A strain of Acinetobacter oliveti AO-06 capable of degrading skatole and its application

Abstract

The present invention relates to a skatole-degrading Acinetobacter olivineus ( Acinetobacter oleivorans )AO-06 and its application can effectively solve the problem of efficient degradation of skatole. The olive Acinetobacter ( Acinetobacter oleivorans )AO‑06 has the ability to quickly and efficiently degrade skatole in pig manure, and can effectively remove odors in various environments through reaction. It has high practical value and good application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microbial technology for treating environmental pollution, in particular to an Acinetobacter olivetii AO-06 capable of degrading skatole and application thereof. Background Art

[0002] With the continuous development of the economy and the improvement of people's living standards, my country's animal husbandry has developed rapidly. In recent years, my country's animal husbandry has gradually developed in the direction of scale, and the number of livestock and poultry on hand and on the market has steadily increased. On the basis of meeting the needs of residents for meat, eggs and milk, a large amount of manure and odors are being discharged, and odor control has become the main challenge facing the livestock and poultry, aquaculture, garbage treatment plants, and sewage treatment plants industries.

[0003] 3-Methylindole (C9H9N), commonly known as skatole (3MI), is a typical nitrogen heterocyclic aromatic compound. It is soluble in organic solvents, insoluble in water, has a bicyclic structure, is one of the main metabolites of tryptophan, and is an indole derivative. Skatole is a natural substance found in animal feces, beetroot, camphor wood, and coal tar. It is also an important precursor for synthetic food additives, agricultural chemicals, and industrial chemicals. A considerable amount of skatole has been detected in the intestines and feces of humans and animals, pig manure, activated sludge, municipal and industrial wastewater, and aquaculture water. Skatole is considered to be one of the three major harmful odor substances emitted by livestock and poultry. It has a strong fecal odor and an odor threshold of less than 0.003 mg / m 3 , it can still be sensed by humans and livestock at extremely low concentrations. In addition to the stench, skatole can cause harm to human and animal health: it can cause acute pulmonary edema and emphysema in cattle and sheep; it can cause lesions in human neuroendocrine and immune functions, and has the risk of causing teratogenicity and carcinogenesis in respiratory cells; skatole is also considered to be an attractant for mosquitoes, flies and beetles, which can promote the spread of insect-borne diseases. Therefore, it is of great significance to solve the environmental pollution caused by skatole.

[0004] The physical adsorption method and chemical oxidation method currently used have certain effects in removing 3-methylindole, but the physical adsorption method is costly, consumes a lot of energy, and is easily affected by the environment. In addition to the above disadvantages, the chemical oxidation method is also prone to secondary pollution. Biodegradation can effectively solve the above problems, so it has received more and more attention. Since Fujioka et al. discovered in 1968 that a certain Gram-positive bacterium could oxidize skatole to 2-oxo-3-methyl-3-hydroxyindole, there have been dozens of reports on skatole-degrading bacteria, such as Clostridium malenominatum A-3 under anaerobic conditions; methanogenic bacteria under sulfur-reducing conditions and Pseudomonas putida LPC24 under oxygen-limited conditions; aerobic bacteria such as Lactobacillus brevis, Rhodopseudomonas palustris WKU-KDNS3, Acinetobacter toweneri NTA1-2A and Acinetobacter gullouiae TAT1-6, Burkholderia sp. IDO3, Rhodococcus sp. DMU1 and Rhodococcus pyridinivorans Rp3, etc. The strains reported so far are either difficult or even unable to degrade high concentrations of skatole. In general, these strains take a long time to degrade skatole and the degradation concentration is low. Obtaining strains with high efficiency and rapid degradation of high concentrations of skatole is still the primary task of livestock and poultry manure treatment and environmental management. They can completely degrade 3-methylindole in a short time; or they have strict growth conditions and cannot be used in actual production, and cannot meet the requirements of efficient degradation. Therefore, it is still necessary to screen new strains of efficient skatole degradation.

