Separated acinetobacter calcoaceticus F18 and application thereof in ethyl acetate degradation

Abstract

The invention belongs to the technical field of biology, and discloses separated acinetobacter calcoaceticus F18 and application thereof in degradation of ethyl acetate, the preservation number of the strain is CCTCC NO: M20252127, the degradation rate of the strain F18 for degrading the ethyl acetate to 0.36 g / L ethyl acetate in a VOCs sampling bag for 24 h reaches 75.5%, and the degradation rate of the strain F18 for degrading the ethyl acetate to 0.36 g / L ethyl acetate in 48 h can reach 91.3%. The F18 strain has good extreme environment and ethyl acetate tolerance, can grow in high-salt 6.0% NaCl at the initial pH value of 5.0 and pH value of 10.0, at the temperature of 20-40 DEG C, can keep 87% removal rate when the ethyl acetate concentration reaches up to 1.44 g / L, and can be applied to a high-concentration ethyl acetate treatment scene.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to an isolated Acinetobacter calcoaceticus F18 and application thereof in ethyl acetate degradation. BACKGROUND

[0002] Ethyl acetate (molecular formula: CH3CO2CH2CH3) is a common ester organic solvent. Ethyl acetate is an industrial chemical widely used. In addition to being used as a solvent, ethyl acetate is often used in the production of resins such as polyurethane and epoxy resin. Every year, a large amount of ethyl acetate is released into the atmosphere during manufacturing, use and disposal. Gaseous ethyl acetate is an irritating and explosive substance. The loss of these substances into the ambient air can adversely affect air quality, thereby endangering public health. Ethyl acetate is highly volatile and has an irritating effect on the eyes, skin, mucous membranes and upper respiratory tract.

[0003] Acinetobacter calcoaceticus belongs to the genus Acinetobacter. Zhao Changle (2023) et al. found that the genus Acinetobacter has the function of degrading polyethylene. Studies have shown that strains of this genus can degrade various environmental pollutants, such as petroleum hydrocarbons, phenolic compounds, and cypermethrin (Li Min 2019).

[0004] Wen Shaofu (2025) et al. found that Acinetobacter calcoaceticus can effectively reduce the content of ion exchange state lead in soil, alleviate the stress of lead in soil on corn, and reduce the accumulation of lead in corn plants; this provides a reference case for the use of Acinetobacter in environmental governance.

[0005] The method of biodegrading VOCs and other harmful substances has the advantages of high cost-effectiveness, less residue or byproduct, and environmental friendliness. Therefore, it is of great environmental protection significance to provide a method for degrading ethyl acetate by using Acinetobacter calcoaceticus through biological fermentation, thereby reducing the harm of ethyl acetate to the human body. SUMMARY

[0006] The purpose of the present application is to provide an Acinetobacter calcoaceticus F18. The preservation number of the strain is CCTCC NO: M20252127.

[0007] Another purpose of the present application is to provide the application of Acinetobacter calcoaceticus F18 in removing ethyl acetate.

[0008] In order to achieve the above purpose, the present application adopts the following technical measures:

[0009] The applicant isolated a strain of bacteria from a biological deodorization treatment system of a company in Hangzhou, and the strain has good ethyl acetate removal capacity and is identified as Acinetobacter calcoaceticus. The strain was sent to the China Center for Type Culture Collection on September 25, 2025, and the address is: China, Wuhan, Wuhan University, the classification name is Acinetobacter calcoaceticus F18, and the preservation number is CCTCC NO: M20252127.

[0010] The Acinetobacter calcoaceticus F18 of the application is cultured on an LB medium plate for 1 day. The colony is round, the front is yellow, the surface is smooth, the edge is neat and clear, and the colony is easy to pick up. The microscopic morphology is short rod-shaped.

[0011] The scope of protection of the application also includes:

[0012] The fermentation broth of Acinetobacter calcoaceticus F18 contains live Acinetobacter calcoaceticus F18.

[0013] A composition comprising Acinetobacter calcoaceticus or / and the fermentation broth of Acinetobacter calcoaceticus F18.

[0014] Application of Acinetobacter calcoaceticus, the fermentation broth of Acinetobacter calcoaceticus F18 or / and the above-mentioned composition in the degradation of ethyl acetate

[0015] Application of Acinetobacter calcoaceticus, the fermentation broth of Acinetobacter calcoaceticus F18 or / and the above-mentioned composition in the preparation of an ethyl acetate degradation agent.

