Deodorizing bacterial strain, deodorizing microbial agent and application of deodorizing bacterial strain and deodorizing microbial agent

Abstract

The invention belongs to the technical field of biological agents and environmental engineering, and particularly relates to a deodorizing strain, a deodorizing agent and application of the deodorizing strain and the deodorizing agent. The deodorizing strain disclosed by the invention has a relatively good deodorizing effect. On the basis, the strains of the compound fungicide obtained by compounding the three strains have no antagonism, and through the synergistic effect of different deodorization strains, the average removal effect on NH3 and H2S is better, and the deodorization effect is stable and lasting; tests find that compared with a single deodorization strain, the composite deodorization bacterial agent composed of the three deodorization strains has the advantages that the sulfur removal rate is increased and is close to 80% on the basis that the high ammonia removal rate is kept, and the problem that the sulfur removal rate is low in previous research is solved. Meanwhile, the deodorization microbial inoculum is simple to operate and low in cost in the application process, and the problems that a traditional deodorization microbial inoculum is not obvious in effect and slow in effect in the traditional stacking and composting treatment process of livestock manure and household garbage are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological microbial agents and environmental engineering thereof, and specifically relates to a deodorizing strain, a deodorizing microbial agent and application thereof. Background Art

[0002] While the livestock and poultry farming industry provides people with high-quality meat, eggs and milk, it also produces a large amount of livestock and poultry manure and sewage, and the odor problem caused by livestock and poultry manure and sewage is also increasing. Animal feces and urine, animal skin, moldy bedding, feed, dust drainage ditches, sewage treatment facilities, etc. are the main sources of odor. The odorous gases generated in the livestock and poultry farming process mainly include volatile sulfur compounds, ammonia and volatile amines, volatile fatty acids, phenols, indoles, etc., which have the characteristics of strong odor and low threshold, posing certain risks to the environment around the farm and human health. NH 3 and H 2 S is the main component of odor produced during livestock and poultry farming. 3 It is mainly produced by bacteria decomposing livestock and poultry feces, bedding and animal intestinal bacteria. 2 S is mainly produced by the degradation of sulfur-containing organic matter in fresh livestock and poultry manure by anaerobic microorganisms. It is colorless, volatile, and has the smell of rotten eggs. Therefore, the treatment of malodorous gas pollution from livestock and poultry manure has become a more serious problem at present.

[0003] The treatment of malodorous gases from livestock and poultry manure includes physical, chemical and microbial treatment methods, but physical and chemical deodorization methods are expensive, require high technology, and are prone to secondary pollution; while microbial deodorization methods are low in cost, simple to operate, and effective, and have become the main way to deodorize livestock and poultry manure. Microbial deodorization technology mainly utilizes the physiological metabolic activities of a variety of beneficial microorganisms to degrade malodorous substances and convert them into carbon dioxide, water, inorganic salts, etc., to reduce the occurrence of malodor.

[0004] There are some functional microorganisms with deodorizing effect in compost, biogas slurry, biogas residue and other media, mainly Bacillus subtilis, Bacillus licheniformis, Bacillus faecalis, Pseudomonas, etc. At present, many countries and regions use a combination of multiple microbial agents to improve the odor reduction effect. Some scholars have shown that the composite deodorizing agent has a certain deodorizing effect on fresh and old feces, and has a certain deodorizing effect on NH 3 , H 2 S and volatile organic acids have obvious removal effects. However, the types of deodorizing strains found so far are relatively few and their effects are relatively simple, making it difficult to meet the current odor-related emission standards. Summary of the invention

[0005] The purpose of the present invention is to provide a deodorizing bacterial strain, a deodorizing bacterial agent and applications thereof. The present invention newly discovered three deodorizing bacterial strains. The three deodorizing bacterial strains and the deodorizing bacterial agent compounded based on the three deodorizing bacterial strains have good deodorizing effects and stable and long-lasting effects.

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

[0007] The present invention also provides a deodorizing bacterial agent, which comprises a deodorizing bacterial strain;

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

[0009] The deposit numbers of the Providencia larvae CM1, Pseudochalobacterium insaccharolyticum CM5 and Providencia CM18 are CGMCC No.31631, CGMCC No.31632 and CGMCC No.31633 respectively.