[0005] Deodorants are widely used in the field of odor (peculiar smell) control because they can remove surface odors by spraying and can also treat concentrated and collected odorous gases. However, most of the deodorants currently sold on the market or the patents for deodorants that have been published use the strong fragrance of deodorants to cover up the odor, but do not remove the odor-causing compounds; some deodorants use strong oxidizing systems such as sodium hypochlorite, hydrogen peroxide / acid / ferrous ions to remove odor-causing compounds through fierce chemical reactions. Such deodorants are usually expensive and will corrode equipment if used for a long time. At the same time, some stabilizers are usually added to the deodorants to ensure the stability of the system. In addition, the removal efficiency of these deodorants for major odor-causing compounds such as sulfur-containing compounds is usually only 80-90%, and they are not suitable for surface odor control occasions that are in direct contact with the human body, and cannot meet the requirements of health and environmental protection. Therefore, it is imperative to invent a deodorant preparation that has no side effects, no secondary pollution, good effect of degrading skatole, safety and environmental protection, and thus greatly improves the quality of the environment. Summary of the invention

[0006] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide an olive bacterium AO-06 for degrading skatole and its application, which can effectively solve the problem of efficient degradation of skatole.

[0007] The technical solution solved by the present invention is that the olive Acinetobacter AO-06 described in the present invention is classified and named as olive Acinetobacter AO-06 (Acinetobacter oleivorans), which has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 24972, a deposit date of May 26, 2022, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The 16S rDNA gene sequence of the strain is shown in SEQ ID NO.1.

[0008] The application of the olive bacterium AO-06 in degrading skatole.

[0009] The application of the olive bacterium AO-06 in the preparation of a microbial environmentally friendly agent for efficiently degrading skatole.

[0010] The preparation method of the olive Acinetobacter AO-06 microbial environmental protection agent comprises the following steps:

[0011] 1) Preparation of seed solution: Take a ring of activated AO-06 strain on the plate and inoculate it into a 250ml Erlenmeyer flask containing 100ml sterilized liquid seed culture medium, place it in a full-temperature shaking incubator, and culture it at 26-35°C and 140-180r / min for 36-48h, then inoculate it into 500ml culture medium containing 150ml liquid seed culture medium for culture, and culture it at 26-35°C and 140-180r / min for 36-48h. After testing, the number of viable bacteria in the seed solution is greater than or equal to 1-2×10 9 CFU / ml, that is, the seed solution was prepared;

[0012] 2) Preparation of fermentation broth: The seed solution obtained in step 1) is transferred to a 1L triangular flask containing 500ml of fermentation medium at a 10-15% inoculation rate, the culture temperature is 26-35°C, the rotation speed is 140-180r / min, the pH is natural, the culture time is 36-48h, and then transferred to a 50L fully automatic fermentation tank for amplification culture, and the tank pressure during the fermentation culture process is maintained at 0.03-0.05MPa; sterile air is introduced during the culture process, the culture temperature is 26°C-35°C, the rotation speed is 200-300r / min, and the time is 25-48h. After testing, the number of viable bacteria in the bacterial solution is greater than or equal to 1-2×10 9 CFU / ml of fermentation culture is the microbial environmental protection agent.

[0013] In the step 1), the liquid seed culture medium is composed of: 4.0 g / L sucrose, 8.0 g / L tryptone, 4.0 g / L yeast powder, 1.0 g / L ammonium sulfate, 0.4 g / L potassium dihydrogen phosphate, 0.05 g / L manganese sulfate, and 6.0 g / L NaCl, and the pH is adjusted to 7.0. After the culture medium is sterilized, skatole is first dissolved in anhydrous ethanol to a final concentration of 0.1 g / L, and then filtered and sterilized with a 0.2 μm organic filter membrane before being added to the liquid seed culture medium.

