Acinetobacter gellinae and application thereof

Abstract

The invention discloses Acinetobacter gerneri and application thereof, the Acinetobacter gerneri is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, the preservation number is CGMCC No.16148, and the preservation time is July 25, 2018. The Acinetobacter gerneri has the advantages that the preservation number is CGMCC No.16148, and the preservation number is CGMCC No.16148; the invention provides a novel acinetobacter gellinae strain, the acinetobacter gellinae strain is high in deodorization efficiency, and the removal rate of ammonium nitrogen reaches 98% or above and even reaches 100%; the method can be applied to biological deodorization only through a single strain, and has the obvious technical advantages of being convenient to use, package and transport and easy to commercialize; according to the acinetobacter gellinae disclosed by the invention, efficient deodorization can be realized, and high-density thalli can be quickly obtained by utilizing heterotrophic conditions, so that quick start of biological deodorization engineering is realized.

Description

Technical Field

[0001] The present application relates to the field of industrial microbial technology, and in particular to Acinetobacter garneri and its application. Background Art

[0002] Malodor is one of the seven major environmental hazards in the world. Malodor substances can be divided into five categories according to their composition: oxygen-containing compounds, nitrogen-containing compounds, hydrocarbons, sulfur-containing compounds, halogens and their derivatives.

[0003] Biological deodorization technology uses the metabolic process of microorganisms to destroy or transform malodorous substances into odorless compounds. Biological deodorization generally uses microorganisms fixed on solid media or in liquids to adsorb and decompose malodorous substances, including bio-trickling filtration, bio-filter tank, bio-spraying, etc. Regardless of the type of biological deodorization technology, the key is the microbial strain that specifically degrades malodorous substances.

[0004] Take ammonia (NH3), a typical component of nitrogen-containing malodorous gases, as an example. Traditional deodorization includes two processes: nitrification and denitrification. Nowadays, with the discovery of heterotrophic nitrifying bacteria Alcaligenes faecalis and aerobic denitrifying bacteria Bacillus subtilis, it has become possible for bacteria that perform two types of reactions to survive efficiently in the same reaction system.

[0005] Acinetobacter gerneri is a strain of the genus Acinetobacter. The prior art has not yet found its industrial use in the field of biological deodorization technology. Summary of the invention

[0006] In view of this, the present application provides Acinetobacter gerneri and its application, aiming to improve the problem that the industrial use of Acinetobacter gerneri in the field of biological deodorization technology has not yet been found in the prior art.

[0007] The embodiment of the present application is implemented as follows: Acinetobacter gerneri, the Acinetobacter gerneri (Acinetobacter gerneri) is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.16148 and a deposit time of July 25, 2018.

[0008] Optionally, in some embodiments of the present application, the 16S rDNA sequence of the Acinetobacter garneri is shown in the sequence listing SEQ ID No: 1.

[0009] Optionally, in some embodiments of the present application, the Acinetobacter gerneri is derived from aerobic composting of the Jingyan County Qiangsheng Livestock and Poultry Breeding Professional Cooperative.

[0010] Optionally, in some embodiments of the present application, the Acinetobacter garneri is used in the preparation of a deodorizing bacterial agent.

[0011] Furthermore, the deodorizing bacterial agent is a liquid deodorizing bacterial agent or a solid deodorizing bacterial agent.

[0012] Optionally, in some embodiments of the present application, the Acinetobacter garneri is used to remove malodorous substances from sludge composting.

[0013] Furthermore, the malodorous substances include nitrogen-containing malodorous gases and / or ammonium ion compounds.

[0014] Furthermore, the nitrogen-containing malodorous gas includes ammonia; and / or

[0015] The ammonium ion-containing compound includes (NH4)2SO4.

[0016] Furthermore, according to the application of Acinetobacter garneri in removing malodorous substances from sludge compost, the application is specifically: aerobically culturing Acinetobacter garneri at 28°C to 32°C for 0.5d to 1.5d to obtain an activated bacterial liquid, and inoculating the bacterial liquid into the sludge compost to be deodorized for deodorization.

[0017] Furthermore, according to the application of Acinetobacter garneri in removing malodorous substances in sludge compost, the application is specifically: Acinetobacter garneri is aerobically cultured at 30°C for 1 day to obtain an activated bacterial liquid, and the bacterial liquid is inoculated into the sludge compost to be deodorized for deodorization.

