Application of Klebsiella variicola in degradation of benzoic acid compounds

Abstract

The application discloses application of Klebsiella variicola in degradation of benzoic acid compounds and belongs to the technical field of microorganisms. A strain is mixed with benzoic acid compounds in a culture medium to obtain a degradation liquid; and the degradation liquid is put into soil or water containing the benzoic acid compounds to be degraded. The strain can utilize 3.4-dihydroxybenzoic acid as a sole carbon source and energy source, and the degradation rate can reach more than 99.12% when removing the toxic harm of 3.4-dihydroxybenzoic acid and other phenolic acid self-toxic substances in plant soil, the upper limit of the tolerance of the strain to 3.4-dihydroxybenzoic acid is high, and the strain can effectively repair the soil environment with relatively serious toxic harm of phenolic acid self-toxic substances.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microbial technology, and particularly relates to application of Klebsiella variicola in degradation of benzoic acid compounds. BACKGROUND

[0002] Allelopathy was first proposed by German scientist Molish in 1937, and was defined by Rice in 1984 as a chemical ecological phenomenon that plants (or microorganisms) release chemicals into the surrounding environment, thereby affecting the growth and development of adjacent plants (or microorganisms). It is the result of extremely complex interactions among plants, microorganisms and soil. Autointoxication is a major type of plant allelopathy, which refers to the release of allelochemicals of plants into the surrounding environment and the inhibitory effect on the growth of the same plants. Autointoxication is widespread in most plants. Autointoxicants can be released into the soil environment through aboveground leaching, root exudation and plant stubble decomposition. With the growth and development process of plants, autointoxicants accumulate in the soil, changing the microenvironment around the rhizosphere, which is not conducive to plant growth. At the same time, autointoxicants can also stimulate the growth of rhizosphere pathogenic bacteria and inhibit the growth of beneficial microorganisms, leading to imbalance of rhizosphere microbial community structure, change of microbial diversity level, increase of pathogenic bacteria proportion and aggravation of soil-borne diseases. Accumulation of autointoxicants in the soil is one of the important reasons for the occurrence of continuous cropping obstacles of plants.

[0003] Previous studies have shown that phenolic acids, glycosides and flavonoids are the main autointoxicants. Among them, phenolic acids are the main components of autointoxicants of tea tree, soybean, rice, cowpea, peanut, watermelon, melon, corn, pepper, asparagus, American ginseng, licorice, Chinese flowering crabapple, peach, poplar, sanchi ginseng, strawberry and lily. Phenolic acids have a benzene ring with an active carboxyl group as the molecular skeleton, and various types and substitution sites of substituents on the benzene ring constitute the diversity of molecular structure and properties, which can affect the membrane system, photosynthesis, enzyme activity, soil microbial activity and soil physical and chemical properties of plants, and produce toxic effects on plant growth and development. Currently reported phenolic acid autointoxicants include phthalic acid, cinnamic acid, ferulic acid, salicylic acid, syringic acid, 3,4-dihydroxybenzoic acid, vanillic acid, coumaric acid, benzoic acid and coumarin. In nature, a plant can produce several to dozens of phenolic acid autointoxicants at the same time, and the main phenolic acid autointoxicants produced by different plants also differ.

[0004] The measures for relieving the autotoxicity include resistant varieties, crop rotation, intercropping, biological carbon adsorption and degradation by using beneficial microorganisms, etc. The microbial degradation has the characteristics of economy, high efficiency and no secondary pollution, and has a wide application prospect. At present, there are few studies on the strains capable of degrading 3,4-dihydroxybenzoic acid at home and abroad. The strains capable of degrading the pollutants reported so far include Pseudomonas putida, Rhizobium and Acinetobacter. The strains can grow by using 3,4-dihydroxybenzoic acid as the sole carbon source, nitrogen source and energy source. So far, there is no related report on Burkholderia and 3,4-dihydroxybenzoic acid. SUMMARY

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present application is to provide an application of Klebsiella variicola in degrading benzoic acid compounds. The strain can use 3,4-dihydroxybenzoic acid as the sole carbon and nitrogen source, and has high degradation efficiency and concentration tolerance limit for 3,4-dihydroxybenzoic acid pollution in soil and water.

[0006] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0007] The application of Klebsiella variicola in degrading benzoic acid compounds, the strain is mixed with benzoic acid compounds in the culture medium to obtain a degradation liquid: the degradation liquid is poured into soil or water containing benzoic acid compounds for degradation, and the strain is Klebsiella variicola LY16.