[0014] In the step 2), the fermentation medium is: 20.0 g / L molasses, 6.0 g / L peptone, 6.0 g / L yeast extract powder, 4.0 g / L ammonium sulfate, 5.0 g / L sodium acetate, 1.0 mL / L Tween 80, 3.0 g / L mineral powder, and the rest is water, and the pH is natural. The above raw materials are mixed, stirred and dissolved in a magnetic stirrer, and the mixed fermentation culture is sterilized at 121° C. for 20 min, then cooled, and the pH is adjusted to 7.0. During the fermentation culture process, the culture temperature is 30° C.

[0015] The raw materials used in the degradation process of the present invention are easy to obtain, the preparation method is simple, and the production cost is low. The screened olive bacterium AO-06 has the ability to efficiently and quickly degrade skatole in pig manure, and can efficiently remove odors in various environments through reaction. The degradation rate of skatole in compost can reach 96.4%. The deodorization product will not cause secondary pollution to the environment, which provides a basis for the application of the strain in the purification of odor-polluted environments, and has high practical value and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the colony diagram of olive bacterium AO-06 of the present invention.

[0017] Figure 2 It is a phylogenetic tree diagram of the strain olive bacterium AO-06 of the present invention.

[0018] Figure 3 It is a growth-skatole degradation curve of the strain Acinetobacter oliveti AO-06 of the present invention.

[0019] Figure 4 This is a graph showing the effects of different conditions of the present invention on the growth and degradation of skatole by strain Acinetobacter oliveti AO-06. DETAILED DESCRIPTION

[0020] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples.

[0021] Example 1

[0022] In the specific implementation of the present invention, the preparation steps of the olive Acinetobacter AO-06 microbial environmental protection agent are as follows:

[0023] 1) Preparation of seed solution: Take a ring of activated AO-06 strain on the plate and inoculate it into a 250ml Erlenmeyer flask containing 100ml sterilized liquid seed culture medium, place it in a full-temperature shaking incubator, and culture it at 26°C and 140r / min for 36h. Then inoculate it into 500ml culture medium containing 150ml liquid seed culture medium and culture it at 26°C and 140r / min for 36h. After testing, the number of viable bacteria in the seed solution is greater than or equal to 1-2×10 9 CFU / ml, that is, the seed solution was prepared;

[0024] 2) Preparation of fermentation broth: The seed solution obtained in step 1) was transferred to a 1L Erlenmeyer flask containing 500ml of fermentation medium at an inoculation rate of 11%, the culture temperature was 26°C, the rotation speed was 140r / min, the pH was natural, the culture time was 36h, and then transferred to a 50L fully automatic fermenter for amplification culture. The tank pressure during the fermentation culture process was maintained at 0.03MPa; sterile air was introduced during the culture process, the culture temperature was 26°C, the rotation speed was 200r·min -1 , the time is 25h. After testing, the number of live bacteria in the bacterial solution is greater than or equal to 1-2×10 9 CFU / ml of fermentation culture is a microbial environmental protection agent.

[0025] Example 2

[0026] In the specific implementation of the present invention, the preparation steps of the olive Acinetobacter AO-06 microbial environmental protection agent are as follows:

[0027] 1) Preparation of seed solution: Take a ring of activated AO-06 strain on the plate and inoculate it into a 250ml Erlenmeyer flask containing 100ml sterilized liquid seed culture medium, place it in a full-temperature shaking incubator, and culture it at 30°C and 160r / min for 42h. Then inoculate it into 500ml culture medium containing 150ml liquid seed culture medium and culture it at 30°C and 160r / min for 42h. After testing, the number of viable bacteria in the seed solution is greater than or equal to 1-2×10 9 CFU / ml, that is, the seed solution was prepared;

[0028] 2) Preparation of fermentation broth: The seed solution obtained in step 1) was transferred to a 1L Erlenmeyer flask containing 500ml of fermentation medium at a 13% inoculation rate, the culture temperature was 30°C, the rotation speed was 160r / min, the pH was natural, the culture time was 42h, and then transferred to a 50L fully automatic fermenter for amplification culture. The tank pressure during the fermentation culture process was maintained at 0.04MPa; sterile air was introduced during the culture process, the culture temperature was 30°C, the rotation speed was 250r / min, and the culture time was 37h. After testing, the number of viable bacteria in the bacterial solution was greater than or equal to 1-2×10 9 CFU / ml of fermentation culture is a microbial environmental protection agent.