[0018] Further, according to the application of the Acinetobacter gerneri in removing malodorous substances from sludge compost, the application is specifically: aerobically culturing the Acinetobacter gerneri at 28°C to 32°C for 0.5d to 1.5d to obtain an activated bacterial liquid, inoculating the bacterial liquid into a deodorization device, and passing malodorous gas containing ammonia from the sludge compost into the deodorization device for deodorization, and the temperature in the deodorization device is 20°C to 40°C.

[0019] Furthermore, according to the application of the Acinetobacter Gorneri in removing malodorous substances from sludge compost, the application is specifically: aerobically culturing the Acinetobacter Gorneri at 30°C for 1 day to obtain an activated bacterial liquid, inoculating the bacterial liquid into a deodorization device, and passing the malodorous gas containing ammonia from the sludge compost into the deodorization device for deodorization, and the temperature in the deodorization device is 30°C.

[0020] Optionally, in some embodiments of the present application, the Acinetobacter garneri is used as a deodorizing ingredient in the preparation of a deodorizing product.

[0021] Beneficial effects:

[0022] The invention provides a novel Acinetobacter garneri strain CGMCC No.16148, which has high deodorization efficiency and an ammonium nitrogen removal rate of more than 98% or even 100%. The invention only requires a single strain (Acinetobacter garneri) to be applied to biological deodorization, and has the obvious technical advantages of being easy to use, convenient to package and transport, and easy to form commercialization. The nutritional type of the Acinetobacter garneri strain is heterotrophic, and can use organic carbon sources such as beef extract and peptone to achieve rapid reproduction of bacterial bodies under heterotrophic conditions, and the logarithmic growth phase ends in 15 to 20 hours, reaching a stable phase, and achieving efficient deodorization. The bacteria can also use heterotrophic conditions to quickly obtain high-density bacterial bodies, thereby realizing rapid start-up of a biological deodorization project. The Acinetobacter garneri strain is screened from environmental samples that are subjected to long-term stress from substances such as ammonia, hydrogen sulfide, and volatile acids, and has no ecological invasion threat to the surrounding environment, and complies with biosafety regulations. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 is a diagram of the growth of Acinetobacter gerneri on a plate of the present application;

[0025] Figure 2 is the phylogenetic tree of the Acinetobacter glnerii strain of the present application;

[0026] Figure 3 It is a growth curve diagram of the Acinetobacter garneri strain of the present application at different temperatures. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. In addition, it should be understood that the specific implementation methods described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0028] In this application, "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0029] In the present application, "at least one" means one or more, and "plurality" means two or more. "One or several", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0030] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0031] The technical solution of this application is as follows:

[0032] See also Figure 1 The present application example provides Acinetobacter gerneri, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.16148 and a deposit date of July 25, 2018.

[0033] The invention provides a novel Acinetobacter garneri strain CGMCC No.16148, which has high deodorization efficiency and an ammonium nitrogen removal rate of more than 98% or even 100%. The invention only requires a single strain (Acinetobacter garneri) to be applied to biological deodorization, and has the obvious technical advantages of being easy to use, convenient to package and transport, and easy to form commercialization. The nutritional type of the Acinetobacter garneri strain is a heterotrophic nutritional type, and rapid reproduction of bacterial bodies is achieved under heterotrophic conditions, and the logarithmic growth phase ends in 15 to 20 hours, reaching a stable phase, and efficient deodorization is achieved. The bacteria can also use heterotrophic conditions to quickly obtain high-density bacterial bodies, thereby realizing rapid start-up of a biological deodorization project. The Acinetobacter garneri strain is screened from environmental samples that are subjected to long-term stress from substances such as ammonia, hydrogen sulfide, and volatile acids, and has no ecological invasion threat to the surrounding environment, and complies with biosafety regulations.

[0034] Optionally, in some embodiments of the present application, the 16S rDNA sequence of Acinetobacter garneri is shown in the sequence listing SEQ ID No: 1.

[0035] In some embodiments, the Acinetobacter gerneri is derived from aerobic composting of the Jingyan County Qiangsheng Livestock and Poultry Breeding Professional Cooperative.

[0036] In some embodiments, the Acinetobacter garneri is used in the preparation of a deodorizing bacterial agent.

[0037] Furthermore, the deodorizing bacterial agent is a liquid deodorizing bacterial agent or a solid deodorizing bacterial agent.

