A deodorizing strain, a deodorizing bactericide and their applications

Abstract

The present invention belongs to the technical fields of biological agents and environmental engineering, and particularly relates to a deodorizing strain, a deodorizing bacterial agent and their applications. The deodorizing strain of the present invention has a good deodorizing effect. On this basis, there is no antagonistic effect among the strains of the compound bacterial agent obtained by compounding three strains. Through the synergistic effect among different deodorizing strains, the average removal effect on NH3 and H2S is better, and the deodorizing effect is stable and lasting. It is found through experiments that, compared with the single deodorizing strain, the composite deodorizing bacterial agent composed of the above three deodorizing strains improves the sulfur removal rate on the basis of maintaining a high ammonia removal rate, and the sulfur removal rate is close to 80%, solving the problem of low sulfur removal rate in previous studies. At the same time, the deodorizing bacterial agent of the present invention is simple to operate and low in cost during the application process, solving the problems of the traditional deodorizing bacterial agent being ineffective and slow to take effect during the stacking and composting treatment of traditional livestock and poultry manure and domestic waste.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological agents and environmental engineering, and particularly relates to a deodorizing strain, a deodorizing agent and their applications. Background Art

[0002] While the livestock and poultry breeding industry provides people with high-quality meat, eggs and milk, it also generates a large amount of livestock and poultry breeding manure and sewage, and the problem of malodor caused by livestock and poultry breeding manure and sewage is also increasing. Animal feces and urine, animal skin, moldy bedding, feed, dust, sewage ditches, sewage treatment facilities, etc. are all the main sources of odor generation. The malodorous gases generated during the livestock and poultry breeding process mainly include volatile sulfur-containing compounds, ammonia and volatile amines, volatile fatty acids, phenols, indoles, etc., which have the characteristics of strong odor and low threshold, bringing certain risks to the surrounding environment of the farm and human health. NH3 and H2S are the main components of the odor generated during the livestock and poultry breeding process. NH3 is mainly produced by the decomposition of livestock and poultry feces and urine by bacteria and the action of bacteria in the animal intestine. H2S is mainly produced by the degradation of sulfur-containing organic matter in fresh livestock and poultry feces under the action of anaerobic microorganisms, and has the characteristics of colorless, volatile and stinky egg odor. Therefore, the treatment of livestock and poultry manure malodorous gas pollution has become a relatively serious problem at present.

[0003] The treatment of livestock and poultry manure malodorous gas includes physical, chemical and microbial treatment methods. However, the physical and chemical methods for deodorization are expensive, require high technology, and are prone to secondary pollution; while the microbial treatment method has low deodorization cost, simple operation and good effect, and has become the main way for livestock and poultry manure deodorization. The microbial deodorization technology mainly uses the physiological metabolism activities of a variety of beneficial microorganisms to degrade malodorous substances and convert them into carbon dioxide, water, inorganic salts, etc., reducing the occurrence of malodor.

[0004] There are some functional microorganisms with deodorizing effects in media such as compost, biogas slurry and biogas residue, mainly including Bacillus subtilis, Bacillus licheniformis, Alcaligenes faecalis, Pseudomonas, etc. At present, at home and abroad, the method of compounding a variety of microbial agents is mostly used to improve the odor reduction effect. Some scholars have shown that the compound deodorizing agent has a certain deodorizing effect on fresh feces and stale feces, and has obvious removal effects on NH3, H2S and volatile organic acids. However, the types of deodorizing strains found at present are few and the action effects are relatively single, making it difficult to meet the requirements of the current odor-related emission standards. Summary of the Invention

[0005] The purpose of the present invention is to provide a deodorizing strain, a deodorizing agent and their applications. The present invention newly discovers three deodorizing strains, and the deodorizing effects of the three deodorizing strains and the deodorizing agent compounded based on the three deodorizing strains are better and the effects are stable and lasting.

[0006] The present invention provides a deodorizing strain, including Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 or Providencia sp. CM18; the preservation numbers of Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18 are CGMCC No. 31631, CGMCC No. 31632 and CGMCC No. 31633 in sequence.

[0007] The present invention also provides a deodorizing agent, which includes the deodorizing strain;

[0008] The deodorizing strain includes one or more of Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18;

[0009] The preservation numbers of Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18 are CGMCC No. 31631, CGMCC No. 31632 and CGMCC No. 31633 in sequence.

[0010] Preferably, the deodorizing agent includes the bacterial suspension of the deodorizing strain and / or the fermentation broth of the deodorizing strain.