[0029] Example 3

[0030] In the specific implementation of the present invention, the preparation steps of the olive Acinetobacter AO-06 microbial environmental protection agent are as follows:

[0031] 1) Preparation of seed solution: Take a ring of activated AO-06 strain on the plate and inoculate it into a 250ml Erlenmeyer flask containing 100ml sterilized liquid seed culture medium, place it in a full-temperature shaking incubator, and culture it at 35°C and 180r / min for 48h. Then inoculate it into 500ml culture medium containing 150ml liquid seed culture medium and culture it at 35°C and 180r / min for 48h. After testing, the number of viable bacteria in the seed solution is greater than or equal to 1-2×10 9 CFU / ml, that is, the seed solution was prepared;

[0032] 2) Preparation of fermentation broth: The seed solution obtained in step 1) was transferred to a 1L Erlenmeyer flask containing 500ml of fermentation medium at a 14% inoculation rate, the culture temperature was 35°C, the rotation speed was 180r / min, the pH was natural, the culture time was 48h, and then transferred to a 50L fully automatic fermenter for amplification culture. The tank pressure during the fermentation culture process was maintained at 0.05MPa; sterile air was introduced during the culture process, the culture temperature was 35°C, the rotation speed was 300r / min, and the culture time was 48h. After testing, the number of viable bacteria in the bacterial solution was greater than or equal to 1-2×10 9 CFU / ml of fermentation culture is a microbial environmental protection agent.

[0033] The olive Acinetobacter AO-06 of the present invention is obtained by separation and screening from a manure sample of a pig farm in Luoshan County, Xinyang City, Henan Province. Through further scientific screening, a microbial environmental protection agent suitable for pig farm composting and efficient degradation of skatole is obtained, wherein the number of viable bacteria of olive Acinetobacter AO-06 can reach 1.0-2.0×10 9 CFU / mL, and sprayed into the pig farm compost through an agricultural sprayer to achieve zero-emission manure treatment. The relevant test data are as follows:

[0034] 1 Materials and methods

[0035] 1.1 Source of the strain: The strain was isolated and screened from manure samples of a pig farm in Luoshan County, Xinyang City, Henan Province.

[0036] 1.2 Culture medium:

[0037] Inorganic salt medium (g / L): (NH4)2SO4 1.0, KH2PO4 0.2, K2HPO4 0.8, MgC l2 7H2O 1.07, FeC l2 ·4H2O0.016, NaCl 5.0, pH7.0±0.2;

[0038] The composition of the culture medium for strain slant and liquid seed is: sucrose 4.0g / L, tryptone 8.0g / L, yeast powder 4.0g / L, ammonium sulfate 1.0g / L, potassium dihydrogen phosphate 0.4g / L, manganese sulfate 0.05g / L, NaCl 6.0g / L, pH is adjusted to 7.0, the culture medium is sterilized, skatole is first dissolved in anhydrous ethanol to make the final concentration of skatole 0.1g / L, filtered and sterilized with a 0.2μm organic filter membrane before adding to the liquid seed culture medium (solid culture medium is supplemented with 15.0g / L agar).

[0039] 1.3 Methods

[0040] 1.3.1 Preparation of skatole stock solution

[0041] Weigh 0.5 g of 3-methylindole and dissolve it in 100 mL of anhydrous ethanol to prepare 5 g / L skatole. Filter and sterilize with a 0.2 μm organic filter membrane and store at 4°C for later use.