[0010] Preferably, the deodorizing bacterial agent comprises a bacterial suspension of the deodorizing bacterial strain and / or a fermentation liquid of the deodorizing bacterial strain.

[0011] Preferably, the deodorizing bacterial agent comprises the fermentation liquid of Providencia larvae CM1, the fermentation liquid of Pseudocobacillus saccharolyticus CM5 and the fermentation liquid of Providencia CM18; the volume ratio of the fermentation liquid of Providencia larvae CM1, the fermentation liquid of Pseudocobacillus saccharolyticus CM5 and the fermentation liquid of Providencia CM18 is 0.5-2.0:0.5-2.0:0.5-2.0.

[0012] Preferably, the effective viable counts of Providencia larvae CM1, Pseudomonas aeruginosa CM5 and Providencia CM18 in the deodorizing bacteria agent are ≥ 10 8 cfu / mL.

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

[0014] Preferably, the odor components include NH 3 , H 2 S and volatile organic acids.

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

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

[0017] Preferably, the odor-containing material includes livestock and poultry excrement and / or domestic garbage.

[0018] Beneficial effects:

[0019] The present invention provides a deodorizing strain, including Providencia larvae CM1, Pseudocrobacterium insaccharolyticum CM5 or Providencia CM18; the deposit numbers of Providencia larvae CM1, Pseudocrobacterium insaccharolyticum CM5 and Providencia CM18 are CGMCC No.31631, CGMCC No.31632 and CGMCC No.31633 respectively. The deodorizing strain of the present invention has a good deodorizing effect. On this basis, there is no antagonism between the composite bacterial agent strains obtained by compounding the three strains, and through the synergistic effect between different deodorizing strains, the deodorizing effect on NH 3 and H 2 The average removal effect of S is the best, and the deodorization effect is stable and lasting; through experiments, it is found that compared with a single deodorization strain, the composite deodorizing bacterial agent composed of the above three deodorizing bacterial 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%, which solves 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, which solves the problem that the traditional deodorizing bacterial agent is not effective and slow to take effect during the stacking and composting of poultry and livestock manure and domestic garbage in the past, and alleviates the environmental pollution in the treatment of waste such as manure.

[0020] Biological Deposit Description

[0021] Providencia vermicola CM1, which belongs to Providencia vermicola in biological taxonomy, was deposited in the China General Microbiological Culture Collection (CGMCC) on August 7, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31631;

[0022] Pseudochrobactrum asaccharolyticum CM5, which belongs to Pseudochrobactrum asaccharolyticum in biological taxonomy, was deposited in the China General Microbiological Culture Collection (CGMCC) on August 7, 2024. The deposit address is 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 CM18, which belongs to Providencia sp. in biological taxonomy, was deposited in the China General Microbiological Culture Collection Center (CGMCC) on August 7, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 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 embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 NH of different strains screened within 8 days in Example 1 3 Removal rate;

[0026] Figure 2 The H of different strains screened within 8 days in Example 1 2 S removal rate;

[0027] Figure 3 is the NH of the composite deodorant at different spraying rates in Example 2 3 Removal rate;

[0028] Figure 4 is the H of the composite deodorant agent at different spraying rates in Example 2 2 S removal rate;

[0029] Figure 5 It is the average removal rate of the composite deodorizing bacteria agent with different spraying amounts in Example 2 within 8 days. DETAILED DESCRIPTION

[0030] The invention provides a deodorizing strain, comprising Providencia sp. CM1, Pseudochrobactrum asaccharolyticum CM5 or Providencia sp. CM18; the preservation numbers of Providencia sp. CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18 are CGMCC No. 31631, CGMCC No. 31632 and CGMCC No. 31633 respectively.

[0031] The deodorizing strain of the present invention is separated from chicken manure compost and pig manure compost samples. Through 16S rRNA sequence determination and homology comparison analysis, it is found that the similarities of Providencia larvae CM1, Pseudochrobactrum insaccharolyticum CM5 and Providencia CM18 with Providencia vermicola, Pseudochrobactrum asaccharolyticum and Providencia sp. are 100%, 100% and 99.929% respectively, thereby confirming their biological classification and carrying out biological preservation respectively.