[0011] Preferably, the deodorizing agent includes the fermentation broth of Providencia vermicola CM1, the fermentation broth of Pseudochrobactrum asaccharolyticum CM5 and the fermentation broth of Providencia sp. CM18; the volume ratio of the fermentation broth of Providencia vermicola CM1, the fermentation broth of Pseudochrobactrum asaccharolyticum CM5 and the fermentation broth of Providencia sp. CM18 is 0.5 - 2.0:0.5 - 2.0:0.5 - 2.0.

[0012] Preferably, the effective viable counts of Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18 in the deodorizing agent are each ≥ 10 8 cfu / mL.

[0013] The present invention also provides the application of the deodorizing strain or the deodorizing agent described in the above technical solution in odor treatment.

[0014] Preferably, the components of the odor include one or more of NH3, H2S and volatile organic acids.

[0015] Preferably, the odor includes the odor generated by livestock and poultry manure and / or domestic waste.

[0016] The present invention also provides a method for removing odor, which is to spray the deodorant bacteria agent described in the above technical solution onto the material containing odor; the spraying amount of the deodorant bacteria agent is 5% - 15% of the mass of the material containing odor.

[0017] Preferably, the material containing odor includes livestock and poultry manure and / or domestic waste.

[0018] Beneficial effects:

[0019] The present invention provides a deodorizing strain, including Providencia vermicola CM1, Pseudogracilibacter asaccharolyticus CM5 or Providencia sp. CM18; the preservation numbers of Providencia vermicola CM1, Pseudogracilibacter asaccharolyticus CM5 and Providencia sp. CM18 are CGMCC No. 31631, CGMCC No. 31632 and CGMCC No. 31633 in sequence. The deodorizing strain described in the present invention has a good deodorizing effect. On this basis, there is no antagonistic effect between the strains in the compound bacteria agent obtained by compounding the three strains. Through the synergistic effect between different deodorizing strains, the average removal effect on NH3 and H2S is optimal, and the deodorizing effect is stable and lasting; through experiments, it is found that compared with the single deodorizing strain, the composite deodorant bacteria agent composed of the above three deodorizing strains improves the sulfur removal rate on the basis of maintaining a high ammonia removal rate, and the sulfur removal rate is close to 80%, solving the problem of low sulfur removal rate in previous studies. At the same time, the deodorant bacteria agent described in the present invention is simple to operate and low in cost during the application process, solving the problems of ineffective and slow-acting traditional deodorant bacteria agents in the stacking and composting treatment processes of livestock and poultry manure and domestic waste in the past, and alleviating the environmental pollution in the treatment of waste such as manure.

[0020] Biological preservation description

[0021] Providencia vermicola CM1, taxonomically belonging to Providencia vermicola, was preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 7, 2024. The preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No. 31631;

[0022] Pseudochrobactrum asaccharolyticum CM5, taxonomically belonging to Pseudochrobactrum asaccharolyticum, was deposited on August 7, 2024 at the China General Microbiological Culture Collection Center (CGMCC). The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31632;

[0023] Providencia sp. CM18, taxonomically belonging to Providencia sp., was deposited on August 7, 2024 at the China General Microbiological Culture Collection Center (CGMCC). The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31633. Brief Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0025] Figure 1 It is the NH3 removal rate of different strains within 8 days during the secondary screening in Example 1;

[0026] Figure 2 It is the H2S removal rate of different strains within 8 days during the secondary screening in Example 1;

[0027] Figure 3 It is the NH3 removal rate of the composite deodorant bacteria agent under different spraying amounts in Example 2;

[0028] Figure 4 It is the H2S removal rate of the composite deodorant bacteria agent under different spraying amounts in Example 2;

[0029] Figure 5 It is the average removal rate of the composite deodorant bacteria agent under different spraying amounts within 8 days in Example 2. Detailed Description of the Invention

[0030] The present invention provides a deodorizing strain, including Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 or Providencia sp. CM18; the deposit numbers of Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18 are CGMCC No. 31631, CGMCC No. 31632 and CGMCC No. 31633 in sequence.

[0031] The deodorizing strains of the present invention are isolated from chicken manure compost and pig manure compost samples. Through 16S rRNA sequence determination and homologous alignment analysis, it is found that the similarity between Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18 and Providencia vermicola, Pseudochrobactrum asaccharolyticum and Providencia sp. is 100%, 100% and 99.929% respectively. Furthermore, their biological classifications are confirmed and they are respectively subjected to biological preservation.

[0032] Pseudochrobactrum and Providencia are mostly seen in the soil remediation and sewage treatment contaminated by pesticides, heavy metals and organic compounds in some studies, and there is no relevant report in the field of deodorization. Therefore, the present invention discovers the new functions of Pseudochrobactrum and Providencia, providing a new direction for the exploration of deodorizing strains to a certain extent.