[0016] Application of Acinetobacter calcoaceticus, the fermentation broth of Acinetobacter calcoaceticus F18 or / and the above-mentioned composition as one of the effective components or the main component in the treatment of ethyl acetate in a water environment.

[0017] Application of Acinetobacter calcoaceticus, the fermentation broth of Acinetobacter calcoaceticus F18 or / and the above-mentioned composition as one of the effective components or the main component in the preparation of a water environment treatment biological agent.

[0018] The application described above, preferably, the pH of the water environment is pH 5-pH 10;

[0019] The application described above, preferably, the temperature of the water environment is 20-40℃;

[0020] The application described above, preferably, the NaCl concentration of the water environment is 1.0%-6.0%.

[0021] Compared with the prior art, the application has the following characteristics:

[0022] The A. calcoaceticus F18 in the application is first reported to have the function of removing ethyl acetate, has rapid growth of bacterial bodies, high viable bacterial count and strong ethyl acetate removal capacity.

[0023] The A. calcoaceticus F18 in the application has rapid growth speed and high bacterial body density at 25 DEG C. The degradation rate of the strain on 0.36 g / L ethyl acetate in a VOCs sampling bag reaches 75.5% in 24 h, and the degradation rate of the strain on 0.36 g / L ethyl acetate reaches 91.3% in 48 h. The strain has good extreme environment tolerance and can grow in initial pH 5.0 and pH 10.0, temperature 25-40 DEG C and high salt 6.0% NaCl. The strain also has good removal rate on ethyl acetate with a concentration of 1.26 g / L, and can be applied to high-concentration ethyl acetate treatment scenarios BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a colony diagram of the A. calcoaceticus F18.

[0025] Figure 2 It is a growth curve diagram of the A. calcoaceticus F18.

[0026] Figure 3 It is an ethyl acetate removal rate of the A. calcoaceticus F18 VOCs test.

[0027] Figure 4 It is a growth change diagram of the A. calcoaceticus F18 under different initial pH values.

[0028] a, b, c, d, e, f, g represent pH, OD 600nm , viable bacterial count of the strain F18; the same letter represents no significant difference (p>0.05) in viable bacterial count of the strain F18; the letter only represents the comparison between pH (OD 600nm or viable bacterial count).

[0029] Figure 5 It is a growth change diagram of the A. calcoaceticus F18 under different temperatures.

[0030] a, b, c, d, e represent pH, OD 600nmSignificant difference (p<0.05) between the viable cell counts of strain F18; the same letter means no significant difference (p>0.05) between the viable cell counts of strain F18; the letter only represents the comparison between pH (OD 600nm or viable cell count.

[0031] Figure 6 The growth change diagram of A. calcoaceticus F18 under different NaCl concentrations;

[0032] a, b, c, d, e, f, g represent the pH, OD 600nm Significant difference (p<0.05) between the viable cell counts of strain F18; the same letter means no significant difference (p>0.05) between the viable cell counts of strain F18; the letter only represents the comparison between pH (OD 600nm or viable cell count.

[0033] Figure 7 The removal rate of A. calcoaceticus F18 under high concentration of ethyl acetate.

[0034] a, b, c, d, e represent significant difference (p<0.05) between the removal rates of strain F18; the same letter means no significant difference (p>0.05) between the viable cell counts of A. calcoaceticus F18. DETAILED DESCRIPTION

[0035] The technical solutions described in the present application are all conventional technologies if not specifically stated; the reagents or materials described in the present application are all from commercial channels if not specifically stated. Each experimental group of the present application is provided with three parallels if not specifically stated.

[0036] Example 1:

[0037] Isolation and identification of A. calcoaceticus F18

[0038] The applicant adds 10.0 g of the filler from a biological trickling filter tower in a biological deodorization treatment system of a certain company in Hangzhou into a conical flask containing 90 ml of sterile normal saline and glass beads, and performs oscillation at 200 r / min for 1 h at 30°C, so that the attachments on the filler are basically removed, and the liquid becomes turbid. 5 mL of supernatant is sucked from the conical flask and transferred into 50 mL of an inorganic salt selective culture medium with an ethyl acetate concentration of 25 mg / L. The bottle opening is sealed with a silica gel plug, and the bottle opening is further sealed by sequentially adding 1 layer of sealing film and 2 layers of plastic film. The sealed culture medium is placed on a constant-temperature shaker at 30°C and 200 r / min for gradient domestication. During the culture process, 5 mL is taken from the culture solution every 1 day and transferred into 50 mL of fresh culture medium of the same kind, and meanwhile the ethyl acetate concentration is increased until 100.0 mg / L, and the domestication is stopped. After the gradient domestication is completed, the final selective culture medium is diluted with sterile water and coated on LB solid culture medium, and single colonies are continuously streaked and separated to obtain single bacteria, which are named F18.