[0032] In some studies, Pseudochalobacterium and Providencia are often found in soil remediation and sewage treatment contaminated by pesticides, heavy metals and organic compounds, but there are no related reports in the field of deodorization. Therefore, the present invention discovered new functions of Pseudochalobacterium and Providencia, which to a certain extent provided a new direction for the exploration of deodorization strains.

[0033] The present invention also provides a deodorizing bacterial agent, 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 bacterial agent of the present invention preferably comprises a bacterial suspension of the deodorizing strain and / or a fermentation broth of the deodorizing strain, and more preferably a fermentation broth of the deodorizing strain. The deodorant agent of the present invention preferably includes a fermentation broth of Providencia larvae CM1, a fermentation broth of Pseudomonas aeruginosa CM5, and a fermentation broth of Providencia CM18; the volume ratio of the fermentation broth of Providencia larvae CM1, the fermentation broth of Pseudomonas aeruginosa CM5, and the fermentation broth of Providencia CM18 is preferably 0.5-2.0:0.5-2.0:0.5-2.0, more preferably 1:1:1; the effective viable counts of Providencia larvae CM1, Pseudomonas aeruginosa CM5, and Providencia CM18 in the deodorant agent are preferably ≥10 8The present invention has no special limitation on the preparation method of the bacterial suspension of the deodorizing strain and the fermentation liquid of the deodorizing strain, and the preparation steps of the fermentation liquid and bacterial suspension of Pseudomonas aeruginosa and Providencia sp. in the art can be adopted.

[0034] The present invention also provides the use of the deodorizing strain or the deodorizing agent described in the above technical solution in odor treatment. The components of the odor described in the present invention preferably include NH 3 , H 2 S and one or more of volatile organic acids, preferably NH 3 and / or H 2 S. The odor of the present invention preferably includes odor generated by livestock and poultry manure and / or domestic garbage; the odor generated by domestic garbage preferably includes odor generated during composting and / or burying of domestic garbage.

[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. -1The 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 NH 3 and H 2 S selective medium, 30℃, 180r·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] NH 3 The selective culture medium was sucrose 50.0 g, ammonia 10.0 mL, KH 2 PO 4 2.0 g, MgSO 4 7H 2 O0.5g, FeSO 4 0.1 g, 1% ZnSO 4 5.0 mL, NaCl 2.0 g, distilled water 1000 mL, pH natural.

[0047] H 2 S selective medium is glucose 5.0g, K 2 HPO 4 0.5 g, KNO 3 1.0 g, MgCl 2 0.5 g, NaCl 0.5 g, NH 4 Cl 0.5g, Na 2 CO 3 1.0 g, FeCl 2 0.01 g, distilled water 1000 mL, pH natural.

[0048] Nutrient broth agar medium (NB medium) consists of 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 a pH of 7.3±0.2.

[0049] 3. Purification of strains

[0050] The plate streak method was used to pick single colonies with good growth, different colors and shapes from the plate with an inoculation loop and inoculate them on the corresponding plate, and then continue to culture at the same temperature. The strain was purified 4 times using the three-zone plate streak method to obtain a pure strain.

[0051] 4. Preliminary screening of deodorizing strains

[0052] Take 100mLNH 3 The selective culture medium was placed in a shake bottle and injected with NH 3 Selective medium 25% ammonia water, the isolated strain was activated, 500 μL of activated bacterial solution was inoculated into a shaking bottle, sealed and placed on a shaking table at 30°C and 180 r / min for continuous constant temperature culture for 5 days, and the changes in the bacterial solution were observed. If the bacterial solution was turbid, it indicated that the strain had the ability to degrade NH 3 If the bacterial solution remains transparent, it means that the strain cannot directly utilize NH 3 .

[0053] Take 200 mL of H 2 S selective medium was placed in a shake flask. After the strain culture was activated, 500 μL of the activated bacterial solution was inoculated into the medium in the shake flask. Next, a 50 mL sterile beaker was placed in the shake flask. 12 mL of 25% H 2 SO 4 , add 4g FeS to the small beaker and seal it quickly. After continuous cultivation at 30℃ and 200r / min on a shaker for 5 days, observe the turbidity of the bacterial solution. If it is turbid, it indicates that the strain has the ability to degrade H 2 If the bacterial solution remains transparent, it indicates that the strain cannot directly utilize H 2 S.