[0038] For example, the Acinetobacter cerna strain CGMCC No. 16148 can be cultured in a liquid culture medium under appropriate conditions to obtain a liquid deodorant, and then the liquid deodorant is mixed with sterilized bran at a mass ratio of 1:1 (W / W), and then dried in a constant temperature box to control the moisture, which is a solid deodorant. The deodorant can be further processed and packaged into a variety of deodorant products.

[0039] In some embodiments, the Acinetobacter garneri is used to remove malodorous substances from sludge composting.

[0040] Furthermore, the malodorous substance is a nitrogen-containing malodorous gas.

[0041] Furthermore, the nitrogen-containing malodorous gas includes ammonia.

[0042] Furthermore, the malodorous substance is an ammonium ion-containing compound.

[0043] Furthermore, the ammonium ion-containing compound includes (NH4)2SO4.

[0044] Furthermore, according to the application of Acinetobacter garneri in removing malodorous substances from sludge compost, the application is specifically: aerobically culturing Acinetobacter garneri at 28°C to 32°C for 0.5d to 1.5d to obtain an activated bacterial liquid, and inoculating the bacterial liquid into the sludge compost to be deodorized for deodorization.

[0045] Furthermore, according to the application of Acinetobacter garneri in removing malodorous substances in sludge compost, the application is specifically: Acinetobacter garneri is aerobically cultured at 30°C for 1 day to obtain an activated bacterial liquid, and the bacterial liquid is inoculated into the sludge compost to be deodorized for deodorization.

[0046] Further, according to the application of the Acinetobacter gerneri in removing malodorous substances from sludge compost, the application is specifically: aerobically culturing the Acinetobacter gerneri at 28°C to 32°C for 0.5d to 1.5d to obtain an activated bacterial liquid, inoculating the bacterial liquid into a deodorization device, and passing malodorous gas containing ammonia from the sludge compost into the deodorization device for deodorization, and the temperature in the deodorization device is 20°C to 40°C.

[0047] Furthermore, according to the application of the Acinetobacter Gorneri in removing malodorous substances from sludge compost, the application is specifically: aerobically culturing the Acinetobacter Gorneri at 30°C for 1 day to obtain an activated bacterial liquid, inoculating the bacterial liquid into a deodorization device, and passing the malodorous gas containing ammonia from the sludge compost into the deodorization device for deodorization, and the temperature in the deodorization device is 30°C.

[0048] In some embodiments, the Acinetobacter garneri is used as a deodorant ingredient in the preparation of a deodorant product.

[0049] The optimal pH of the Acinetobacter garneri of the present invention is 7.0, and it can grow rapidly at 20° C. to 35° C., and the optimal temperature is 30° C. The bacterial nutrition type is heterotrophic, and it can utilize organic carbon sources such as beef extract and peptone for heterotrophic rapid growth.

[0050] Test Example 1

[0051] Strain screening and identification:

[0052] 1. Strain screening

[0053] 1.1 Samples and culture medium

[0054] Samples: Four sampling points were selected from the aerobic composting tank of the Qiangsheng Livestock and Poultry Breeding Professional Cooperative in Jingyan County. The sampling points covered the heating stage, high temperature stage and cooling stage of the composting. 300g of composting materials were collected from each sampling point, and then the composting materials collected from the four points were mixed. After mixing, 300g was collected from them as samples for standby use. The raw materials for aerobic composting include chicken manure, sludge, sawdust and other raw materials.

[0055] Enriched acclimation medium: (NH4)2SO4 4.5g, sodium succinate 12.35g, glucose 4.5g, sodium acetate 4.5g, K2HPO4 0.5g, MgSO4·7H2O 0.1g, NaCl 0.2g, FeSO4·7H2O 0.01g, MnSO4 0.005g, add water to 1000ml, pH 7.0. High temperature steam sterilization at 121℃, 20min.

[0056] Isolation medium: (NH4)2SO4 2.5g, sodium succinate 8.5g, glucose 2.5g, sodium acetate 2.5g, K2HPO40.5g, MgSO4·7H2O 0.1g, NaCl 0.2g, FeSO4·7H2O 0.01g, MnSO40.005g, agar powder 18g, add water to 1000ml, pH 7.0. High temperature steam sterilization at 121℃, 20min.