[0033] The present invention also provides a deodorizing bactericide, which comprises one or more of the deodorizing strains described in the above technical solution, preferably two or three of the deodorizing strains, and more preferably three of the deodorizing strains. The deodorizing bactericide of the present invention preferably comprises the bacterial suspension of the deodorizing strain and / or the fermentation broth of the deodorizing strain, and more preferably the fermentation broth of the deodorizing strain. The deodorizing bactericide of the present invention preferably comprises the fermentation broth of Providencia vermicola CM1, the fermentation broth of Pseudochrobactrum asaccharolyticum CM5 and the fermentation broth of Providencia sp. CM18; the volume ratio of the fermentation broth of Providencia vermicola CM1, the fermentation broth of Pseudochrobactrum asaccharolyticum CM5 and the fermentation broth of Providencia sp. CM18 is preferably 0.5-2.0:0.5-2.0:0.5-2.0, and more preferably 1:1:1; the effective viable counts of Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18 in the deodorizing bactericide are respectively preferably ≥10 8 cfu / mL. The present invention has no special limitation on the preparation methods of the bacterial suspension of the deodorizing strain and the fermentation broth of the deodorizing strain, and the preparation steps of the fermentation broth and bacterial suspension of Pseudochrobactrum and Providencia in the art can be adopted.

[0034] The present invention also provides the application of the deodorizing strain described in the above technical solution or the deodorizing bactericide described in the above technical solution in odor treatment. The components of the odor of the present invention preferably include one or more of NH3, H2S and volatile organic acids, and further preferably include NH3 and / or H2S. The odor of the present invention preferably includes the odor generated by livestock and poultry manure and / or domestic waste; the odor generated by the domestic waste preferably includes the odor generated during the composting and / or burial of domestic waste.

[0035] The present invention also provides a method for removing odors, spraying the deodorizing bacteria agent described in the above technical solution onto a material containing odors for deodorization treatment; the spraying amount of the deodorizing bacteria agent is 5% to 15% of the mass of the material containing odors. The spraying amount of the deodorizing bacteria agent described in the present invention is preferably 5% to 10% of the mass of the material containing odors, and more preferably 10%. The odor-containing material described in the present invention preferably includes livestock and poultry manure and / or domestic garbage. The duration of the deodorization treatment described in the present invention is preferably ≤8 days. The present invention does not specifically limit the spraying method and specific process, and the conventional spraying method and specific process in this field can be used.

[0036] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] 1. Screening of deodorizing microbial strains

[0039] 1. Strain source

[0040] The bacteria source used in the present invention comes from chicken manure compost and pig manure compost samples.

[0041] 2. Enrichment and isolation of strains

[0042] 10 g of chicken manure compost sample from Shangzhuang Experiment Station of China Agricultural University was added into a sterile conical flask containing 90 mL of sterile water and glass beads and placed at 30 °C and 180 r / min. -1 The enrichment culture was shaken for 30 minutes. The enrichment culture solution was diluted 10 times, and 0.1 mL of the sample dilution solution was pipetted onto each solid plate (NB culture medium) and spread evenly. After standing for 20 minutes, the culture was placed at 30°C for 24 hours and the results were observed.

[0043] 10 g of pig manure compost sample from Shangzhuang Experimental Station of China Agricultural University was inoculated into 100 mL of NH3 and H2S selective culture medium respectively, and the mixture was incubated at 30 °C and 180 r·min. -1 The culture was shaken to obtain the acclimated culture fluid. After 2 days, the culture medium was replaced by taking 10 mL of the acclimated culture fluid and adding it to 100 mL of fresh selective culture medium for the second generation acclimation. The enrichment and acclimation were repeated 4 times. Finally, the enrichment culture fluid of each sample was diluted by 10 times, and 0.1 mL of the sample dilution was taken and spread evenly on each solid plate. The plate was placed at 30°C for constant temperature culture for 24 hours and the results were observed.

[0044] A total of 46 strains were isolated, of which 33 were isolated from chicken manure compost samples and 13 were isolated from pig manure compost samples.

[0045] The culture medium formula used is as follows:

[0046] The NH3 selective medium is 50.0 g of sucrose, 10.0 mL of ammonia water, 2.0 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.1 g of FeSO4, 5.0 mL of 1% ZnSO4, 2.0 g of NaCl, 1000 mL of distilled water, and the pH is natural.

[0047] The H2S selective medium is 5.0 g of glucose, 0.5 g of K2HPO4, 1.0 g of KNO3, 0.5 g of MgCl2, 0.5 g of NaCl, 0.5 g of NH4Cl, 1.0 g of Na2CO3, 0.01 g of FeCl2, 1000 mL of distilled water, and the pH is natural.