[0008] Klebsiella variicola LY16 has been preserved in the China General Microbiological Culture Collection Center, the address is No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, the preservation date is January 28, 2023, and the preservation number is CGMCC 1.61611.

[0009] Klebsiella variicola LY16 is obtained by screening from the soil of a tea garden in Wuyishan City, Nanping City, Fujian Province; the screening method is: first, analyze and separate harmful substances in the soil or water, combine with the original microbial community structure and metabolic pathway analysis of the soil or water, and then select suitable culture medium to separate and purify each strain, and transfer to MSM medium with benzoic acid compounds as the sole carbon source, and screen the above strain by measuring the content change of benzoic acid compounds in the culture medium.

[0010] The biological characteristics of Klebsiella variicola LY16 are shown in Table 1.

[0011] The 16S rDNA sequence of Klebsiella variicola LY16 is subjected to blast comparison in NCBI, and the result shows that the 16S rDNA sequence of LY16 has the highest homology of 100% with Burkholderia latens strain AU17928 isolate AU17928 chromosome 1, complete sequence (accession number: CP013435.1), and in combination with morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, it is identified as Klebsiella variicola.

[0012] In the application, the benzoic acid compound is 3,4-dihydroxybenzoic acid.

[0013] In the application, the degradation temperature is 25-30 DEG C, the degradation pH value is 5-8, preferably 6-8, and further preferably 7-8.

[0014] In the application, the concentration of 3,4-dihydroxybenzoic acid in the culture medium is 0.1-4 g / L, preferably 1-3.5 g / L.

[0015] In the application, one of glucose or yeast extract is added to the culture medium, and the concentration of glucose or yeast extract is 0.5 g / L.

[0016] The Klebsiella variicola LY16 described above is applied to the preparation of a plant continuous cropping obstacle biological bacterial agent.

[0017] In the application, the bacterial agent contains Klebsiella variicola LY16 strain as an active ingredient.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] 1. The strain provided by the application can grow by utilizing 3,4-dihydroxybenzoic acid as the only carbon and nitrogen source and energy source, and can rapidly and effectively degrade 3,4-dihydroxybenzoic acid, so that the effect of rapid detoxification can be achieved when removing 3,4-dihydroxybenzoic acid pollution in the environment.

[0020] 2. The strain provided by the present application has a high upper limit of tolerance to benzoic acid compounds, and can degrade 5 g / L of 3,4-dihydroxybenzoic acid pollutants, which proves that the strain of the present application can effectively repair the environment with more serious pollution.

[0021] 3. The growth and 3,4-dihydroxybenzoic acid degradation of the strain provided by the present application are optimized, which can more effectively and sufficiently degrade benzoic acid compounds in the environment and solve the pollution problem.

[0022] 4. The present application adopts a microbial method to degrade pollutants in the environment, which has the advantages of high efficiency, no secondary pollution and low cost, and has a good application prospect in the treatment of benzoic acid compounds. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure is a detection result diagram of the degradation characteristics of the strain LY16;

[0024] Figure 2 Figure is a colony morphology (a) and strain morphology diagram (b);

[0025] Figure 3 Figure is a 16S r DNA electrophoresis diagram (a) and a 16S phylogenetic tree (b) of the strain;

[0026] Figure 4 Figure is a self-toxic substance standard regression curve diagram;

[0027] Figure 5 Figure is a maximum tolerance capacity verification result diagram of the strain LY16;

[0028] Figure 6 Figure is a proportion of the root length of the lettuce seedling cultured for 72 h (CK, not inoculated, inoculated);

[0029] Figure 7 Figure is an average root length of the lettuce seedling cultured for 72 h. DETAILED DESCRIPTION

[0030] The present application will be further described below in combination with specific examples.

[0031] Example 1

[0032] The purpose strain is separated and identified from the tea garden soil in Wuyishan City, Nanping City, Fujian Province.

[0033] LB medium components include: Tryptone (Tryptone) 10 g, Yeast Extract (yeast extract): 5 g, NaCl (sodium chloride) 10 g, the above compounds are dissolved with double distilled water and constant volume to 1000 mL, adjust pH to 7.0, high pressure sterilization at 121℃ for 20 min. If the solid medium is configured, 15 g of agar is added.