[0057] 1.2 Isolation and purification of strains

[0058] Add 5g of sample to 100ml of enrichment medium, enrich and acclimate for 3 days at 30℃ and 180r / min; acclimate the enrichment solution for the second time with an inoculum size of 10%; after culturing for 3 days; then take 1ml from the acclimatization solution for 10-fold gradient dilution, and then take 0.1mL of the diluted bacterial solution from each dilution gradient, spread it on the separation medium plate for separation, and single colonies will be formed on the plate after 1d to 2d. Pick out suitable single colonies from the single colonies formed on the plate, dilute and spread them again, and repeat more than 3 times to obtain pure bacteria.

[0059] 2. Strain identification

[0060] 2.1 Strain morphology, growth characteristics and 16S rDNA sequence analysis

[0061] Take the above strains and observe them under an optical microscope. The cell morphology of the strain is rod-shaped. The colony is milky white, with a moist and smooth surface, a convex center, a round shape and smooth edges. Figure 1 As shown (colony diagram after 2 days of growth). Gram staining was negative. The strain was inoculated into a 96-well plate and placed in a fully automatic growth curve tester. Medium-speed shaking culture was performed at 20°C, 25°C, 30°C, 35°C, and 40°C. The instrument automatically measured the OD value of the strain at different times. According to the growth curve diagram of the strain at different temperatures (as shown in Figure 2 The growth temperature range of the above strains was determined to be 20°C to 35°C, and the optimum growth temperature was 30°C.

[0062] The whole genome of the pure strain was extracted using a bacterial whole genome rapid extraction kit, and the 16S rDNA sequence was amplified using universal primers 27F / 1429R. The amplified PCR product was tested by agarose gel electrophoresis and UV spectrophotometer for concentration and purity, and then sequenced. The sequencing results were compared with BLAST on NCBI, and the sequence homology with Acinetobacter gerneri (MTCC 9824) was as high as 99.57%. The phylogenetic tree is shown in Figure 3 , A01 in the phylogenetic tree is the Acinetobacter garneri of the present invention, and the 16S rDNA sequence of the Acinetobacter garneri is shown in the sequence listing SEQ ID No: 1.

[0063] 27F: 5′-AGAGTTTGATCCTGGCTC AG-3′

[0064] 1429R:5′-CGGCTACCTTGTTACGAC-3′

[0065] 2.2 Strain naming

[0066] Based on the morphological characteristics, physiological and biochemical properties and 16S rDNA sequence analysis (such as SEQ ID NO.1) of the purified strain, the purified strain was named Acinetobacter gerneri.

[0067]

[0068] The above strains are deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101; deposit date July 25, 2018, deposit number CGMCC No.16148.

[0069] Test Example 2

[0070] The Acinetobacter gerneri of the present invention is used for removing nitrogen-containing malodorous substances.

[0071] 1. Culture medium

[0072] Beef extract peptone medium was used as growth and storage medium.

[0073] High concentration ammonium ion culture medium: (NH4)2SO4 3.8g, sodium succinate 13.5g, glucose 6.0g, K2HPO4 0.5g, MgSO4·7H2O 0.1g, NaCl 0.2g, FeSO4·7H2O 0.01g, MnSO4 0.005g, add water to 1000ml, pH 7.0. Dispense 150ml each into 250ml Erlenmeyer flasks, and sterilize at 121℃ for 20min.

[0074] The low-concentration ammonium ion culture medium reduces the first three components of the high-concentration ammonium ion culture medium by 80%, while the other conditions remain unchanged.

[0075] 2. Experimental methods

[0076] The strain obtained in Test Example 1 was inoculated into a test tube containing 5 ml of growth and preservation medium and cultured at 30° C. for 1 day under aerobic conditions. When the bacterial solution grew well, an activated bacterial solution was obtained.

[0077] Inoculate the bacterial solution at an inoculum volume of 1% (V / V) into high-concentration ammonium ion culture medium and low-concentration ammonium ion culture medium (250ml Erlenmeyer flasks), respectively. Immediately after inoculation, take samples from the Erlenmeyer flasks containing the bacterial solution to determine the total nitrogen, nitrite nitrogen, nitrate nitrogen, and ammonium nitrogen content in the liquid. Culture at 30°C. Take samples once every 12 hours for the low ammonium ion concentration group and once every 24 hours for the high ammonium ion concentration group to determine the total nitrogen, nitrite nitrogen, nitrate nitrogen, and ammonium nitrogen content in the culture solution. Stop the determination when the ammonium ion concentration in each Erlenmeyer flask is very low. Set up three parallels for each experimental group.