[0054] The initial screening results are shown in Table 1.

[0055] Table 1 Preliminary screening results

[0056]

[0057] NH after initial screening 3 Selective medium and H 2The turbidity of the S selective culture medium was graded (mild turbidity, moderate turbidity, and severe turbidity), where mild turbidity means that the culture medium is slightly light yellow or translucent, and the strain growth ability is weak; moderate turbidity means that the culture medium is light yellow, and the strain growth ability is moderate; severe turbidity means that the culture medium is yellow or yellow-white, obviously turbid, and the strain growth ability is strong. The results are shown in Tables 2 and 3. 3 According to the turbidity classification of the selective culture medium, 5 strains were slightly turbid, 11 strains were moderately turbid, and 8 strains were severely turbid. 2 According to the turbidity classification of S selective medium, 3 strains were slightly turbid, 10 strains were moderately turbid, and 6 strains were severely turbid. 3 There are many strains with good removal effects, H 2 S is less.

[0058] Table 2NH 3 Turbidity Fractionation of Selective Culture Media

[0059] Mild 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 3H 2 Turbidity classification of S selective culture medium

[0061] Mild 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] Comprehensive Table 2-3 NH 3 and H 2 The turbidity of S medium, the morphology of strains, and the growth conditions were different. It was found that 13 strains had good NH 3 and H 2 S removal ability.

[0063] 5. Rescreening of deodorizing strains:

[0064] The 13 strains selected from the initial screening were subjected to a re-screening culture test using pig manure for 8 days, and NH 3 and H 2 S absorption liquid determines the deodorizing effect of the strain.

[0065] The specific operation was to culture different strains in NB liquid medium at 30°C and 180 r·min -1 Culture until the number of viable cells is ≥10 8 cfu / mL, add the bacterial solution to a 1L covered plastic bucket containing 200g of pig manure at a volume ratio of 10%, and mix thoroughly with a glass rod. Place a 50mL small beaker containing 20mL of 2% boric acid absorption solution in each large beaker to absorb NH 3The barrel was sealed with a double layer of plastic wrap and then covered with a lid. The control group (CK) was an equal amount of sterile water, with 3 replicates per group. A 50 mL small beaker containing 20 mL of alkaline zinc ammonium complex salt solution was placed in the barrel to absorb H 2 S. Determination of NH by boric acid absorption titration 3 The release amount was calculated based on the NH 3 The removal rate of H was determined by zinc ammonium complex colorimetry. 2 S release, calculate H 2 S removal rate. Each treatment was repeated 3 times. NH 3 and H 2 The release of S was measured over a period of 8 days.

[0066] NH 3 Determination method: boric acid absorption Kjeldahl method was used to determine NH 3 First, 20 mL of 2.0% boric acid solution was used to absorb the NH 3 Then, add two drops of methyl red-bromocresol green indicator and titrate with sulfuric acid standard solution until the color changes from blue-green to light red, and record the consumption of sulfuric acid solution. According to the formula: C = (c × v × 2 × 1000 × 17) / V, NH 3 concentration. Where C represents NH 3 concentration, in mg / L; c is the molar concentration of sulfuric acid solution, in mol / L; v represents the volume of sulfuric acid solution consumed, in mL; 17 is NH 3 The mass fraction of is g / mol; V represents the volume of 2% boric acid absorption solution, in mL.

[0067] H 2 Determination method of S: Determination of H by zinc ammonium complex salt absorption colorimetry 2 The release of S, the absorption of H by zinc ammonium complex salt 2 After S gas, add the mixed display liquid, shake well, let stand for 30 minutes, add 2 drops of 40% diammonium phosphate solution, and measure the absorbance at a wavelength of 665nm. The calculation formula is as follows: C = (AA 0 )Bs / Vs. Where C is H 2 S concentration, in mg / m 3 ; A is the absorbance of the sample color solution; A 0 is the absorbance of the blank solution; Bs is the reciprocal of the slope, and its unit is μg / absorbance; Vs is converted to the sampling volume under standard conditions, and its unit is L.

