, with the continuous improvement of the scale of livestock and poultry farming, environmental problems in the breeding process are becoming increasingly serious. In particular, the odor from livestock and poultry farms has not been properly handled due to complex components and difficulty in treatment, which seriously affects the surrounding environment and residents. The main components of odor from livestock and poultry farms are: ammonia, hydrogen sulfide and volatile organic compounds (VOCs). Ammonia and hydrogen sulfide are the odor components that have been studied more, while research on VOCs is relatively less, and the emission of VOCs poses a serious threat to human health and the environment. VOCs can damage the central nervous system and cause symptoms such as headaches, nausea, and vomiting; VOCs are fine particulate matter (PM 2.5 ), PM 2.5 It can harm the human respiratory system and is also the main cause of haze; VOCs will produce ozone through repeated photochemical reactions in the atmosphere, forming photochemical smog that harms human health and the environment. Therefore, it is urgent to strengthen research on VOCs control.

[0003] Polluted gas treatment technologies are divided into two categories: recovery and decomposition. Recovery is mainly physical, while decomposition is mainly chemical and biological. Recovery technology is suitable for gaseous pollutants with high concentrations and recovery value; decomposition technology decomposes gaseous pollutants into basically harmless small molecules. VOCs in livestock and poultry farms have complex components, high dust content and continuous emissions. Considering the construction cost and operation cost of the treatment equipment, biological methods are the most suitable treatment method for VOCs in livestock and poultry farms. Therefore, there is an urgent need to screen microorganisms that can effectively degrade VOCs in livestock and poultry farms.

[0004] The inventors of the present disclosure screened and obtained a new strain of Klebsiella aerogenes N1, with a preservation number of CGMCC NO.30271, which can effectively degrade VOCs 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 disclosure provides a new strain of Klebsiella aerogenes, which is Klebsiella aerogenes N1, with a preservation number of CGMCC NO.30271. The strain was deposited on April 7, 2024 at the General Microbiology Center of China Culture Collection Administration at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The aerogenes Klebsiella N1 provided in the present invention 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 N1 was isolated and purified. Experiments found that N1 can degrade VOCs, and identification showed that N1 is Klebsiella aerogenes.

[0009] In a second aspect, the present disclosure also provides a bacterial agent prepared from the above-mentioned Klebsiella aerogenes N1, 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 1×10 8 -1×10 9 CFU / mL.

[0011] Preferably, the method for preparing the bacterial solution includes: using a sterile inoculation loop to pick a single colony of Klebsiella aeruginosa N1 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 of the NB liquid culture medium is as follows (g / L): 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 second aspect, the present disclosure also provides the use of the above-mentioned Klebsiella aeruginosa N1 in degrading VOCs in livestock and poultry farms.

[0013] Preferably, the application comprises: preparing the bacterial liquid of the aerogenes Klebsiella N1; and adding the bacterial liquid into the 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 -1×10 9 CFU / mL.

[0016] Preferably, the volume-to-weight ratio of the bacterial liquid to the feces is 0.1-0.3 mL / g (eg, 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 gas-producing Klebsiella N1 bacterial liquid is: picking a single colony of gas-producing Klebsiella N1 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.) until the logarithmic growth phase; the formula of the NB liquid culture medium is as follows (g / L): peptone 10.0, beef extract powder 3.0, sodium chloride 5.0, the balance is water, pH 7.2±0.2, 25°C.

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

[0020] Preferably, the VOCs in livestock and poultry farms are VOCs in the odor of pig farms. The VOCs in pig farms have complex components, mainly including volatile fatty acids (VFA), olefins, alkanes, aromatic hydrocarbons, halogenated hydrocarbons, esters, ethers, phenols, indoles, volatile organic sulfides (VSCs), aldehydes and ketones. The Klebsiella aeruginosa N1 provided in the present disclosure is particularly suitable for removing VOCs in the odor of pig farms.

[0021] The beneficial effects of the present invention compared to the prior art include but are not limited to:

[0022] First, the gas-producing Klebsiella N1 provided by the present disclosure is a new strain that can degrade VOCs in livestock and poultry farms. In recent years, the scale of my country's livestock and poultry farming industry has continued to increase, and the treatment of polluted gases from livestock and poultry farming is particularly important. Among them, VOCs in livestock and poultry farms have complex components, high dust content, and continuous emissions, making them difficult to treat. Therefore, it is very important to screen out strains that can degrade VOCs in livestock and poultry farms. The gas-producing Klebsiella N1 provided by the present disclosure provides a good foundation for degrading VOCs in livestock and poultry farms.