[0042] 1.3.2 Isolation and screening of skatole-degrading bacteria

[0043] Weigh 1g of the collected pig manure compost soil and add it to the sterilized liquid seed medium. Cultivate it in a shaking incubator at 30℃ and 180r / min for 24h. Inoculate it in an inorganic salt medium containing 100mg / L skatole at a 5% inoculum and culture it in a shaking incubator at 30℃ and 180r / min for 24h. After continuous transfer cultivation for 4 times under the same conditions, take 1mL of the enrichment solution and dilute it to 10 at different dilutions. -5 , 10 -6 , 10 -7 , 100 μL of each was spread on the enriched solid medium. After cultivation in a constant temperature incubator, the colony morphology, size, and color were observed, and a single colony was picked for microscopic examination to confirm whether its cell morphology was single, and streaked on beef extract peptone solid medium for further purification and cultivation until a single cell morphology was obtained. Finally, several strains that could grow with skatole as the only carbon source were isolated and inoculated into slant medium and stored at 4°C.

[0044] After repeated dilution and plate screening and coating, three strains that can grow with 3-methylindole as the sole carbon source were isolated and screened, indicating that the three strains have a good degradation effect on 3-methylindole. Then, the degradation effect of skatole by these three strains was analyzed by high-performance liquid chromatography. The results showed that the degradation effect of one of the strains was significantly higher than that of the other two strains, and this strain was named AO-06. Based on this, the present invention selected AO-06 as the test strain for the next step of experimental exploration of degradation of 3-methylindole.

[0045] 1.3.3 Morphological, physiological and biochemical characteristics of the degradation bacteria and molecular biological identification results

[0046] After multi-line plate streaking, the colony surface of strain AO-06 on LB solid medium was smooth, small and round, white, with protruding, moist and shiny surface, and neat edges (such as Figure 1 Microscopic observation showed that the bacteria were rod-shaped, Gram-staining was negative, and there were no spores.

[0047] According to the Manual of Identification of Common Bacterial Systems, experiments such as starch hydrolysis, gelatin hydrolysis, glucose and mannose utilization were carried out on strain AO-06, and the results of physiological and biochemical experiments are shown in Table 1.

[0048] Table 1 Physiological and biochemical test results of strain AO-06

[0049]

[0050]

[0051] The 16S rRNA gene product of the strain was 1434 bp in size. It was compared with the known sequences in GenBank on the NCBI website and a phylogenetic tree was constructed using the neighbor-joining method. The sequence similarity between the strain and Acinetobacter oleivorans was 99.9% ( Figure 2 The sequence homology was 99%. After comprehensive consideration, the strain was preliminarily determined to be Acinetobacter oleivorans.

[0052] 1.3.4 16S rRNA gene sequence determination

[0053] The 16S rRNA gene PCR amplification primers of the strain used universal primers: forward primer 27F: 5′-AGAGTTTGATCATGGCTCAG-3′, reverse primer 1492R: 5′-ACGGTTACCTTACCTTGTTACGACTT-3′, PCR reaction system (50μL): genomic DNA 2μL, upstream and downstream primers (10μmol / L) 2μL each, 2×Taq PCR Mix 25μL, purified water 19μL. PCR reaction conditions: 98℃2min; 98℃10s, 55℃15s, 72℃90s, 38 cycles; 72℃5min. The amplified product was sent to a sequencing company for sequencing, and the sequence information was compared with the known sequences in GenBank on the NCBI website. The phylogenetic tree was constructed by the neighbor-joining method using MEGA 6 software to identify the strain.