[0048] The nutrient broth agar medium (NB medium) is 10.0 g of peptone, 0.5 g of sodium chloride, 3.0 g of beef extract powder, 15.0 g of agar, 1000 mL of distilled water, and the pH is 7.3 ± 0.2.

[0049] 3. Purification of strains

[0050] Using the streak plate method, pick single colonies with good growth, different colors and morphologies in each environment from the plate with an inoculation loop and inoculate them onto the corresponding plate, and then continue to culture at the same temperature. Purify the strain 4 times with the three-zone streak plate method to obtain pure strains.

[0051] 4. Primary screening of deodorizing strains

[0052] Take 100 mL of the NH3 selective medium and place it in a shaking flask, inject ammonia water with a volume of 25% of the NH3 selective medium to activate the isolated strain. Take 500 μL of the activated bacterial liquid and inoculate it into the shaking flask, seal it and place it on a shaker at 30 °C and 180 r / min for continuous constant-temperature culture for 5 days, and then observe the change of the bacterial liquid. If the bacterial liquid becomes turbid, it indicates that the strain has the ability to degrade NH3. If the bacterial liquid remains transparent, it indicates that the strain cannot directly utilize NH3.

[0053] Take 200 mL of the H2S selective medium and place it in a shaking flask. After culturing and activating the strain, take 500 μL of the activated bacterial liquid and inoculate it into the medium in the shaking flask. Next, place a 50 mL sterile small beaker in the shaking flask, add 12 mL of 25% H2SO4 to the small beaker, add 4 g of FeS to the small beaker and quickly seal it, and continuously culture it on a shaker at 30 °C and 200 r / min for 5 days, and then observe the turbidity of the bacterial liquid. If it is turbid, it indicates that the strain has the ability to degrade H2S. If the bacterial liquid remains transparent, it indicates that the strain cannot directly utilize H2S.

[0054] The results of the primary screening are shown in Table 1.

[0055] Table 1 Primary screening results

[0056]

[0057] The turbidity of the NH3 selective medium and the H2S selective medium after primary screening was graded (slightly turbid, moderately turbid, severely turbid). Slightly turbid means the medium shows a slightly pale yellow or semi-transparent state, and the growth ability of the strain is weak; moderately turbid means the medium shows a pale yellow color, and the growth ability of the strain is moderate; severely turbid means the medium shows a yellow or yellowish-white color, is significantly turbid, and the growth ability of the strain is strong. The results are shown in Table 2 and Table 3. From the turbidity grading of the NH3 selective medium, 5 strains were slightly turbid, 11 strains were moderately turbid, and 8 strains were severely turbid; from the turbidity grading of the H2S selective medium, 3 strains were slightly turbid, 10 strains were moderately turbid, and 6 strains were severely turbid. It can be found that there are more strains with good removal effect on NH3, and fewer on H2S.

[0058] Table 2 Turbidity grading of NH3 selective medium

[0059] Slight turbidity Moderate turbidity Severe turbidity CM12 CM4 CM1 CM22 CM5 CM2 CM26 CM6 CM3 CM29 CM8 CM7 CM35 CM9 CM17 - CM15 CM18 - CM21 CM23 - CM28 CM34 - CM29 - - CM30 - - CM32 -

[0060] Table 3 Turbidity grading of H2S selective medium

[0061] Slight turbidity Moderate turbidity Severe turbidity CM1 CM2 CM4 CM10 CM3 CM5 CM36 CM6 CM9 - CM12 CM23 - CM16 CM28 - CM17 CM32 - CM18 - - CM21 - - CM22 - - CM34 -

[0062] Based on the turbidity of the NH3 and H2S media in Tables 2 - 3 and the differences in the morphology and growth status of the strains, it was found that 13 strains had good removal abilities for both NH3 and H2S.

[0063] 5. Rescreening of deodorizing strains:

[0064] The 13 strains selected by primary screening were used for a rescreening culture test with pig manure for 8 days, and the NH3 and H2S absorption solutions were measured every 2 days to determine the deodorizing effect of the strains.

[0065] The specific operation is to incubate different strains in NB liquid medium at 30 °C and 180 r·min -1 until the viable cell count ≥ 10 8cfu / mL, add the bacterial solution to a 1-L plastic bucket with a lid containing 200 g of pig manure at an inoculation amount of 10% by volume, and mix well with a glass rod. Place one 50-mL small beaker containing 20 mL of 2% boric acid absorption solution in each large beaker to absorb NH3. Seal the bucket with double-layer plastic wrap and then cover the bucket lid. The control group (CK) is an equal amount of sterile water, with 3 replicates in each group; place one 50-mL small beaker containing 20 mL of alkaline zinc ammonium complex solution to absorb H2S. The boric acid absorption titration method is used to determine the NH3 release amount, and the removal rate of NH3 under laboratory culture conditions is calculated. The zinc ammonium complex colorimetric method is used to determine the H2S release amount, and the H2S removal rate is calculated. Each treatment has 3 replicates. The release amounts of NH3 and H2S are measured every 2 days, and the measurement period is 8 days.