[0023] Second, adding the bacterial liquid prepared by Klebsiella gas-producing N1 to the feces for treatment can effectively degrade VOCs in pig farms, with a degradation rate of up to 85.37%. Klebsiella gas-producing N1 also has different degrees of degradation effects on odor, ammonia and hydrogen sulfide in livestock and poultry farms, and has good industrial application prospects.

[0024] The deposit date of the Klebsiella aerogenes N1 disclosed in the present invention is April 7, 2024, the deposit number is CGMCCNO.30271, the classification name is: Klebsiella aerogenes N1, the name of the depository is: General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short), the address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The VOCs removal rate of the strains with a VOCs removal rate exceeding 50% in the strain screening experiment of Example 1 is shown;

[0026] Figure 2 A colony photograph of Klebsiella aerogenes N1 in Example 2 is shown;

[0027] Figure 3 The phylogenetic tree of Klebsiella aerogenes N1 in Example 2 is shown;

[0028] Figure 4 The growth curve of Klebsiella aerogenes N1 in Example 3 is shown. DETAILED DESCRIPTION

[0029] 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.

[0030] The technical solution of the present disclosure will be described below in conjunction with exemplary embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] Example 1 Isolation and screening of Klebsiella aerogenes N1

[0034] (1) Preparation of strains and culture medium:

[0035] The strain originated from a filter element sample of a biotrickling filter in a pig farm in Chongqing.

[0036] Select NB medium, YPD medium, and MRS medium to isolate strains from samples. The components of each medium are as follows:

[0037] 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;

[0038] 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;

[0039] 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;

[0040] The solid culture medium corresponding to the above three liquid culture media was prepared by adding 16 g / L agar to the above formula.

[0041] (2) Separation and purification:

[0042] The strains in the sample obtained in step (1) are separated and purified. Under a sterile operating environment, 10 g of the sample is added to 90 mL of physiological saline and mixed, and gradient dilution is performed with sterile distilled water to obtain a bacterial suspension. 100 μL of the bacterial suspension is spread on solid culture media (NB culture media, YPD culture media, and MRS culture media, respectively), inverted in an incubator, and cultured at 37°C for 24 hours. In an ultra-clean workbench, a sterile inoculation loop is used to pick single colonies of different morphologies and streak them on the solid culture media. After repeating the streaking on the plate 3 times or more, the purified strains are obtained, and the strains are stored in -20°C glycerol for later use. A total of 50 strains are isolated and preserved, numbered N1-N50.

[0043] (3) Screening of strain degradation performance of VOCs:

[0044] The above strains were inoculated into the corresponding liquid culture medium described in step (1) 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-foamed feces. 50g of water-foamed feces was taken in 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 exhaust the gas, the air bag was sealed after being inflated uniformly with a pump for 30s. It was cultured at a constant temperature of 30°C for 24 hours, and the VOCs concentration was directly detected by an odor concentration detector (the manufacturer and model is AIRSENSE OlfoSense ModuleV1.02). Calculate the VOCs removal rate after 24 hours, and select strains with a VOCs removal rate of more than 50% for plotting, such as Figure 1 As shown. Figure 1 It can be seen that strain N1 (isolated and purified from NB culture medium) had the highest average VOCs removal rate after 24 hours, which was 72.36%. Therefore, strain N1 was identified.

[0045] Example 2 Identification and preservation of Klebsiella aerogenes N1

[0046] (1) Strain identification

[0047] Morphological identification: strain N1 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 N1 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.

[0048] Identification by molecular biology methods: The genomic DNA of strain N1 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 16S rDNA region sequence of the N1 strain. The amplified size was about 1.5 bp, and the 16S rDNA 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 N1 was identified as Klebsiella aerogenes. The phylogenetic tree was constructed using the Neighbor Joining method of MAGE6 software, as shown in Figure 2. Figure 3The 16S rDNA sequence of Klebsiella aerogenes N1 is as follows:

[0049]

[0050]

[0051] (2) Preservation of strains

[0052] The inventor named strain N1 as Klebsiella aerogenes N1 and submitted it for preservation. The preservation date is April 7, 2024, the preservation number is CGMCC NO.30271, the classification name is: Klebsiella aerogenes N1, the name of the preservation unit is: General Microbiology Center of China Microorganism Culture Collection Administration (referred to as CGMCC), the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0053] Example 3 Growth curve determination of Klebsiella aerogenes N1

[0054] The growth curve of the new strain Klebsiella aerogenes N1 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 Klebsiella aerogenes N1 as shown in the figure. Figure 4 shown.