[0034] The components of the MSM medium include: sodium hydrogen phosphate (Na2HPO4·12H2O) 14.3 g, potassium dihydrogen phosphate (KH2PO4) 3 g, magnesium sulfate (MgSO4·7H2O) 0.06 mg, ferrous sulfate (FeSO4·7H2O) 0.3 mg, manganese sulfate (MnSO4·H2O) 0.28 mg, copper sulfate (CuSO4) 0.05 mg, zinc sulfate (ZnSO4) 0.05 mg, boric acid (H3BO3) 0.05 mg; the above compounds are dissolved with double distilled water and constant volume to 1000 mL, adjust pH to 7.0, high pressure sterilization at 121℃ for 20 min. 3,4-dihydroxybenzoic acid stock solution is set to 1 g / mL, and bacteria filter is used for filtration and sterilization. e -1 The configuration of MSM solid medium: 2 g of agar powder is added to 100 mL of the above liquid inorganic salt medium.

[0035] The configuration of MSM solid medium: 2 g of agar powder is added to 100 mL of the above liquid inorganic salt medium.

[0036] Take 5 g of the collected soil sample to prepare soil suspension, inoculate 2% into 100 mL of LB medium liquid medium. Place the conical flask in a shaking bed at 180 rpm, and cultivate at 30℃ for 3 days.

[0037] Inoculate 2% into 100 mL of fresh MSM medium, add 3.4-dihydroxybenzoic acid to make its final concentration 3 g / L, and transfer every 5 days, repeat 3 times.

[0038] Take the last time of the culture solution to 10 -1 to 10 -4 Gradient dilution, and the dilution is coated on LB solid medium, placed in an incubator at 30℃ for 2 days, then inoculated into LB liquid medium with a concentration of 2%, placed in a shaking bed at 180 rpm, 30℃ for 3 days, then inoculated into 3.4-dihydroxybenzoic acid MSM liquid medium, and the turbidity of the culture solution was observed. Finally, 3,4-dihydroxybenzoic acid degrading strains were screened. The physiological and biochemical characteristics and basic biological characteristics of the strains obtained by isolation and screening are shown in Table 1.

[0039] Table 1 Basic biological characteristics of the strains obtained by isolation and screening

[0040]

[0041] Note: "+" indicates that the carbon source (or nitrogen source) can be utilized, "-" indicates that the carbon source (or nitrogen source) cannot be utilized

[0042] The DNA of the strain was extracted by using Ezup column bacterial genomic DNA extraction kit, and PCR amplification was carried out by using 16S rRNA universal primers (5'-AGTTTGATCMTGGCTCAG-3', 5'-GGTTACCTTGTTACGACTT-3') (94°C pre-denaturation for 4 min; 94°C denaturation for 45 s, 55°C annealing for 45 s, 72°C extension for 1 min, 30 cycles; 72°C extension for 10 min; 4°C termination reaction), and the amplification product was sequenced. The sequencing was sent to Shenguo Bioengineering (Shanghai) Co., Ltd. to obtain 1436 nucleotides (sequences in the sequence listing). The sequence was subjected to blast comparison in NCBI, and the results showed that the 16S rDNA sequence of the above strain had the highest homology with Klebsiella variicola H1 (accession number: CP013435.1), up to 100%, and combined with the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, it was named Klebsiella variicola LY16. The phylogenetic tree of the sequencing results is as follows Figure 3 The 16S rDNA sequence is:

[0043]

[0044]

[0045] Example 2

[0046] Degrading property detection of strain Klebsiella variicola LY16

[0047] The active strain LY16 single colony was inoculated in a volume ratio of 4:1 of inorganic salt and LB liquid medium, and was shaken and cultured, and the concentration of 3,4-dihydroxybenzoic acid in the medium was 1 g / L. After overnight culture, the bacterial cells were collected, washed with inorganic salt medium for 3 times and resuspended in inorganic salt medium as a degradation liquid for use;

[0048] In the inorganic salt medium containing 3,4-dihydroxybenzoic acid (1 g / L), the degradation liquid was inoculated according to the initial OD600 = 0.8 inoculation amount, and was placed in a 180 rpm shaker, and was cultured at 30°C and was sampled at intervals.

[0049] In addition, 3,4-dihydroxybenzoic acid standard solutions were prepared using inorganic salt basal culture medium at concentrations of 0, 0.004, 0.008, 0.012, 0.016, and 0.020 mg / mL. The absorbance was measured at 260 nm, and a standard curve was plotted. The results are shown below. Figure 4 The curve intercept is 9.66667 × 10⁻⁶. -4 ±0.00392, the slope is 76.31667±0.5909, R 2 =0.99976.