[0066] Determination method of NH3: The boric acid absorption Kjeldahl method is used to determine the NH3 release amount. First, after 20 mL of 2.0% boric acid solution absorbs NH3 in the experimental group, add two drops of methyl red-bromocresol green indicator, and titrate it with a sulfuric acid standard solution until the color changes from blue-green to light red, and record the consumption of the sulfuric acid solution. Calculate according to the formula: C = (c × v × 2 × 1000 × 17) / V to obtain the NH3 concentration. In the formula, C represents the NH3 concentration, with the unit of mg / L; c is the molar concentration of the sulfuric acid solution, with the unit of mol / L; v represents the volume of the consumed sulfuric acid solution, with the unit of mL; 17 is the mass fraction of NH3, with the unit of g / mol; V represents the volume of the 2% boric acid absorption solution, with the unit of mL.

[0067] Determination method of H2S: The zinc ammonium complex absorption colorimetric method is used to determine the H2S release amount. After the zinc ammonium complex absorbs H2S gas, add the mixed display solution, shake well, let it stand for 30 min, add 2 drops of 40% diammonium hydrogen phosphate solution, and measure the absorbance at a wavelength of 665 nm. The calculation formula is as follows, C = (A - A0)Bs / Vs. In the formula, C is the H2S concentration, with the unit of mg / m 3 ; A is the absorbance of the sample color-developing solution; A0 is the absorbance of the blank solution; Bs is the reciprocal of the slope, with the unit of μg / absorbance; Vs is the sampling volume converted to the standard condition, with the unit of L.

[0068] Calculation formulas for NH3 removal rate and H2S removal rate: Removal rate = (blank concentration - treatment concentration) / blank concentration. The average NH3 removal rate and the average H2S removal rate are the averages of the removal rates within 8 days.

[0069] The reagents used are:

[0070] 2% boric acid: Weigh 10 g of boric acid powder and make up to 500 mL with distilled water.

[0071] 0.02 mol / L Hydrochloric Acid: Take 1.67 mL of concentrated hydrochloric acid, add it to distilled water, stir and mix well, and make up the volume to 1000 mL.

[0072] 1 mol / L Sodium Hydroxide Solution: Dissolve 20 g of NaOH solid in distilled water and make up the volume to 500 mL.

[0073] 0.1% Bromocresol Green Indicator: Dissolve 1 g of bromocresol green in 1000 mL of 95% ethanol.

[0074] 0.2% Methyl Red Indicator: Dissolve 2 g of methyl red in 1000 mL of 95% ethanol.

[0075] Methyl Red - Bromocresol Green Indicator: Mix 0.1% bromocresol green indicator and 0.2% methyl red indicator in a ratio of 3:1.

[0076] Zinc - Ammonium Complex Salt Solution: Take 5 g of zinc sulfate ZnSO4 and dissolve it in 500 mL of distilled water. Take 6 g of NaOH and dissolve it in 300 mL of distilled water. Mix the above zinc sulfate solution and NaOH solution. During stirring, add 70 g of ammonium sulfate (NH4)2SO4. After dissolution, add 50 g of glycerol and make up the volume to 1 L with distilled water.

[0077] p - Aminodimethylaniline Solution Stock Solution: Add 50 mL of H2SO4 to 30 mL of distilled water. After the solution stops heating, add 12 g of p - aminodimethylaniline hydrochloride to the solution.

[0078] The formula for the p - aminodimethylaniline solution working solution is: Measure 2.5 mL of the stock solution and dilute it to 100 mL with 1:1 sulfuric acid solution.

[0079] Ferric Chloride Solution: Weigh 100 g of FeCl3·6H2O and dissolve it in water, then make up the volume to 100 mL.

[0080] Mixed Display Solution: Mix according to the ratio of 1 mL of p - aminodimethylaniline working solution and 40 μL of ferric chloride solution.

[0081] 40% Diammonium Hydrogen Phosphate Solution: Weigh 40 g of (NH4)2HPO4 and dissolve it in distilled water, then make up the volume to 100 mL.

[0082] Experiments found that there are certain differences in the deodorization effects of different strains. Strains that showed good deodorization ability in the primary screening also had a certain deodorization ability in the re - screening experiment, such as Figures 1 - 2 and shown in Table 4.

