A Lawrence estuarine Pseudomonas D13 and its application

Abstract

The invention provides a Lawrence estuarine Pseudomonas D13 and an application thereof. The Pseudomonas D13 has a strong ability to degrade n-hexanal and can effectively remove n-hexanal. The bacterium also has good acid-base tolerance and osmotic pressure tolerance and can grow and reproduce in extreme acid-base stress environments and salt stress environments.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, relates to Lawrence Estuarine Pseudomonas D13 and application thereof. Background Art

[0002] Hexanal is an organic compound belonging to the class of saturated fatty aldehydes. Some foods and natural products contain trace amounts of hexanal, which is generally considered safe, so it can also be used as a fragrance and solvent. However, when it comes to industrial use or accidental exposure, higher concentrations of hexanal harm the respiratory system, irritate the skin and eyes, and affect the central nervous system, causing certain harm to the human body. At present, research on the degradation of hexanal focuses on the catalytic degradation of metal compounds, which catalyzes the degradation of hexanal through metal oxides such as titanium, manganese, and cobalt.

[0003] Pseudomonas spp. belongs to the genus Pseudomonas. GF ​​Rafikova et al. (2020) found that Pseudomonas laurentiana ( P. laurentiana ) has antifungal activity and can decompose phosphates and synthesize plant hormone substances. Elena K (2022) et al. Pseudomonas laurentiana It can increase the germination rate of some leguminous seeds, enhance the growth of stems, leaves and roots, improve the nitrogen nutrition of plants, and stimulate the formation of nodules; it can also enhance the growth of leguminous seeds in oil-contaminated soil and accelerate the biodegradation process of oil. Pseudomonas laurentiana Related reports on the degradation of n-hexanal.

[0004] Therefore, a method for utilizing Pseudomonas laurensis ， It is of great significance to degrade n-hexanal through biological fermentation and reduce the harm of n-hexanal to human body. Summary of the invention

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a Pseudomonas estuarine D13 for degrading n-hexanal. The Pseudomonas estuarine D13 was deposited in the China Center for Type Culture Collection on July 12, 2024 and was classified and named Pseudomonas estuarine D13 ( Pseudomonas laurentiana D13), the deposit number is CCTCC No: M 20241537.

[0006] Wherein, the Pseudomonas estuarineus D13 has a nucleotide sequence as shown in SEQ ID NO.1.

[0007] The Pseudomonas laurensis D13 described in this application ( Pseudomonas laurentianaD13) The colonies are round, white, smooth, with clear edges, easy to pick up, and short rod-shaped under microscopic conditions.

[0008] The Lawrence estuarine Pseudomonas D13 provided by the invention has good acid-base tolerance and osmotic pressure tolerance, and can grow and reproduce in extreme acid-base stress environments and salt stress environments.

[0009] Regarding pH tolerance, the Pseudomonas spp. D13 has growth activity in a culture medium with a pH range of 5.0 to 12.0, and its growth is significantly inhibited when the pH is lower than 4.0 or higher than 12.0.

[0010] Regarding NaCl tolerance, the Lawrence Estuarine Pseudomonas D13 has growth activity in a culture medium with a NaCl mass ratio concentration of no more than 4%. When the NaCl mass ratio concentration is 5% to 6%, the growth of the strain is significantly inhibited, and the growth amount increases but the growth amount is small. When the NaCl mass ratio concentration is greater than or equal to 7%, the strain no longer grows.

[0011] The culture medium refers to a medium that can provide the necessary carbon source, nitrogen source and other trace elements for the growth of the Lawrence Estuarine Pseudomonas D13. The optional carbon source includes, but is not limited to, any one or more combinations of glucose, sucrose, fructose, maltose and starch. The optional nitrogen source includes, but is not limited to, any one or more combinations of tryptone, yeast extract and beef extract.

[0012] As a preferred embodiment, the culture medium is BPY culture medium, and its specific ingredients are: 5.0 g / L glucose, 10.0 g / L tryptone, 5.0 g / L yeast extract, 5.0 g / L beef extract, 5.0 g / L sodium chloride, pH 7.0, sterilized at 115°C for 30 min.