[0050] The specific results of the degradation characteristics of strain LY16 are as follows: Figure 1 As shown, from Figure 1 It can be seen that strain LY16, in inorganic salt medium (supplemented with 1 g / L 3,4-dihydroxybenzoic acid), can degrade 94.5% of 1 g / L 3,4-dihydroxybenzoic acid within 12 hours, while the OD600 value of the degrading strain increases. In the control group without the degrading strain, the concentration of 3,4-dihydroxybenzoic acid did not change significantly. This indicates that strain LY16 can utilize 3,4-dihydroxybenzoic acid as its sole carbon and nitrogen source for growth. Furthermore, the concentration of 3,4-dihydroxybenzoic acid in tea plant soil is typically around 50 mg / kg. In this invention, strain LY16 degraded 1000 mg / L of 3,4-dihydroxybenzoic acid within 12 hours, demonstrating its high application potential.

[0051] Example 3

[0052] Optimization of degradation conditions for strain LY16

[0053] Strain LY16 was inoculated into an inorganic salt medium containing 1 g / L of 3,4-dihydroxybenzoic acid. The pH of the inorganic salt medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. The inoculation amount of strain LY16 was initially OD600 = 0.8. The medium was then placed in a shaker at 180 rpm and cultured at 30 °C with interval sampling. The medium was centrifuged at 12,000 rpm, and the supernatant was collected. The concentration of 3,4-dihydroxybenzoic acid in the supernatant was measured by spectrophotometry to obtain a pH-tolerant degradation bacterium.

[0054] When the pH is between 4 and 7, the degradation rate increases with increasing pH. After 12 hours of treatment, the degradation rate is 9.1% at pH 4, 58.2% at pH 5, 87.4% at pH 6, and 94.5% at pH 7.0. When the pH is between 8 and 10, the degradation rate decreases with increasing pH. The degradation rate is 69.4% at pH 8, 28.4% at pH 9, and 7.4% at pH 10. The results indicate that the optimal pH for degradation by the strain is 7.

[0055] Example 4

[0056] Validation of the maximum tolerance of strain LY16

[0057] The selected strain LY16 was inoculated into inorganic salt medium with 3,4-dihydroxybenzoic acid concentrations of 0.985 g / L, 1.800 g / L, 2.677 g / L, 3.629 g / L and 4.172 g / L, respectively. The medium pH was 7.0. The medium was incubated in a shaker at 30℃ and 180 rpm. Samples were taken at intervals, and the concentration of the strain in the medium was tested using a spectrophotometer.

[0058] Depend on Figure 5 It can be seen that when the initial concentrations of 3,4-dihydroxybenzoic acid were 0.985 g / L, 1.800 g / L, and 2.677 g / L, after 24 hours of cultivation, the 3,4-dihydroxybenzoic acid in the culture medium was almost completely consumed, and the number of strains was close to its maximum. However, when the initial concentration of 3,4-dihydroxybenzoic acid reached 3.629 g / L, although 24 hours of cultivation were performed, the content of 3,4-dihydroxybenzoic acid in the culture medium did not change significantly, and the number of strains did not change significantly. But after 48 hours of cultivation, the concentration of 3,4-dihydroxybenzoic acid in the culture medium increased significantly. The content of 3,4-dihydroxybenzoic acid decreased significantly, while the number of strains in the culture medium increased significantly. In the culture medium with an original concentration of 4.172 g / L of 3,4-dihydroxybenzoic acid, the content of 3,4-dihydroxybenzoic acid decreased significantly after 72 h of culture, while the number of strains increased only slightly. When the culture time was 96 h, the amount of 3,4-dihydroxybenzoic acid degradation in the culture medium was almost the same as that on the third day. Therefore, it can be concluded that the strain's tolerance concentration of 3,4-dihydroxybenzoic acid in the culture medium is about 4 g / L.

[0059] Example 5

[0060] Effect of organic matter on the degradation efficiency of strain LY16

[0061] The selected strain LY16 was inoculated into an inorganic salt medium with a 3,4-dihydroxybenzoic acid concentration of 1 g / L and a pH of 7.0. Lactic acid, glucose, and succinic acid were added to the medium to make the concentrations of each organic compound 0.5 g / L. The initial inoculum size of strain LY16 was OD0.05. 600 =0.8, and then placed in a shaker at 180 rpm and incubated at 30°C. The remaining amount of autotoxic substance was determined by spectrophotometry after 6 hours, and the results are shown in Table 2.