[0083] Table 4 Average Deodorization Rates of Different Strains within 8 Days of Re - screening

[0084] Strain number <![CDATA[NH3 average removal rate / %]]> <![CDATA[Average H2S removal rate / %]]> CM1 77.79a 33.12c CM2 59.31abc 31.81c CM4 34.19de 76.77ab CM5 56.38bcd 76.06ab CM9 50.15a 68.08ab CM12 30.75e 28.81c CM17 42.10cde 24.23c CM18 73.41a 76.63ab CM21 28.45de 37.31c CM23 62.12ab 83.59ab CM28 43.69cde 93.26a CM32 48.79bcd 93.82a CM34 72.56bcde 79.92b

[0085] Note: Different letters in the same column of Table 4 indicate significant differences with P < 0.05.

[0086] From Figures 1 - 2 and Table 4, it can be concluded that: The removal rates of most strains for H2S are higher than those for NH3. In terms of NH3 removal, the NH3 removal rates of 7 strains can reach more than 50%, and among them, 2 strains can even reach more than 70%, showing an obvious effect in inhibiting NH3 release. During the entire culture period, the deodorization ability of most strains shows a trend of first increasing and then decreasing with the extension of time. In terms of H2S removal, the H2S removal rates of 8 strains can reach more than 50%, and among them, 2 strains can even reach more than 90%, showing an extremely obvious effect in inhibiting H2S release.

[0087] 6. Molecular identification of deodorizing strains:

[0088] PCR amplification and sequencing were performed on the gene fragment. The primer sequences used were 27F: 5'-AGAGTTTGATC CTGGCTCAG-3' (SEQ ID NO.1) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.2). The sequencing was entrusted to Shanghai Majorbio for 16S rRNA sequence determination, and the sequencing results were subjected to homology comparison and analysis with the sequences in the NCBI database. The results are shown in Table 5. The final results show that a total of 3 strains, Pseudochrobactrum asaccharolyticum, Providencia sp., and Providencia vermicola, were isolated and belong to the genera Pseudochrobacterium and Providencia.

[0089] Table 5 Molecular identification of 16 strains

[0090] Strain number Similarity Most homologous type strain 16S rRNA gene sequence CM4 99.854％ Pseudochrobactrum asaccharolyticum 1369bp CM5 100.000％ Pseudochrobactrum asaccharolyticum 1361bp CM2 99.929％ Providencia sp. 1407bp CM9 99.930％ Providencia sp. 1419bp CM12 99.929％ Providencia sp. 1417bp CM17 100.000％ Providencia sp. 1413bp CM18 99.929％ Providencia sp. 1417bp CM21 99.929％ Providencia sp. 1416bp CM28 99.929％ Providencia sp. 1412bp CM32 99.929％ Providencia sp. 1416bp CM34 99.929％ Providencia sp. 1415bp CM1 100.000％ Providencia vermicola 1415bp CM23 99.929％ Providencia vermicola 1410bp

[0091] Example 2

[0092] Preparation of compound high-efficiency deodorizing bactericide

[0093] 1. Optimization of deodorizing strains: By comparing the deodorization rates of different strains in the rescreening, strains with better removal abilities for both NH3 and H2S were selected.

[0094] According to the results of the second screening in Example 1, among the 9 strains of Providencia sp., CM18 showed the best deodorization performance, with the removal rates of NH3 and H2S both as high as over 70%, being 73.41% and 76.63% respectively; followed by CM34 with relatively good deodorization effect, and the removal rates of NH3 and H2S were 72.56% and 79.92% respectively, showing a good deodorization effect. In addition, the removal rates of NH3 and H2S of CM9 were also relatively good, being 50.15% and 68.08% respectively. CM2 only had a relatively good NH3 removal effect, with a removal rate of 59.31%. The H2S removal rates of CM28 and CM32 were over 90%, but the NH3 removal rates were low, both remaining at 40%. The deodorization effects of NH3 and H2S of other strains including CM12, CM17, and CM21 were average, all lower than 50%. Therefore, CM18 and CM34 were preferably selected for the next antagonistic experiment.

[0095] 2. Antagonistic experiment: After the selected strains were cultured in liquid at 30 °C and 180 r·min -1 for 24 h, 5 μL of each bacterial liquid was taken and spotted on the nutrient broth agar medium plate for pairwise antagonistic experiments. Observe the growth of the strains. If the two can grow normally without affecting each other, it is non-antagonistic; if there is an obvious growth boundary between the two, it is antagonistic. Select multiple non-antagonistic deodorizing bacteria. At the same time, according to the deodorization effect of a single strain, select the strain combination with the best NH3 and H2S removal effects.