[0078] The control group (CK) used an equal amount of sterile water instead of bacterial solution and was cultured, sampled and measured in the same way.

[0079] 3. Experimental results

[0080] The changes in the concentrations of total nitrogen, nitrite nitrogen, nitrate nitrogen and ammonium nitrogen in the high and low ammonium ion concentration culture solutions inoculated with the strain of the present invention are shown in Table 1.

[0081] Table 1 Changes of nitrogen concentration in various forms over time (mg / L, in terms of nitrogen)

[0082]

[0083] As shown in Table 1, in the low ammonium ion concentration group, the ammonium ion conversion rate reached 81.4% in 12 hours, 100% in 24 hours, and the total nitrogen loss rate was about 6.3%; in the high ammonium ion concentration group, the ammonium ion conversion rate reached 98.6% after 6 days of cultivation, the daily converted ammonium ions were 134.0 mg / L, and the total nitrogen loss rate was about 8.9%.

[0084] Test Example 3

[0085] The Acinetobacter garneri CGMCC No. 16148 of the present invention is applied to aerobic composting of sludge to reduce the amount of ammonia produced.

[0086] 1. Culture medium

[0087] The growth and storage medium is the same as in Example 2.

[0088] 2. Experimental methods

[0089] The strain obtained in Test Example 1 was inoculated into a test tube containing 5 ml of culture medium and cultured aerobically at 30°C for 1 day. The activated bacterial solution was then inoculated into a conical flask containing 250 ml of growth and preservation medium and cultured aerobically at 30°C for 1 day. After the bacterial solution has grown well, it is ready for use.

[0090] The residual sludge from the sewage treatment plant was selected as the raw material and sawdust as the auxiliary material for the experiment, with a mass ratio of 95:5. The raw and auxiliary materials were mixed evenly and heated to 1m 3 The proportion of spraying 100ml of bacterial solution on the compost raw materials is to make the Acinetobacter gerneri strains and the compost raw materials evenly mixed. The total volume of compost is 1.5m 3 The bottom of the pile was laid with porous plastic pipes, and a turbocharger fan was used to deliver oxygen to the pile for aerobic fermentation. No activated bacteria were added to the control group. Then a composite high-precision gas analyzer (model PGD5-C-M5) was used to measure the concentration of ammonia in the gas in the pile at different fermentation times.

[0091] 3. Experimental results

[0092] The changes in ammonia concentration in the aerobic compost pile inoculated with the strain of the present invention are shown in Table 2.

[0093] Table 2 Changes of ammonia concentration in sludge aerobic composting pile over time (ppm)

[0094] Time / d 1 3 5 10 15 20 25 Control group 146.7 201.8 246.5 236.1 221.6 211.4 208.3 Experimental Group 28.3 45.0 49.5 40.2 36.1 25.3 20.4

[0095] As can be seen from Table 2, the ammonia concentration in the aerobic compost pile inoculated with the strain of the present invention is significantly lower than that in the control group.

[0096] The above is a detailed introduction to the Acinetobacter gerneri and its application provided in the examples. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method and core idea of ​​the present application. At the same time, for those skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. Acinetobacter garneri, characterized in that: The Acinetobacter gerneri is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.16148 and the deposit time being July 25, 2018.

2. Acinetobacter garneri according to claim 1, characterized in that: The 16SrDNA sequence of Acinetobacter garneri is shown in the sequence table SEQ ID No:

1.

3. Use of Acinetobacter garneri according to claim 1 in the preparation of a deodorizing bacterial agent.

4. The use according to claim 3, characterized in that: The deodorizing bacterial agent is a liquid deodorizing bacterial agent or a solid deodorizing bacterial agent.

5. Use of Acinetobacter garneri according to claim 1 in removing malodorous substances from sludge composting.

6. The use according to claim 5, characterized in that: The malodorous substances include nitrogen-containing malodorous gases and / or ammonium ion compounds.

7. The use according to claim 6, characterized in that: The nitrogen-containing malodorous gas includes ammonia, and / or The ammonium ion-containing compound includes (NH4)2SO4.

8. Use of the Acinetobacter garneri according to claim 1 as a deodorizing ingredient in the preparation of a deodorizing product.

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