[0068] NH 3 Removal rate and H 2 S removal rate calculation formula: removal rate = (blank concentration - treatment concentration) / blank concentration. NH3 Average removal rate and H 2 The average removal rate of S is the average removal rate within 8 days.

[0069] The reagents used are:

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

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

[0072] 1 mol / L sodium hydroxide solution: Dissolve 20 g of NaOH solid in distilled water and make up to 500 mL.

[0073] 0.1% bromocresol green indicator: 1g bromocresol green is dissolved in 1000mL 95% ethanol.

[0074] 0.2% methyl red indicator: 2g methyl red is dissolved in 1000mL 95% ethanol.

[0075] Methyl red bromocresol green indicator: 0.1% bromocresol green indicator and 0.2% methyl red indicator are mixed in a ratio of 3:1.

[0076] Zinc ammonium complex salt solution: Take 5g zinc sulfate ZnSO 4 Dissolve in 500 mL of distilled water, take 6 g of NaOH and dissolve in 300 mL of distilled water, mix the above zinc sulfate solution and NaOH solution, and add 70 g (NH 4 ) 2 SO 4 Dissolve ammonium sulfate, add 50g glycerol, and make up to 1L with distilled water.

[0077] p-Aminodimethylaniline solution stock solution: 50mL H 2 SO 4 , add 30 mL of distilled water, and after the solution stops heating, add 12 g of p-aminodimethylaniline hydrochloride into the solution.

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

[0079] Ferric chloride solution: weigh 100g FeCl 3 6H 2 O was dissolved in water and the volume was made up to 100 mL.

[0080] Mixed display solution: Mix 1 mL of p-aminodimethylaniline working solution and 40 μL of ferric chloride solution.

[0081] 40% diammonium hydrogen phosphate solution: weigh 40g (NH 4 ) 2 HPO 4 Dissolve in distilled water and make up to 100 mL.

[0082] The experiment found that there were certain differences in the deodorization effects of different strains. The strains that showed good deodorization ability in the initial screening also had certain deodorization ability in the re-screening experiment, such as Figures 1-2 And as shown in Table 4.

[0083] Table 4 Average deodorization rate of different strains within 8 days of rescreening

[0084] Strain number <![CDATA[NH 3 Average removal rate / %]]> <![CDATA[H 2 S average 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 in Table 4 indicate significant differences (P < 0.05).

[0086] Depend on Figures 1-2 As shown in Table 4, most strains are sensitive to H 2 The removal rate of S was higher than that of NH 3 The removal rate of NH 3 In terms of removal, NH 3 The removal rate can reach more than 50%, and two strains can even reach more than 70%, inhibiting NH 3 During the entire culture period, the deodorization ability of most strains showed a trend of first increasing and then decreasing with the extension of time. 2 In terms of S removal, H 2 The S removal rate can reach more than 50%, and two strains can even reach more than 90%, inhibiting H 2 The effect of S release is extremely obvious.

[0087] 6. Molecular identification of deodorizing strains:

[0088] The gene fragment was amplified and sequenced by PCR, and the primer sequences were 27F: 5'-AGAGTTTGATC CTGGCTCAG-3' (SEQ ID NO.1), 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.2). The sequencing was carried out by Shanghai Meiji Biological for 16S rRNA sequence determination, and the sequencing results were compared with the sequences in the NCBI database for homology analysis. The results are shown in Table 5. The final results showed that a total of 3 bacteria Pseudochrobactrum asaccharolyticum, Providencia sp. and Providencia vermicola were isolated, belonging to the genera Pseudochrobactrum and Providencia.