[0096] Combining CM18 and CM34 of Providencia sp., CM1 and CM23 of Providencia vermicola, and CM4 and CM5 of Pseudochrobactrum asaccharolyticum, pairwise combinations of the three types of bacteria were used for antagonistic experiments. The results showed that there was no obvious antagonistic phenomenon among the bacteria. However, the growth rates of CM4 and CM34 were relatively slow, and the H2S removal rate of CM1 was only 33.12%. Finally, CM1, CM5, and CM18 were preferably selected for compounding, and were respectively named Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5, and Providencia sp. CM18.

[0097] 3. Optimization of the composite deodorant bacteria combination: The selected CM1, CM5, and CM18 were combined in pairs and in threes, and a combination optimization test was carried out using pig manure for 8 days. The NH3 and H2S absorption solutions were measured every 2 days to determine the deodorization effect of the strains. The specific method was the same as the rescreening method of the deodorant strains in Example 1. When inoculating two or three kinds of bacteria, the two kinds of bacteria were inoculated according to the inoculation volume ratio of 1:1, and the total inoculation amount was 10% of the culture medium volume. The three kinds of bacteria were inoculated according to the inoculation volume ratio of 1:1:1, and the total inoculation amount was 10% of the total culture medium volume. The results showed that the average removal rates of NH3 and H2S of the combination of the three strains CM1, CM5, and CM18 were higher than those of other combinations. Therefore, CM1, CM5, and CM18 were selected as the final composite deodorant bacteria combination.

[0098] Table 6 NH3 removal rates of different combinations

[0099] Time (days) / Combination CM1 and CM5 CM1 and CM18 CM5 and CM18 CM1, CM5 and CM18 2 45.22％ 52.07％ 44.77％ 55.00％ 4 56.74％ 60.46％ 52.74％ 64.67％ 6 49.44％ 52.81％ 40.33％ 62.33％ 8 42.57％ 43.59％ 39.26％ 56.84％ <![CDATA[Average NH3 removal rate]]> 49.58％ 52.23％ 44.28％ 59.71％

[0100] Table 7 H2S removal rates of different combinations

[0101] Time (days) / Combination CM1 and CM5 CM1 and CM18 CM5 and CM18 CM1, CM5 and CM18 2 67.33％ 69.67％ 80.44％ 83.00％ 4 60.91％ 66.75％ 76.25％ 81.27％ 6 54.29％ 53.25％ 70.57％ 72.77％ 8 53.78％ 59.85％ 64.38％ 81.09％ <![CDATA[Average H2S removal rate]]> 59.08％ 62.38％ 72.91％ 79.53％

[0102] 4. Verification of the deodorization effect of the composite deodorant bacteria and optimization of process parameters

[0103] The three selected deodorant bacteria were cultured in NB liquid medium at 30 °C and 180 r·min -1 until the viable bacteria count ≥ 10 8 cfu / mL, and then were compounded in equal volume to form a composite deodorant bacteria and sprayed onto pig manure. By setting different spraying amounts, the influence of process parameters on the deodorization effect was explored to determine the spraying scheme with the best deodorization effect.

[0104] The specific operation method is as follows: 5 spraying amounts were set in the experiment, which were 2.5%, 5%, 10%, 15%, and 20% of the pig manure quality respectively. The whole spraying cycle was 8 days, and the generation amounts of NH3 and H2S under different spraying amounts were measured every 2 days. The boric acid absorption titration method was used to measure the NH3 release amount, and the zinc ammonium complex salt absorption method was used to measure the H2S release amount, so as to compare and analyze the removal rates of NH3 and H2S. Each treatment was set with 3 replicates, and the results are as Figures 3 - 5 shown in Table 8 - 10.

[0105] Table 8 NH3 removal rates of the composite deodorant bacteria under different spraying amounts

[0106] Time (days) / Inoculum volume (%) 2.5 5 10 15 20 2 56.67％ 82.59％ 55.00％ 53.33％ 20.00％ 4 25.00％ 65.19％ 64.67％ 60.00％ 15.00％ 6 20.00％ 65.18％ 62.33％ 60.00％ 16.67％ 8 36.84％ 54.19％ 56.84％ 58.42％ 36.84％

[0107] Table 9 H2S removal rates of the composite deodorant bacteria under different spraying amounts

[0108] Time (days) / Inoculum volume (%) 2.5 5 10 15 20 2 65.67％ 70.67％ 83.00％ 77.33％ 68.00％ 4 58.68％ 76.96％ 81.27％ 75.15％ 89.88％ 6 46.08％ 76.44％ 72.77％ 54.43％ 50.31％ 8 69.25％ 58.86％ 81.09％ 48.82％ 40.16％