[0054] 1.3.5 HPLC method for determination of 3-methylindole concentration

[0055] In the test, the content of 3-methylindole in the sample was determined by high performance liquid chromatography. First, 10 mg of skatole standard sample was weighed and placed in a 10 mL volumetric flask. It was dissolved in anhydrous ethanol to prepare a 1 mg / mL standard stock solution. The standard series solutions of 50, 100, 200, 300, and 500 μg / mL were diluted in sequence, filtered through a 0.2 μm organic filter membrane, and the peak area was determined by HPLC. Each concentration was repeated 3 times. A linear regression equation was established based on the injection concentration and the obtained peak area.

[0056] Chromatographic conditions: chromatographic column is Agilent 5TC-C18 column (250mm×4.6mm, 5μm); column temperature: 30℃; UV detection wavelength: 260nm; mobile phase is 1% acetic acid: water 1:1; flow rate is 1.0mL / min; injection volume is 20μL.

[0057] 1.3.6 Strain growth test

[0058] The test selected 3-methylindole efficient degrading bacteria AO-06 as the test strain. The strain was cultured using inorganic salt medium at an inoculum dosage of 10%, a shaking speed of 140r / min, a temperature of 30°C, and a natural pH. The carbon source was 3-methylindole at a concentration of 100mg / L. The strain was cultured for 72h, and the growth was measured every 12h starting from 0h. There were 3 replicates for each culture group, and the blank treatment was set as the inorganic salt culture group with 3-methylindole added, and the blank treatment group was not inoculated with bacteria. The growth of the strain was measured by using an ultraviolet spectrophotometer to determine the absorbance value of the inorganic salt culture medium containing bacteria cultured to the measurement time at a wavelength of 600nm. When measuring the growth, the culture medium was mixed evenly with a vortex oscillator, and distilled water was used as a control. The OD600 value of the liquid culture medium of the strain was measured with an ultraviolet spectrophotometer, and the data was recorded. Figure 3 As shown, with the bacterial OD 600 The value increased, AO-06 was in logarithmic growth phase, and the 3MI concentration gradually decreased; the bacterial growth reached a peak at 48h, at which time the 3MI degradation rate reached 100%, and then the bacterial growth slowly decreased.

[0059] 1.3.7 Effects of different environmental factors on skatole degradation by strain AO-06

[0060] The effects of different environmental factors on the degradation of skatole by the strain were investigated. The basic conditions for inorganic salt culture were a skatole concentration of 100 mg / L, a temperature of 30°C, a shaking speed of 180 r / min, a natural pH, and an inoculation size of 5%. Samples were taken regularly to determine the growth of the strain (OD600) and the concentration of residual skatole in the culture medium, and the skatole removal rate was taken as the investigation target.

[0061] Skatole degradation rate (%) = (skatole content in uninoculated medium - skatole content in inoculated medium) / skatole content in uninoculated medium × 100%

[0062] The effects of initial concentration of 3MI, pH, temperature and rotation speed on the degradation of skatole by the strain were investigated respectively. Including different initial concentrations of 3MI: 100, 150, 200, 250, 300 mg / L; different pH: 4.0, 5.0, 6.0, 8.0, 9.0; different temperatures: 20, 25, 30, 37, 42℃, different rotation speeds: 0, 60, 120, 180, 240 r / min, and the sampling time point was 12h. The effects on strain growth and skatole degradation were investigated, with the skatole content of 100 mg / L.

[0063] (1) Initial concentration

[0064] Depend on Figure 4 A shows that when the initial concentration of 3MI is 100 mg / L and the strain is cultured for 48 hours, the AO-06 strain has a significant degradation effect on 3MI. It can also be seen that the higher the concentration of 3MI added to the culture medium, the longer the degradation time of the strain, which may be due to the hysteresis of the degradation time formed by the strain constantly adapting to the toxic environment during the growth process.

[0065] (2) pH

[0066] Depend on Figure 4 B shows that Acinetobacter oleivorans AO-06 has the best degradation effect on 3MI at pH 8.0, and it is basically degraded within 36 hours. Outside this range, excessive acid or alkali will significantly inhibit the degradation effect of AO-06, and the degradation rate is less than 50% in 120 hours.