[0089] Table 5 Molecular identification of 16 strains

[0090] Strain number Similarity The most homologous model bacteria 16S rRNA gene sequence CM4 99.854％ Pseudochrobactrumasaccharolyticum 1369bp CM5 100.000％ Pseudochrobactrumasaccharolyticum 1361bp CM2 99.929％ Providenciasp. 1407bp CM9 99.930％ Providenciasp. 1419bp CM12 99.929％ Providenciasp. 1417bp CM17 100.000％ Providenciasp. 1413bp CM18 99.929％ Providenciasp. 1417bp CM21 99.929％ Providenciasp. 1416bp CM28 99.929％ Providenciasp. 1412bp CM32 99.929％ Providenciasp. 1416bp CM34 99.929％ Providenciasp. 1415bp CM1 100.000％ Providenciavermicola 1415bp CM23 99.929％ Providenciavermicola 1410bp

[0091] Example 2

[0092] Preparation of composite high-efficiency deodorizing bacteria agent

[0093] 1. Optimization of deodorizing strains: By comparing the deodorizing rates of different strains, NH 3 and H 2 The strains with better S removal ability.

[0094] According to the results of the re-screening in Example 1, it was found that among the 9 strains of Providencia sp., CM18 showed the best deodorizing performance, NH 3 and H 2 The removal rates of S were all above 70%, 73.41% and 76.63% respectively. CM34 had a better deodorization effect, and NH 3 and H 2 The removal rates of S were 72.56% and 79.92% respectively, showing good deodorization effect. 3 and H 2 The S removal rates were also good, 50.15% and 68.08% respectively. 3 The removal effect was good, with a removal rate of 59.31%; the H 2 The S removal rate is as high as over 90%, but NH 3 The removal rate was lower, all maintained at 40%. Other strains included CM12, CM17, and CM21 for NH 3 and H 2 The deodorizing effect of S was average, all lower than 50%. Therefore, CM18 and CM34 were preferred for the next antagonistic experiment.

[0095] 2. Antagonism test: The selected strains were cultured at 30℃ and 180r / min -1 After 24 hours of liquid culture, 5 μL of bacterial liquid was spotted on nutrient broth agar plates for pairwise antagonism test to observe the growth of strains. If the two strains did not affect each other's normal growth, they were non-antagonistic. If there was a clear growth boundary between the two strains, they were antagonistic. The non-antagonistic deodorizing bacteria were selected. At the same time, according to the deodorizing effect of a single strain, the strain that removed NH 3 and H 2 The best strain combination.

[0096] Antagonistic experiments were conducted by combining CM18 and CM34 of Providencia sp., CM1 and CM23 of Providencia vermicola, and CM4 and CM5 of Pseudochrobactrum asaccharolyticum. The results showed that there was no obvious antagonism between the bacteria. However, the growth rates of CM4 and CM34 were slower, and the H of CM1 was higher than that of CM4. 2 The S removal rate was only 33.12%. Finally, CM1, CM5 and CM18 were selected for compounding and named Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18, respectively.

[0097] 3. Optimization of the combination of composite deodorant bacteria: The selected CM1, CM5 and CM18 were combined in pairs or threes, and pig manure was used to conduct an 8-day combination optimization test. The NH 3 and H 2 S absorption liquid was used to determine the deodorizing effect of the strains. The specific method was the same as the re-screening method of the deodorizing strains in Example 1. When two or three strains were inoculated, the two strains were inoculated at a volume ratio of 1:1, and the total volume of the inoculated strains was 10% of the volume of the culture medium. The three strains were inoculated at a volume ratio of 1:1:1, and the total volume of the inoculated strains was 10% of the total volume of the culture medium. The results showed that the NH 3 and H 2 The average removal rates of S were higher than those of other combinations, so CM1, CM5 and CM18 were selected as the final composite deodorant bacterial agent combination.

[0098] Table 6 NH in different combinations 3 Removal rate

[0099] Time (day) / 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[NH 3 Average removal rate]]> 49.58％ 52.23％ 44.28％ 59.71％

[0100] Table 7 Different combinations of H 2 S removal rate

[0101] Time (day) / 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[H 2 S average removal rate]]> 59.08％ 62.38％ 72.91％ 79.53％

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

[0103] The three selected deodorizing bacteria were cultured in NB liquid medium at 30°C and 180 r·min. -1 Culture until the number of viable cells is ≥108 cfu / mL, and compounded into a composite deodorizing bacterial agent in equal volumes and sprayed on pig manure. By setting different spraying volumes, the influence of process parameters on the deodorizing effect was explored, and the spraying scheme with the best deodorizing effect was determined.