[0013] In a second aspect of the present invention, there is provided a method for treating the above-mentioned Pseudomonas estuarineis D13 ( Pseudomonas laurentiana D13) Application in the degradation of n-hexanal.

[0014] As a preferred embodiment, Pseudomonas estuarineis D13 is used to degrade n-hexanal in the gas phase.

[0015] As a preferred embodiment, the application achieves the purpose of degrading n-hexanal by culturing Pseudomonas estuarine D13.

[0016] The third aspect of the present invention provides a bacterial preparation, which contains the Pseudomonas estuarineis D13 as described above.

[0017] As a preferred embodiment, the bacterial preparation also includes a bacterial protective agent and a bacterial nutrient solution.

[0018] As a preferred embodiment, the bacterial preparation is a dry preparation.

[0019] The purpose of degrading n-hexanal is achieved by culturing the Pseudomonas estuarine D13 bacteria in the preparation and utilizing the Pseudomonas estuarine D13.

[0020] The fourth aspect of the present invention provides a method for degrading n-hexanal, using the Pseudomonas estuarineis D13 to degrade n-hexanal.

[0021] The method for degrading n-hexanal includes inoculation and culturing steps. Specifically, the Pseudomonas estuarineus D13 is inoculated into a liquid culture medium, and the liquid culture medium is placed in an environment containing n-hexanal to remove the n-hexanal. The liquid culture medium contains a necessary carbon source, a nitrogen source and other trace elements that can provide the growth of the Pseudomonas estuarineus D13.

[0022] As a preferred embodiment, the liquid culture medium is BPY culture medium, and its specific ingredients are: 5.0 g / L glucose, 10.0 g / L tryptone, 5.0 g / L yeast extract, 5.0 g / L beef extract, 5.0 g / L sodium chloride, pH 7.0, sterilized at 115°C for 30 min.

[0023] As a preferred embodiment, the removal of n-hexanal is carried out in a closed environment at 30° C., and the removal rate of n-hexanal is not less than 99% within 48 hours.

[0024] The invention provides a Lawrence estuarine Pseudomonas D13 with the function of removing n-hexanal, which has rapid bacterial growth, high viable bacterial count and good tolerance to adverse environments. The strain has a strong ability to degrade n-hexanal and can effectively remove n-hexanal; the strain also has good acid-base tolerance and osmotic pressure tolerance and can grow and reproduce in extreme acid-base stress environments and salt stress environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the colony picture of Pseudomonas spp. D13 inoculated on LB solid medium;

[0026] Figure 2 This is the microscopic morphology of Pseudomonas spp. D13 inoculated on LB solid medium;

[0027] Figure 3 It is the removal rate of n-hexanal in the Pseudomonas D13 VOCs test in the Lawrence River Estuary;

[0028] Figure 4This is the growth change diagram of Pseudomonas D13 in Lawrence River Estuary at different initial pH;

[0029] Figure 5 This is a graph showing the growth changes of Pseudomonas spp. D13 in Lawrence Estuary under different sodium chloride concentrations;

[0030] The letters a, b, c, d, e, f, g, h, and i are different, indicating the same type of test results (pH or OD 600nm or viable count) were significantly different (p<0.05); the same letters represent the same type of test results (pH or OD 600nm There was no significant difference in the number of viable bacteria (p>0.05). DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] 1.1 Strain isolation

[0034] Take 10.0g of filler from the biotrickling filter and add it to a conical flask containing 90ml of sterile saline and glass beads. Oscillate at 200rmp for 1h at 30℃ to make the attachments on the filler basically fall off and the liquid becomes turbid. Take 5mL of supernatant from the conical flask and transfer it to 50mL of inorganic salt selection medium with a hexanal concentration of 8.0 mg / L. Seal the bottle mouth with a silicone plug, and add 1 layer of sealing film and 2 layers of plastic film to the bottle mouth in turn for further sealing. The sealed culture medium is placed on a constant temperature shaker at 30℃ and 200 r / min for gradient acclimation. During the culturing process, 5mL of the culture medium is taken out every day and transferred to 50mL of fresh culture medium of the same kind. At the same time, the concentration of hexanal is increased to 24.0 mg / L, and the acclimation is stopped. After the gradient acclimation is completed, the final selection medium is diluted with sterile water and applied on the LB solid culture medium. The grown single colonies are continuously streaked and separated to obtain a single strain of bacteria, named D13.