[0109] Table 10 Average removal rates of the composite deodorizing bactericide with different spraying amounts within 8 days

[0110] Time (days) / Inoculum volume (%) 2.5 5 10 15 20 <![CDATA[Average NH3 removal rate]]> 34.63％ 66.79％ 59.71％ 57.94％ 22.13％ <![CDATA[Average H2S removal rate]]> 59.92％ 70.73％ 79.53％ 63.93％ 62.09％

[0111] Comprehensive Figures 3 - 5 By observing the removal rates of NH3 and H2S in Tables 8 - 10, it is found that when the spraying amounts are 5% and 10%, the removal rates of NH3 and H2S within 8 days of cultivation are relatively high. However, under the condition of a 10% spraying amount, the deodorizing effect is the most stable, and the downward trend of the deodorizing ability with the extension of time is relatively slow. Therefore, when the spraying amount is 10%, the deodorizing effect of the composite deodorizing bactericide is relatively good, and the average removal rates of NH3 and H2S can reach 59.71% and 79.53% respectively.

[0112] From the above embodiments, it can be concluded that: three new deodorizing strains are screened in the present invention, and the three strains are compounded into a composite deodorizing bactericide, which has a high ammonia removal rate and sulfur removal rate, and can be applied to the treatment of waste in various scenarios such as livestock and poultry farms, domestic waste composting plants, and landfills. The deodorizing effect is good and stable and long-lasting.

[0113] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A deodorizing strain, characterized in that, including Providencia larvae CM1, Pseudochrobactrum asaccharolyticum CM5, or Providencia CM18; the preservation numbers of Providencia larvae CM1, Pseudochrobactrum asaccharolyticum CM5, and Providencia CM18 are CGMCC No. 31631, CGMCC No. 31632, and CGMCC No. 31633, respectively; the components of the malodorous gas removed by the deodorizing strain include NH3 and H2S. Providencia vermicola ) CM1, Pseudochrobactrum asaccharolyticum ( Pseudochrobactrum asaccharolyticum ) CM5, or Providencia ( Providencia sp .) CM18; 2. A deodorant bactericide, characterized in that, The deodorizing bactericide comprises deodorizing strains; The deodorizing strain includes one or more of Providencia larvae ( Providencia vermicola ), Pseudogracilibacter asaccharolyticus ( Pseudochrobactrum asaccharolyticum ), and Providencia sp. ( Providencia sp ), namely CM1, CM5, and CM18 respectively; Providencia vermicola ), CM1, Pseudogracilibacter asaccharolyticus ( Pseudochrobactrum asaccharolyticum Pseudochrobactrum asaccharolyticum ), CM5, and Providencia sp. ( Providencia sp Providencia sp ). CM18; The preservation numbers of the Providencia larvae CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp CM18 are CGMCC No.31631, CGMCC No.31632 and CGMCC No.31633 respectively; the components of the odor removed by the deodorizing strains include NH3 and H2S.

3. The deodorant bactericide according to claim 2, characterized in that, The deodorizing bactericide comprises a bacterial suspension of the deodorizing strains and / or a fermentation broth of the deodorizing strains.

4. The deodorant bacterial agent according to claim 3, characterized in that, The deodorizing bactericide comprises a fermentation broth of Providencia larvae CM1, a fermentation broth of Pseudochrobactrum asaccharolyticum CM5 and a fermentation broth of Providencia sp CM18; the volume ratio of the fermentation broth of Providencia larvae CM1, the fermentation broth of Pseudochrobactrum asaccharolyticum CM5 and the fermentation broth of Providencia sp CM18 is 0.5-2.0:0.5-2.0:0.5-2.

0.

5. The deodorant bactericide according to claim 4, characterized in that, The volume ratio of the fermentation broth of Providencia larvae CM1, the fermentation broth of Pseudochrobactrum asaccharolyticum CM5 and the fermentation broth of Providencia sp CM18 is 1:1:

1.

6. The deodorant bactericide according to claim 4, characterized in that The effective viable counts of Providencia larvae CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18 in the deodorizing bactericide are respectively ≥ 10 8 cfu / mL.

7. Use of the deodorizing strains according to claim 1 or the deodorizing bactericide according to any one of claims 2-6 in odor treatment; the components of the odor include NH3 and H2S.

8. A method for removing odors, characterized in that, Spray the deodorizing bactericide according to any one of claims 2-6 onto the material containing the odor; the spraying amount of the deodorizing bactericide is 5%-15% of the mass of the material containing the odor; the components of the odor include NH3 and H2S.

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