A new strain of Klebsiella aerogenes and its application in degrading VOCs in livestock and poultry farms

Abstract

The present disclosure provides a new strain of Klebsiella aerogenes, which is Klebsiella aerogenes N1, with a deposit number of CGMCC NO.30271. Also provided is a bacterial agent prepared from the above-mentioned Klebsiella aerogenes N1, which is a bacterial liquid cultured to the logarithmic growth phase. In addition, a use of the above-mentioned Klebsiella aerogenes N1 in degrading VOCs in livestock and poultry farms is provided. The Klebsiella aerogenes N1 provided in the present disclosure is a new bacterial strain that can degrade VOCs in livestock and poultry farms. The bacterial liquid prepared from Klebsiella aerogenes N1 is added to water-soaked feces for treatment, which can effectively degrade VOCs in pig farms, with a degradation rate of up to 85.37%. At the same time, it has different degrees of degradation effects on the odor, ammonia and hydrogen sulfide in livestock and poultry farms, and has good application prospects.

Description

Technical Field

[0001] The present disclosure belongs to the field of environmental microbial technology, and particularly relates to a new strain of Klebsiella aeruginosa and its application in degrading VOCs in livestock and poultry farms. Background Art

[0002] The 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, as the scale of livestock and poultry farming continues to increase, the 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 its complex composition 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 ) precursors, PM 2.5 VOCs can harm the human respiratory system and are a major cause of smog. Repeated photochemical reactions in the atmosphere produce ozone, forming photochemical smog that harms human health and the environment. Therefore, strengthening research on VOC control is urgent.

[0003] Polluted gas treatment technologies are categorized into two main categories: recovery and decomposition. Recovery is primarily physical, while decomposition is primarily chemical and biological. Recovery technology is suitable for gaseous pollutants with high concentrations and potential for recovery; decomposition technology breaks down gaseous pollutants into largely harmless small molecules. VOCs from livestock and poultry farms have complex compositions, high dust content, and continuous emissions. Considering the construction and operating costs of treatment equipment, biological methods are the most suitable treatment method for VOCs from livestock and poultry farms. Therefore, there is an urgent need to identify microorganisms that can effectively degrade VOCs from livestock and poultry farms.

[0004] The inventors of the present disclosure screened and obtained a new strain of Klebsiella aerogenes N1, with a deposit 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 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 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, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The Klebsiella aerogenes N1 provided in the present disclosure 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 isolated and purified to obtain pure bacteria N1. Experiments found that N1 can degrade VOCs, and identification showed that N1 is Klebsiella aerogenes.

[0009] In a second aspect, the present disclosure further 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 preparation method of the bacterial liquid comprises: picking a single colony of Klebsiella aeruginosa N1 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.), and shaking culture for 10-24h (such as 12h, 15h, 18h, 20h, 22h, etc.); the NB liquid culture medium has the following formula (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 further provides the use of the above-mentioned Klebsiella aerogenes N1 in degrading VOCs in livestock and poultry farms.

[0013] Preferably, the application comprises: preparing the bacterial liquid of Klebsiella aerogenes N1; and adding the bacterial liquid into water-soaked 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 9 CFU / 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 aerogenes Klebsiella N1 bacterial liquid is: picking a single colony of 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.) to 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. Pig farm VOCs have a complex composition, primarily including volatile fatty acids (VFAs), alkenes, alkanes, aromatic hydrocarbons, halogenated hydrocarbons, esters, ethers, phenols, indoles, volatile sulfur compounds (VSCs), aldehydes, and ketones. The Klebsiella aeruginosa N1 provided herein is particularly suitable for removing VOCs in the odor of pig farms.

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

[0022] First, the Klebsiella aeruginosa N1 provided by the present disclosure is a new strain capable of degrading VOCs from livestock and poultry farms. In recent years, the scale of my country's livestock and poultry farming industry has continued to increase, making the treatment of polluted gases from livestock and poultry farming particularly important. VOCs from livestock and poultry farms present complex compositions, high dust content, and persistent emissions, making their treatment difficult. Therefore, screening for strains capable of degrading VOCs from livestock and poultry farms is crucial. The Klebsiella aeruginosa N1 provided by the present disclosure provides a good foundation for degrading VOCs from livestock and poultry farms.

[0023] Second, adding the bacterial liquid prepared by Klebsiella gas-producing N1 to the water-soaked 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 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

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

[0026] Figure 2 shows a colony photograph of Klebsiella aerogenes N1 in Example 2;

[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 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.

[0030] The technical solutions 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] NB medium, YPD medium, and MRS medium were used to isolate the strains from the 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%, balance water. The pH of the prepared medium at 25°C is 7.2±0.2.

[0038] 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.