[0039] The LB culture medium has the following components: 10.0 g / L of tryptone, 5.0 g / L of yeast extract powder, 10.0 g / L of sodium chloride, pH 7.0-pH 7.2, and sterilization at 121°C for 30 min. The inorganic salt selective culture medium has the following components: 4.72 g of potassium nitrate, 0.7 g of potassium dihydrogen phosphate, 2.52 g of sodium hydrogen phosphate, 0.0244 g of magnesium sulfate heptahydrate, and 0.015 g of calcium chloride, and the volume is made up to 1000 mL with distilled water, pH 7.0, and sterilization at 121°C for 30 min.

[0040] Strain F18 strain identification

[0041] (1) Morphological characteristics

[0042] The colony morphology and microscopic morphology of strain F18 on LB solid culture medium are shown in Figure 1 , the colony is round, the front is yellow, the surface is smooth, the edge is neat and clear, and the microscopic morphology is short rod-shaped.

[0043] (2) Gene identification of strain F18

[0044] The bacterial DNA extraction kit is used to extract the genomic DNA of the strain to be tested, and the specific operation steps are referred to the instruction manual. The extracted bacterial genomic DNA is used as a template to amplify the 16S rRNA gene of strain F18, and the primers are as follows:

[0045] 27F: 5´-AGAGTTTGATCCTGGCTCAG-3´

[0046] 1492R: 5´-TACGACTTAACCCCAATCGC-3´;

[0047] The spliced sequence was found to belong to Acinetobacter calcoaceticus by Blast homologous sequence retrieval, and was deposited with the China Center for Type Culture Collection on September 25, 2025, at an address in Wuhan, China, Wuhan University, classified and named as Acinetobacter calcoaceticus F18, and the deposit number is CCTCC NO: M20252127.

[0048] (3) Growth curve of strain F18

[0049] A single colony of strain F18 was picked into LB medium and cultured for 18 h to obtain fresh seed liquid, with an inoculation amount of 2%, a liquid volume of 20%, inoculation into LB base medium, and culture in a 30°C, 200 r / min shake flask, with 2 parallel repeats. OD 600nm and viable cell count were measured every 2 h to explore the growth characteristics of strain F18.

[0050] The results are shown in Figure 2 , which entered the logarithmic growth phase at 4 h, and reached a maximum value of 89.8 x 10 8 CFU / mL at 18 h, and then entered the decline phase, so the subsequent culture time was determined to be 18 h.

[0051] Example 2:

[0052] Growth performance of Acinetobacter calcoaceticus F18 and ethyl acetate removal test

[0053] Acinetobacter calcoaceticus F18 was cultured in liquid LB seed medium at 30°C for 18 h, and the viable cell concentration was 8.98 x 10 9 CFU / mL. 2.5 mL of bacterial solution was centrifuged at 5000 rpm for 5 minutes, the supernatant was removed, and the bacterial solution was resuspended with sterile water. After washing and centrifuging again, the bacterial solution was resuspended and washed twice, and the bacterial solution at the bottom of the centrifuge tube was added to a 1L VOCs sampling bag containing 50 mL of inorganic salt selective medium. The formula of the inorganic salt selective medium is: potassium nitrate 4.72 g, potassium dihydrogen phosphate 0.7 g, sodium hydrogen phosphate 2.52 g, magnesium sulfate heptahydrate 0.0244 g, calcium chloride 0.015 g, distilled water to 1000 mL, pH 7.0, sterilized at 121°C for 30 min.

[0054] In 1L VOCs sampling bag, add 20 μl of ethyl acetate; while set up no bacteria, only add ethyl acetate and inorganic salt medium blank control. Place 1L VOCs sampling bag in constant temperature shaker, temperature 30℃, rotation speed 200 r / min, culture for 12h, 24h, 36h, 48h, detect ethyl acetate content of control group and experimental group by portable gas detector. After measuring ethyl acetate content according to the above method, calculate ethyl acetate removal rate according to the following formula.