[0104] The specific operation method is as follows: the experiment set 5 spraying rates, which were 2.5%, 5%, 10%, 15% and 20% of the mass of pig manure, respectively. The whole spraying cycle was 8 days. The NH under different spraying rates was measured every 2 days. 3 and H 2 The production of S was determined by boric acid absorption titration. 3 The release amount was determined by zinc ammonium complex salt absorption method. 2 S release, thus comparing and analyzing NH 3 and H 2 The removal rate of S was 3 replicates per treatment. The results are shown in Figures 3 to 5 And as shown in Tables 8 to 10.

[0105] Table 8 NH of composite deodorant under different spraying rates 3 Removal rate

[0106] Time (days) / inoculation amount (%) 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 H of composite deodorant under different spraying rates 2 S removal rate

[0108] Time (days) / inoculation amount (%) 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 rate of composite deodorant with different spraying amounts within 8 days

[0110] Time (days) / inoculation amount (%) 2.5 5 10 15 20 <![CDATA[NH 3 Average removal rate]]> 34.63％ 66.79％ 59.71％ 57.94％ 22.13％ <![CDATA[H 2 S average removal rate]]> 59.92％ 70.73％ 79.53％ 63.93％ 62.09％

[0111] comprehensive Figures 3 to 5 and NH in Tables 8 to 10 3 and H 2 The removal rate of S was found to be 5% and 10% for NH 3 and H 2 The removal rates of S were all high, but the deodorization effect was the most stable under the spraying rate of 10%, and the deodorization capacity decreased slowly over time. Therefore, when the spraying rate was 10%, the deodorization effect of the composite deodorant was better. 3 and H 2 The average removal rates of S can reach 59.71% and 79.53% respectively.

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

[0113] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A deodorizing strain, characterized in that: The invention comprises 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 respectively.

2. A deodorizing bacterial agent, characterized in that: The deodorizing bacterial agent includes a deodorizing bacterial strain; The deodorizing strain includes one or more of Providencia vermicola CM1, Pseudochrobactrum asaccharolyticum CM5 and Providencia sp. CM18; The deposit numbers of the Providencia larvae CM1, Pseudochalobacterium insaccharolyticum CM5 and Providencia CM18 are CGMCC No.31631, CGMCC No.31632 and CGMCC No.31633 respectively.

3. The deodorizing bacterial agent according to claim 2, characterized in that The deodorizing bacterial agent comprises a bacterial suspension of the deodorizing bacterial strain and / or a fermentation liquid of the deodorizing bacterial strain.

4. The deodorizing bacterial agent according to claim 3, characterized in that The deodorizing bacteria agent comprises fermentation liquid of Providencia larvae CM1, fermentation liquid of Pseudocobacillus saccharolyticus CM5 and fermentation liquid of Providencia CM18; the volume ratio of the fermentation liquid of Providencia larvae CM1, fermentation liquid of Pseudocobacillus saccharolyticus CM5 and fermentation liquid of Providencia CM18 is 0.5-2.0:0.5-2.0:0.5-2.

0.

5. The deodorizing bacterial agent according to claim 4, characterized in that The effective viable bacteria counts of Providencia larvae CM1, Pseudomonas insaccharolyticus CM5 and Providencia CM18 in the deodorizing bacteria agent are ≥10 8 cfu / mL.

6. Use of the deodorizing bacterial strain according to claim 1 or the deodorizing bacterial agent according to any one of claims 2 to 5 in odor treatment.

7. The use according to claim 6, characterized in that: The components of the odor include one or more of NH3, H2S and volatile organic acids.

8. The use according to claim 6 or 7, characterized in that: The odor includes odor generated by livestock and poultry excrement and / or domestic garbage.

9. A method for removing odor, characterized in that: The deodorizing bacteria agent according to any one of claims 2 to 5 is sprayed onto the material containing the odor; the spraying amount of the deodorizing bacteria agent is 5% to 15% of the mass of the material containing the odor.

10. The method according to claim 9, characterized in that The odor-containing materials include livestock and poultry excrement and / or domestic garbage.

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