[0067] (3) Temperature

[0068] Depend on Figure 4 C shows that Acinetobacter oleivorans AO-06 has the highest degradation efficiency for 3MI, and the degradation effect is significant within 48 hours. Within 120 hours, the suitable temperature range for Acinetobacter oleivorans AO-06 to achieve 100% degradation rate is 25℃-37℃. Other temperatures have an effect on the degradation efficiency of Acinetobacter oleivorans AO-06. This shows that the suitable growth temperature of strain AO-06 is 30℃.

[0069] (4) Speed

[0070] Depend on Figure 4D shows that when the speed is between 80-200r / min, 3MI is completely degraded. During the microbial culture process, when the strain is cultured for 48h, the speed is 160r / min, and the degradation rate reaches 100%, and the degradation effect is significant.

[0071] 1.3.8 Degradation of skatole in pig manure compost by AO-06 microbial environmental protection agent

[0072] On-site treatment at the pig farm: prepare a microbial environmental protection agent with a ratio of 10:90 (m / m) of the original liquid of the microbial environmental protection agent and pure water. Pour the prepared microbial environmental protection agent into an agricultural sprayer, and use an atomizing nozzle to spray the microbial environmental protection agent directly into the pig farm compost in an atomized state, spraying it fully and evenly in place. The spraying amount is 15% of the mass of pig manure. This test is done in 3 parallels, with untreated fresh pig manure as the control. Record the odor intensity of the pig farm and the control group samples every two hours, and sample and analyze the odor lightness level and skatole concentration of the samples. Spray the environmental protection agent of the present invention on-site at the pig farm to detect its deodorizing effect.

[0073] Laboratory treatment: The materials used in this test are fresh pig manure collected on site. Weigh 300g of pig manure and put it into a 1L beaker. Pour it into a spray pot and evenly spray it into the pig manure in the beaker according to the ratio of fermentation stock solution: sterile water = 10:90 (m / m). The spraying amount is 15% of the mass of the pig manure. The test is carried out in parallel 3 times, and the fresh pig manure collected at the same time without treatment is used as a control. The odor intensity of the test group and the control group samples is recorded every two hours, and the odor light level and skatole concentration of the sample are sampled and analyzed. Spray the environmentally friendly bacterial agent of the present invention in the laboratory to detect its deodorizing effect.

[0074] The above two treatment methods both use HPLC to determine the degradation rate of skatole. The odor concentration is determined by the three-point comparison odor bag method; human sense of smell: divided according to the six-level odor intensity classification method of Japan, the specific test results are shown in Table 2.

[0075] Table 2 Performance comparison table

[0076] Test items On-site spraying at pig farms Control trial 1 Laboratory spraying Control trial 2 Degradation rate of skatole 92.6％ 14.6％ 96.5％ 15.8％ Odor intensity / level 1 5 1 5 <![CDATA[Odor concentration / ×10 3 > 3 21 4 23

[0077] The results showed that after one week of compost treatment, the odor level of the control group was level 5, and the odor levels of the compost on site and the feces collected in the laboratory that were treated with microbial deodorizers with strong irritating odors dropped to level 1, significantly reducing the odor release of the compost. The degradation rate of skatole in the compost determined by HPLC could reach 92.6%, and the degradation rate of skatole in the laboratory determined by HPLC could reach 96.5%.

[0078] The invention separates and screens a skatole-efficient degradation strain AO-06 from pig manure compost soil, studies the degradation ability of the strain on skatole, and the degradation effect and growth performance of the strain on skatole under different culture conditions (inoculation amount, temperature, pH value, initial skatole concentration). It is found that the strain is cultured at 30°C for 48 hours under the optimal culture conditions of 3MI initial concentration of 100 mg / L, an oscillation speed of 160 rpm, and 48 hours. In a test of the degradation effect of the AO-06 microbial environmental protection bacterial agent on skatole in pig manure compost, it is found that when the strain AO-06 microbial environmental protection bacterial agent is sprayed on pig farm compost, the degradation rate of the skatole can reach 92.6%, and when it is sprayed on pig manure in a laboratory beaker, the degradation rate of the skatole can reach 96.5%.