[0035] The LB medium components are: tryptone 10.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 10.0 g / L, pH 7.0 to pH 7.2, sterilized at 121°C for 30 min. The inorganic salt selection medium components are: potassium nitrate 4.72 g, potassium dihydrogen phosphate 0.7 g, disodium 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.

[0036] 1.2 Strain identification

[0037] (1) Morphological characteristics

[0038] The colony morphology of strain D13 on LB solid medium is as follows Figure 1 As shown, the microscopic morphology Figure 2 As shown, the colonies are round, white, smooth, with clear edges, easy to pick up, and short rod-shaped in microscopic morphology.

[0039] (2) Genetic identification of strain D13

[0040] Single colony PCR was used, and the primers were universal primers 27F and 1492R, the sequence of 27F was 5´-AGAGTTTGATCCTGGCTCAG-3´, and the sequence of 1492R was 5´-TACGACTTAACCCCAATCGC-3´;

[0041] The spliced ​​sequence was searched by Blast homology sequence and found to belong to Pseudomonas spp. Pseudomonas laurentiana ), which was deposited in the China Center for Type Culture Collection on July 12, 2024, with the deposit address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and was classified and named Pseudomonas spp. Lawrence Estuarinae D13 ( Pseudomonas laurentiana D13), the deposit number is CCTCC No: M 20241537.

[0042] Example 2

[0043] 1.1 Test methods

[0044] Pseudomonas spp. D13 from Lawrence River was cultured in liquid LB seed medium at 30℃ for 20h. The fresh seed liquid was used as the inoculum. The inoculum volume ratio was 5% and centrifuged at 5000 rpm for 5 minutes. The supernatant was removed and resuspended with sterile water. After washing, it was centrifuged again. After continuous resuspending and washing twice, it was inoculated into a 1L VOCs sampling bag containing 50 mL of inorganic salt selective medium. The VOCs sampling bag model was 1L. Hexanal was added to the VOCs sampling bag to make the concentration of hexanal 24.0mg / L. At the same time, a blank control was set without adding bacteria, only adding hexanal and inorganic salt medium. The 1L VOCs sampling bag was placed in a constant temperature shaker for culture at 30℃ and a speed of 200r / min for 12h, 24h, 36h, and 48h. After the cultivation is completed, the portable VOC gas detector is first adjusted to the pause state, and the gas port of the VOCs sampling bag is connected to the gas inlet of the portable VOC gas detector with a glass tube. After unscrewing the valve of the VOCs sampling bag, the portable VOC gas detector is turned on to read the value, and the n-hexanal content of the control group and the experimental group at different times is detected respectively. After the n-hexanal content is measured according to the above method, the n-hexanal removal rate is calculated according to the following formula.

[0045]

[0046] 1.2 Test results and analysis

[0047] The results are as follows Figure 3 As shown in the figure, Pseudomonas spp. D13 from the Lawrence River has a high removal rate for n-hexanal. After 12 hours of cultivation, the removal rate reached 62.8%; after 24 hours of cultivation, the removal rate reached 96.6%; after 48 hours of cultivation, the removal rate reached 99.1%, and n-hexanal was basically completely removed. The above test results show that Pseudomonas spp. D13 from the Lawrence River has a strong ability to degrade n-hexanal, and the effect is stable and the removal rate is high.

[0048] Example 3

[0049] 1.1 Test methods

[0050] BPY medium was used as the basic medium, and the pH gradient was set to pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, pH 10.0, pH 11.0, and pH 12.0. The seed solution of Pseudomonas spp. D13 from Lawrence River was inoculated into the medium at a volume ratio of 1%, and cultured at 30°C and 200 r / min for 20 hours.

[0051] The components of BPY medium are: glucose 5.0 g / L, tryptone 10.0 g / L, yeast extract 5.0 g / L, beef extract 5.0 g / L, sodium chloride 5.0 g / L, pH 7.0, sterilized at 115°C for 30 min.