[0055] Ethyl acetate removal rate =

[0056] The results are shown in Figure 3 The strain can achieve 37.1% of ethyl acetate removal rate at 12h, with the adaptation of the strain to ethyl acetate and the reduction of ethyl acetate concentration, the degradation rate begins to be fast, the strain can achieve 58.6% of ethyl acetate removal rate at 24h; at 48h, most of the ethyl acetate is almost degraded, reaching 91.3% removal rate.

[0057] Example 3:

[0058] pH tolerance test of A. calcoaceticus F18

[0059] Prepare 13 kinds of LB liquid medium with different initial pH gradients, respectively pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, pH 10.0, pH 11.0. Inoculate the seed liquid of A. calcoaceticus F18 into the medium according to 1%, culture at 30℃, 200 r / min for 18h, take sample to measure the pH, OD 600nm and viable cell count of each medium, analyze the influence of different initial pH on the growth of A. calcoaceticus F18.

[0060] The growth change of A. calcoaceticus F18 under different initial pH is shown in Figure 4 . Figure 4 It is shown that A. calcoaceticus F18 can grow normally at pH 5.0-pH 10.0, and the viable cell count is above 2.50×10 9 CFU / mL; among them, the initial pH value is 7.0, the viable cell count is the largest, reaching 6.92×10 9CFU / mL. With the pH value being acidic or alkaline, the viable cell count gradually decreased. When the initial pH value was pH 4.0 and below, the growth of A. calcoaceticus F18 was greatly inhibited and could not grow; when the pH value was pH 11.0 and above, the growth of the strain was significantly inhibited and was significantly lower than the normal level. The above test results showed that A. calcoaceticus F18 had good pH tolerance.

[0061] Example 4:

[0062] Temperature tolerance test of A. calcoaceticus F18

[0063] Eight different culture temperatures were set, which were 20℃, 25℃, 30℃, 35℃, 40℃, 45℃. The seed liquid of A. calcoaceticus F18 was inoculated in the culture medium at an amount of 1%, and cultured at 30℃, 200 r / min for 18h. The pH, OD 600nm and viable cell count of each culture medium were determined, and the effect of different temperatures on the growth of A. calcoaceticus F18 was analyzed.

[0064] The growth change of A. calcoaceticus F18 at different temperatures is shown in Figure 5 .

[0065] Figure 5 It showed that A. calcoaceticus F18 could grow at 20℃-40℃, and had the maximum viable cell count of 6.00x10 9 CFU / mL at 25℃; at 20℃, the activity of strain F18 was inhibited, and the growth activity was greatly reduced, with a viable cell count of 1.50x10 8 CFU / mL; at 45℃ and above, the activity of the bacteria was greatly inhibited and the bacteria could not grow normally and died.

[0066] Example 5:

[0067] NaCl tolerance test of A. calcoaceticus F18

[0068] Thirteen kinds of LB liquid culture media containing different NaCl concentration gradients were prepared, and the NaCl concentration was 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%. The seed liquid of A. calcoaceticus F18 was inoculated in the culture medium at an amount of 1%, and cultured at 30℃, 200 r / min for 18h. The pH, OD 600nmand viable cell count, the effect of different NaCl concentrations on the growth of A. calcoaceticus F18 was analyzed.

[0069] The growth of A. calcoaceticus F18 under different NaCl concentrations is shown in Figure 6

[0070] Figure 6 It is shown that A. calcoaceticus F18 can grow when the NaCl concentration is 1.0%~6.0%. When the NaCl concentration is 1%, the viable cell count is the largest, reaching 7.30×10 9 CFU / mL; as the NaCl concentration rises, the growth rate of A. calcoaceticus F18 begins to decrease. When the NaCl concentration is 1%~3%, the strain grows normally, and the viable cell count can be maintained at more than 3.00×10 9 CFU / mL; when the NaCl concentration is 4%~6%, the growth of the strain is obviously inhibited, and the viable cell count increases slowly. When the NaCl concentration is greater than or equal to 7%, the strain no longer grows. The above test results show that A. calcoaceticus F18 has good osmotic pressure tolerance.