[0079] The AO-06 environmentally friendly microbial agent of the present invention has a significant effect on the degradation of skatole, and the deodorizing effect is less affected by environmental conditions such as temperature and open-air locations. The product is easy to use and can be sprayed on pig farm compost using an agricultural sprayer. It provides an efficient bacterial strain resource for the efficient degradation of skatole, provides a basis for the application of the strain in the purification of odor-polluted environments, and has good economic and social benefits.

[0080] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A strain of Acinetobacter oliveri that degrades skatole Acinetobacter oleivorans )AO-06, characterized in that It has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.24972.

2. The olive Acinetobacter according to claim 1 ( Acinetobacter oleivorans )Application of AO-06 in degradation of skatole.

3. The olive Acinetobacter according to claim 1 ( Acinetobacter oleivorans )Application of AO-06 in the preparation of environmentally friendly microbial agents for efficient degradation of skatole.

4. The use according to claim 3, characterized in that: The method for preparing the environmentally friendly microbial agent comprises the following steps: 1) Preparation of seed solution: Take the activated olive bacterium ( Acinetobacter oleivorans )AO-06 strain was inoculated into a 250ml Erlenmeyer flask containing 100ml sterilized liquid seed culture medium, placed in a full-temperature shaking incubator, cultured at 26-35°C, 140-180r / min for 36-48h, and then inoculated into 500ml culture medium containing 150ml liquid seed culture medium for culture, and cultured at 26-35°C, 140-180r / min for 36-48h. After testing, the number of viable bacteria in the seed liquid was greater than or equal to 1×10 9 CFU / ml, that is, the seed solution was prepared; 2) Preparation of fermentation broth: The seed liquid obtained in step 1) is transferred to a 1L Erlenmeyer flask containing 500ml of fermentation medium at a 10-15% inoculation rate, the culture temperature is 26-35°C, the rotation speed is 140-180r / min, the pH is natural, the culture time is 36-48h, and then transferred to a 50L fully automatic fermenter for amplification culture. The tank pressure during the fermentation culture process is maintained at 0.03-0.05MPa; sterile air is introduced during the culture process, the culture temperature is 26°C-35°C, the rotation speed is 200-300r / min, and the culture time is 25-48h. After testing, the number of viable bacteria in the bacterial liquid is greater than or equal to 1×10 9 CFU / ml of fermentation culture is the microbial environmental protection agent.

5. The use according to claim 4, characterized in that: In the step 1), the liquid seed culture medium is composed of: 4.0 g / L sucrose, 8.0 g / L tryptone, 4.0 g / L yeast powder, 1.0 g / L ammonium sulfate, 0.4 g / L potassium dihydrogen phosphate, 0.05 g / L manganese sulfate and 6.0 g / L NaCl, and the pH is adjusted to 7.

0. After the culture medium is sterilized, skatole is first dissolved in anhydrous ethanol to a final concentration of 0.1 g / L, and then filtered and sterilized with a 0.2 μm organic filter membrane before being added to the liquid seed culture medium.

6. The use according to claim 4, characterized in that in the step 2), the fermentation medium is: molasses 20.0 g / L, peptone 6.0 g / L, yeast extract powder 6.0g / L, ammonium sulfate 4.0g / L, sodium acetate 5.0g / L, Tween 80 1.0mL / L and mineral powder 3.0g / L, the rest is water, pH is natural. The above raw materials are mixed, and fully stirred and dissolved in a magnetic stirrer. The mixed fermentation culture solution is sterilized at 121°C for 20min, and then cooled. The pH is adjusted to 7.

0. During the fermentation culture process, the culture temperature is 30°C.

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