[0062] Table 2. Results of the effect of organic matter on the degradation efficiency of strain LY16

[0063] MSM medium MSM plus glucose MSM plus yeast extract MSM plus lactic acid Concentration (g / L) 0.754 0.562 0.604 0.974 Effect Control Promote Promote Inhibit

[0064] Example 6

[0065] Effects of strain LY16 on lettuce seed growth

[0066] Lettuce seeds were soaked in water for 24 hours to allow them to absorb moisture, then placed in a clean bench and sterilized with a 10% NaClO aqueous solution for 15 minutes. They were then rinsed 3-4 times with sterile water and soaked again until they sank to the bottom. The sunken lettuce seeds were then picked up with tweezers and inoculated onto plates containing a 3,4-dihydroxybenzoic acid aqueous solution. The experiment was divided into three groups, with two replicates per group.

[0067] Group 1: Add 10 mL of an aqueous solution containing 3,4-dihydroxybenzoic acid allergen to a final concentration of 300 mg / L;

[0068] Group 2: Based on Group 1, add bacterial cells (total aqueous solution volume is 10 mL, final bacterial cell concentration OD). 600 =0.8);

[0069] Group 3: Add 10 mL of sterile water as a blank culture medium control.

[0070] After inoculation, the hydroponic plates were placed in a 25℃ light-controlled tissue culture room for light culture, and the seed germination was observed and recorded every 24 hours (for a continuous 72 hours). The appearance of the root tips and the root length of the lettuce seeds after 72 hours of culture were recorded.

[0071] Lettuce seeds are a good model species for studying the allelopathic effects of phenolic acid autotoxicity. 72 hours after inoculation, the roots of uninoculated lettuce seeds in hydroponic plates containing 3,4-dihydroxybenzoic acid showed almost no change in color compared to the previous day, although a few still successfully developed into seedlings. In contrast, the roots of lettuce seeds in inoculated plates were almost unaffected. The germination rate of lettuce seeds in the sterile water negative control plates was significantly better than that in the uninoculated hydroponic plates.

[0072] Depend on Figure 6 It can be seen that after 72 hours of cultivation, the root length of the three groups of lettuce seedlings was measured and recorded. It was found that 80% of the hydroponic lettuce seedlings without the strain had a root length of less than 1 cm, while in the hydroponic solution plate with the strain, nearly 70% of the lettuce seedlings had a root length of 1-2 cm.

[0073] Depend on Figure 7 It was found that the root growth of lettuce seedlings in hydroponic plates inoculated with the strain was significantly better than that in uninoculated plates. This indicates that the selected 3,4-dihydroxybenzoic acid degrading strain LY16 can alleviate the toxic effects of 3,4-dihydroxybenzoic acid on lettuce seedlings, and its presence does not adversely affect the root growth and development of lettuce seedlings. The results suggest that this strain can effectively alleviate the growth inhibition caused by 3,4-dihydroxybenzoic acid in plants in practical applications.

Claims

1. A strain of Klebsiella heterotropha LY16, characterized in that, It was deposited on January 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 1 .61611.

2. The application of Klebsiella pneumoniae LY16 according to claim 1, characterized in that, The degradation of benzoic acid compounds is specifically carried out by: mixing and culturing strain Klebsiella heterotrophus LY16 with 3,4-dihydroxybenzoic acid in a culture medium to obtain a degradation solution; and then immersing the degradation solution in soil or water containing 3,4-dihydroxybenzoic acid for degradation; the concentration of 3,4-dihydroxybenzoic acid in the culture medium is 0.1~4 g / L.

3. The application of Klebsiella pneumoniae LY16 according to claim 2, characterized in that, The degradation temperature is 25~30℃, and the degradation pH is 5~8.

4. The application of Klebsiella pneumoniae LY16 according to claim 2, characterized in that, Add either glucose or yeast extract to the culture medium at a concentration of 0.5 g / L.

5. The application of Klebsiella pneumoniae LY16 according to claim 1, characterized in that, A biological agent for preventing continuous cropping obstacles in plants was prepared, wherein the agent contains Klebsiella pneumoniae strain LY16 as an active ingredient.

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