[0071] Example 6:

[0072] Removal rate test of A. calcoaceticus F18 under high concentration of ethyl acetate

[0073] A. calcoaceticus F18 was cultured in liquid LB seed medium at 30°C for 18 h, and the viable cell concentration was 8.98×10 9 CFU / mL. 2.5 mL of bacterial solution was centrifuged at 5000 rpm for 5 min, the supernatant was removed, and the bacterial solution was resuspended with sterile water. After washing and centrifuging again, the bacterial solution was resuspended and washed twice, and the bacterial solution at the bottom of the centrifuge tube was added to a 1L VOCs sampling bag containing 50 mL of inorganic salt selective medium. The formula of the inorganic salt selective medium is: potassium nitrate 4.72 g, potassium dihydrogen phosphate 0.7 g, sodium hydrogen phosphate 2.52 g, magnesium sulfate heptahydrate 0.0244 g, calcium chloride 0.015 g, and distilled water to 1000 mL, pH 7.0, sterilized at 121°C for 30 min.

[0074] ​Ethyl acetate was added to 1L VOCs sampling bags at concentrations of 0.36 g / L, 0.54 g / L, 0.72 g / L, 0.90 g / L, 1.08 g / L, 1.26 g / L, 1.44 g / L, and 1.62 g / L, respectively. A blank control group was also included, containing only ethyl acetate and inorganic salt culture medium without any bacteria. The 1L VOCs sampling bags were incubated in a constant-temperature shaker at 30℃ and 200 r / min for 48 h. The ethyl acetate content in both the control and experimental groups was measured using a portable gas detector.

[0075] The results are as follows Figure 7 As shown, the strain achieved a maximum removal rate of 91.65% of ethyl acetate at a concentration of 0.36 g / L after 48 hours. At substrate concentrations ranging from 0.36 g / L to 1.44 g / L, the removal rate remained above 87.00% after 48 hours. However, at a substrate concentration of 1.62 g / L, the ethyl acetate removal efficiency decreased significantly to 74.18% after 48 hours. The concentration of 1.44 g / L represents a relatively high tolerance level in engineering applications, indicating that the strain has good tolerance to ethyl acetate and can treat high-concentration ethyl acetate waste gas and wastewater.

[0076] In summary, A. calcoaceticus F18 in this invention can be applied to the removal of ethyl acetate. Due to its strong tolerance to high salt and strong acid and alkali conditions, its application scenarios are also more extensive. In addition to the treatment of ethyl acetate in ordinary waste gas and wastewater, it can also be used for the treatment of ethyl acetate in strong acid and alkali wastewater and high-concentration ethyl acetate waste gas, with stable effect and high removal rate.

Claims

1. An isolated strain of Acinetobacter acetoacetate ( Acinetobacter calcoaceticus F18, accession number: CCTCC NO: M20252127.

2. The fermentation broth of Acinetobacter calcitrate F18 according to claim 1, wherein the fermentation broth contains live Acinetobacter calcitrate F18 bacteria.

3. A composition comprising the fermentation broth of Acinetobacter calciacetate as claimed in claim 1 and / or Acinetobacter calciacetate F18 as claimed in claim 2.

4. The use of the fermentation broth of Acinetobacter calcifera according to claim 1, Acinetobacter calcifera F18 according to claim 2, or / and the composition according to claim 3 in the degradation of ethyl acetate.

5. The use of the fermentation broth of Acinetobacter calciacetate of claim 1, Acinetobacter calciacetate F18 of claim 2, or / and the composition of claim 3 in the preparation of an ethyl acetate degrading agent.

6. The use of the fermentation broth of Acinetobacter calciacetate of claim 1, Acinetobacter calciacetate F18 of claim 2, or / and the composition of claim 3 as one of the active ingredients or the main active ingredient in the treatment of ethyl acetate in an aquatic environment.

7. The use of the fermentation broth of Acinetobacter calcifera according to claim 1, the Acinetobacter calcifera F18 according to claim 2, or / and the composition according to claim 3 as one of the active ingredients or the main active ingredient in the preparation of biological agents for water environment treatment.

8. The application according to claim 6 or 7, wherein the pH of the water environment is 5-10.

9. The application according to claim 6 or 7, wherein the temperature of the water environment is 20-40°C.

10. The application according to claim 6 or 7, wherein the NaCl concentration in the aquatic environment is 1.0% to 6.0%.

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