[0052] After the culture is completed, the pH of each culture medium is measured using a pH meter, and the OD of each culture medium is measured using a spectrophotometer. 600nm .

[0053] The dilution plate method was used to count the number of viable bacteria. The specific method is: take 0.5 ml of fermentation liquid and mix it in 4.5 ml of sterile water to dilute it 10 times, repeat the dilution several times to the appropriate concentration, take 100 μL of the dilution liquid and spread it on the LB solid plate, incubate it in a 30°C incubator for about 16 hours to count the number of colonies, calculate the number of viable bacteria in the fermentation liquid based on the number of colonies, and analyze the effect of different initial pH on the growth of Pseudomonas D13 in Lawrence River Estuary.

[0054] 1.2 Test results and analysis

[0055] The growth changes of Pseudomonas spp. D13 in Lawrence Estuarine at different initial pH values ​​are shown in Figure 2. Figure 3 As shown in Figure 2, Pseudomonas spp. D13 in the Lawrence River estuary can grow normally at pH 5.0 to pH 11.0, with a viable count of 20.00×10 8 CFU / mL; when the initial pH value was 7.0, the number of viable bacteria was the largest, reaching 39.03×10 8 CFU / mL. As the pH value becomes more acidic or alkaline, the number of viable bacteria will gradually decrease. When the initial pH value is 4.0 or below, the growth of Pseudomonas estuarine D13 is greatly inhibited and cannot grow; when the pH value is 12.0 or above, the growth of the strain is significantly inhibited. Although the number of viable bacteria will still increase, it is significantly lower than the normal level. The above test results show that Pseudomonas estuarine D13 has good pH tolerance.

[0056] Example 4

[0057] 1.1 Test methods

[0058] BPY medium was used as the base medium, and the mass ratio concentration gradient of sodium chloride was set to 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, and 7.0%. The seed solution of Pseudomonas spp. D13 was inoculated into the medium at a volume ratio of 1%, and cultured at 30°C and 200 r / min for 20 hours. The pH and OD of each culture medium were measured. 600nm and viable bacterial counts to analyze the effects of different sodium chloride concentrations on the growth of Pseudomonas sp. D13 in the Lawrence estuary.

[0059] 1.2 Test results and analysis

[0060] The growth changes of Pseudomonas sp. D13 in Lawrence estuary under different sodium chloride concentrations are shown in Figure 4 and Figure 5 shown.

[0061] When the mass ratio concentration of sodium chloride was 1.0%~6.0%, D13 could grow. When the mass ratio concentration of sodium chloride was 1%, the number of viable bacteria was the largest, reaching 27.75×10 8 CFU / mL; As the concentration of sodium chloride increases, the growth rate of Pseudomonas spp. D13 in Lawrence River estuary begins to decrease. When the mass ratio of sodium chloride is 1%~4%, the strain growth is in a normal state, and the number of viable bacteria can be maintained at 15.00×10 8 CFU / mL or more; when the mass ratio concentration of sodium chloride is 5%~6%, the growth of the strain is significantly inhibited, and the number of viable bacteria increases, but the growth rate is slow. When the mass ratio concentration of sodium chloride is greater than or equal to 7%, the strain no longer grows. The above test results show that Pseudomonas spp. D13 in the Lawrence River estuary has good osmotic pressure tolerance.

[0062] In summary, the Pseudomonas spp. D13 in the present invention can be applied to the removal of n-hexanal, and has a stable effect and a high removal rate.

[0063] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A Pseudomonas spp. D13 for degrading n-hexanal, characterized in that: The Lawrence Estuarine Pseudomonas D13 was deposited in the China Center for Type Culture Collection on July 12, 2024, with a deposit number of CCTCC No: M20241537.

2. Use of the Pseudomonas estuarineis D13 for degrading n-hexanal as claimed in claim 1 in degrading n-hexanal.

3. A bacterial preparation, characterized in that: The method comprises the Pseudomonas estuarineis D13 as claimed in claim 1.

4. A method for degrading n-hexanal, characterized in that: The Pseudomonas estuarineis D13 described in claim 1 is used to degrade n-hexanal.

5. The method for degrading n-hexanal according to claim 4, characterized in that: The method comprises the steps of inoculation and cultivation.

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