[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, balance water. The pH of the medium at 25°C was 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) 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 media (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 solid culture medium. After repeating the streaking on the plate 3 or more times, the purified strains were obtained and the strains were stored in glycerol at -20°C for later use. A total of 50 strains were 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 shaken for 12 hours to obtain bacterial solution. Pig feces and pig sewage were mixed at a mass ratio of 1:1 to obtain water-soaked feces. 50g of water-soaked feces was 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 parallel experiments were performed for each strain. After being placed in a 3L air bag and sealed to discharge the gas, the air bag was uniformly inflated for 30s with a pump and then sealed. The air bag was cultured at a constant temperature of 30°C for 24 hours, and the VOCs concentration was directly detected by an odor concentration detector (manufacturer and model is AIRSENSE OlfoSense ModuleV1.02). The VOCs removal rate after 24 hours was calculated, and strains with a VOCs removal rate of more than 50% were selected for plotting, as shown below. Figure 1 As shown. Figure 1 It can be seen that strain N1 (isolated and purified from NB culture medium) has the highest average VOCs removal rate after 24 hours, which is 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 was as shown in the figure below. 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 biological 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 the following figure. Figure 3The 16S rDNA sequence of Klebsiella aerogenes N1 is as follows:

[0049]

[0050]

[0051] (2) Preservation of strains

[0052] The inventor named the strain N1 Klebsiella aerogenes N1 and submitted it for deposit. The deposit date was April 7, 2024, the deposit number was CGMCC NO.30271, the classification name was: Klebsiella aerogenes N1, the name of the depository was: General Microbiology Center of China Culture Collection Administration (CGMCC for short), the address was: 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 from 0 to 6 hours, Klebsiella aerogenes N1 is in the delay period of the growth stage, and its growth rate is slow; from 6 to 14 hours, Klebsiella aerogenes N1 enters the logarithmic growth period, and its growth rate reaches the maximum; after 14 hours, Klebsiella aerogenes N1 enters the stable period, and the growth state basically maintains a dynamic balance.

[0056] Example 4 Performance Testing of Klebsiella aerogenes N1

[0057] (1) Strain non-toxicity test

[0058] Klebsiella aeruginosa N1 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 (LD50). 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.

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

[0060] The strain's VOC degradation performance was tested using the same method as in Example 1, with three replicates. The results are shown in Table 1.

[0061] (3) Detection of odor degradation performance of strains

[0062] Strain N1 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 pig 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. The bag was then inflated with a pump for 30 seconds, sealed, and incubated at a constant temperature of 30°C for 24 hours. Odor concentration was measured and the odor removal rate was calculated after 24 hours. Results are shown in Table 1.

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

[0064] Strain N1 was inoculated into NB liquid medium and cultured with shaking at 37°C, 150 rpm, for 12 hours to obtain a bacterial suspension. Pig feces and pig 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. The bag was then inflated with a pump for 30 seconds, sealed, and incubated at a constant temperature of 30°C for 24 hours. NH3 concentrations were measured. NH3 removal rates were calculated after 24 hours. Results are shown in Table 1.

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

[0066] Strain N1 was inoculated into NB liquid medium and cultured with shaking at 37°C and 150 rpm for 12 hours to obtain a bacterial suspension. Pig feces and pig 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. The bag was then inflated with a pump for 30 seconds, sealed, and incubated at a constant temperature of 30°C for 24 hours. H2S concentration was measured. The H2S removal rate after 24 hours was calculated. The results are shown in Table 1.

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

[0068]

[0069] In summary, the Klebsiella aerogenes N1 in the embodiments disclosed herein has the highest removal rates of 85.37%, 68.22%, 63.95%, and 53.68% for VOCs, odor, NH3, and H2S in livestock and poultry farms, respectively. This indicates that the Klebsiella aerogenes N1 in the embodiments disclosed herein has varying degrees of degradation effects on VOCs, odor, NH3, and H2S in livestock and poultry farms. Previous studies have not found examples of Klebsiella aerogenes degrading VOCs. The Klebsiella aerogenes N1 in the embodiments disclosed herein is particularly suitable for treating complex mixed gases such as VOCs in livestock and poultry farms, and also has varying degrees of degradation effects on odor, ammonia, and hydrogen sulfide in livestock and poultry farms, showing promising application prospects.

[0070] 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.

[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 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 new strain of Klebsiella aerogenes, 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 the logarithmic growth phase or the stationary 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 bacterial liquid preparation method includes: using a sterile inoculating loop to pick a single colony of Klebsiella aeruginosa N1 and inoculate it into NB liquid culture medium, and shaking culture at 30-37°C, 120-180 rpm for 10-24 hours; the NB liquid culture medium has the following formula: 10.0 g / L peptone, 3.0 g / L beef extract powder, 5.0 g / 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 aerogenes N1 according to claim 1 in degrading volatile organic compounds (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 2 to 5; and adding the bacterial agent to water-soaked feces for treatment.

8. The use according to claim 7, characterized in that The water-soaked feces is 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 20-30 hours.

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