[0055] from Figure 4 It can be seen that at 0-6h, Klebsiella aerogenes N1 is in the delay period of the growth stage, and its growth rate is slow; at 6-14h, Klebsiella aerogenes N1 enters the logarithmic growth phase, and its growth rate reaches the maximum; after 14h, Klebsiella aerogenes N1 enters the stable period, and the growth state basically maintains a dynamic balance.

[0056] Example 4 Performance test of Klebsiella aerogenes N1

[0057] (1) Strain non-toxicity test

[0058] Klebsiella aerogenes N1 was inoculated into NB liquid culture medium and cultured at 37°C, 150rpm, and shaken for 12 hours. The culture medium 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 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.

[0059] (2) Testing of strain degradation performance of VOCs

[0060] The method for testing the performance of the strain in degrading VOCs was the same as in Example 1, with three parallel tests. The results are shown in Table 1.

[0061] (3) Testing of the odor degradation performance of strains

[0062] Strain N1 was inoculated into NB liquid culture medium, and cultured at 37℃, 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-foaming feces. 50g of water-foaming 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 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℃ for 24h, and the odor concentration was detected. The odor removal rate after 24h was calculated. The results are shown in Table 1.

[0063] (4) Detection of NH3 degradation performance of strains

[0064] The strain N1 was inoculated into NB liquid culture medium, and cultured at 37℃, 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-foaming feces. 50g of water-foaming feces was taken into 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 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℃ for 24h, and the NH3 concentration was detected. The NH3 removal rate after 24h was calculated. The results are shown in Table 1.

[0065] (5) Testing of H2S degradation performance of strains

[0066] The strain N1 was inoculated into NB liquid culture medium, and cultured at 37℃, 150rpm, and shaken for 12h to obtain bacterial solution. Pig feces and pig sewage were mixed in a mass ratio of 1:1 to obtain water-foaming feces. 50g of water-foaming feces was taken into 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 placed in a 3L air bag and sealed to discharge 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℃ for 24h, and the H2S concentration was detected. The H2S removal rate after 24h was calculated. The results are shown in Table 1.

[0067] Table 1 Removal rate of VOCs, odor, NH3 and H2S by Klebsiella aeruginosa N1 in Example 4

[0068]

[0069] In summary, in the embodiments of the present disclosure, the highest removal rates of Klebsiella aeruginosa N1 for VOCs, odor, NH3 and H2S in livestock and poultry farms are 85.37%, 68.22%, 63.95% and 53.68%, respectively, indicating that Klebsiella aeruginosa N1 in the present disclosure has different degrees of degradation effects on VOCs, odor, NH3 and H2S in livestock and poultry farms. In previous studies, no examples of Klebsiella aeruginosa degradation of VOCs have been found. Klebsiella aeruginosa N1 in the present disclosure is particularly suitable for treating complex mixed gases such as VOCs in livestock and poultry farms, and at the same time has different degrees of degradation effects on odor, ammonia and hydrogen sulfide in livestock and poultry farms, and has good application prospects.

[0070] 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.

[0071] 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 Klebsiella aerogenes, which is Klebsiella aerogenes N1, with a deposit number of CGMCC NO.30271.

2. A bacterial agent prepared from the Klebsiella aerogenes N1 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, characterized in that The bacterial liquid contains at least 1×10 8 CFU / mL.

4. The bacterial 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 1 to 3, characterized in that The preparation method of the bacterial liquid comprises: picking a single colony of Klebsiella aeruginosa N1 with a sterile inoculation loop and inoculating it into a NB liquid culture medium, culturing at 30-37°C, 120-180rpm, and shaking for 10-24h; the formula of the NB liquid culture medium is as follows: 10.0g / L peptone, 3.0g / L beef extract powder, 5.0g / L 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.

6. Use of the Klebsiella aeruginosa N1 according to claim 1 in degrading VOCs in livestock and poultry farms.

7. The use according to claim 6, characterized in that: The application comprises: preparing the bacterial agent according to any one of claims 1 to 5; and adding the bacterial agent to water-soaked feces for treatment.

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

9. The use according to claim 8, 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, characterized in that: The processing time is 20-30h.

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