Pseudomonas putida SD-1 and its application in degradation of organic pollutants

Abstract

The present invention discloses a kind of Pseudomonas putida SD-1 and its application in degrading organic pollutants, and the application is to inoculate Pseudomonas putida SD-1 into an inorganic salt culture solution with pH=5-10 containing organic pollutants, and culture it under the conditions of 25-35℃ and 100-200rpm to achieve degradation of organic pollutants. The Pseudomonas putida SD-1 provided by the present invention is taken from the aeration tank of a sewage treatment station of a kitchen waste treatment plant and the biological section circulation section of a kitchen waste odor treatment facility, and has an efficient degradation effect on organic pollutants such as ethanol, and can relatively completely convert pollutants into harmless substances; at the same time, the strain can also degrade other common pollutants in kitchen waste odor such as acetaldehyde and ammonia water to varying degrees, and thus has broad application prospects in the biological purification technology in the treatment of odor generated by kitchen waste composting.

Description

(I) Technical field

[0001] The invention relates to a new strain--Pseudomonas putida SD-1 and application thereof in degrading organic pollutants. (II) Background technology

[0002] Ethanol is an organic compound, a type of alcohol, commonly known as alcohol. Ethanol is a flammable, volatile, slightly irritating, colorless, transparent liquid at room temperature and pressure. It is an isomer of methyl ether. It is miscible with water and soluble in most organic solvents such as ether, chloroform, glycerol, and formaldehyde. Ethanol is a commonly used fuel, solvent, and disinfectant. It is widely used in the defense industry, medical and health care, organic synthesis, food industry, and industrial and agricultural production. It is also often used in organic synthesis.

[0003] Ethanol is a volatile substance with low toxicity and certain irritation. However, due to its volatility, ethanol will burn when exposed to open flames, high heat, or chemical reactions with oxidants. At the same time, as a good solvent, ethanol can dissolve many organic and inorganic substances, has reducing properties, and will be oxidized to form acetaldehyde. Ethanol is addictive and carcinogenic, but ethanol is not a substance that directly causes cancer, but carcinogens are generally soluble in ethanol. During the production process, long-term exposure to high concentrations of ethanol can cause symptoms of irritation of the nose, eyes, and mucous membranes; long-term alcoholism can cause multiple neuropathy, chronic gastritis, fatty liver, cirrhosis, etc.

[0004] In the early stage of kitchen waste degradation, ethanol, acetaldehyde, limonene and ammonia are among the typical pollutants produced. Therefore, it is necessary to study the efficient degradation of ethanol in the environment to improve the living environment of human beings. Through literature search, it was found that Lu Chenchen et al. screened a strain of Acetobacter pasteurianus VII with high ethanol degradation ability from sour wine lees in 2011. No reports were found about Pseudomonas putida using ethanol as the only carbon source to achieve efficient degradation. (III) Summary of the invention

[0005] The present invention aims to provide a Pseudomonas putida SD-1 and its application in degrading organic pollutants. The strain has high efficiency and strong removal capacity for degrading organic pollutants (especially acetaldehyde and ethanol), can achieve degradation with ethanol as the only carbon source, and has a mild growth environment and is easy to expand and cultivate. The discovery of the degrading bacteria is of great significance for the efficient purification of common pollutants such as ethanol in kitchen waste and wastewater and waste gas of the brewing industry.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a strain - Pseudomonas putida SD-1, which is deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: M2022949, a deposit date of June 23, 2022, and an address of Wuhan University, Wuhan, China, 430072.

[0008] The basic characteristics of the Pseudomonas putida SD-1 of the present invention are: the colony is white, opaque, easy to pick, and the bacterial lawn grows along the streaking line; the morphology under the electron microscope is oval, has flagella, and is aerobic.

[0009] The present invention also provides an application of the Pseudomonas putida SD-1 in degrading organic pollutants. The specific application is to inoculate the Pseudomonas putida SD-1 into an inorganic salt culture solution with a pH of 5-10 (preferably pH of 6-8) containing organic pollutants, and culture the solution at 25-35°C and 100-200rpm to achieve degradation of the organic pollutants.

[0010] Preferably, the organic pollutants include ethanol, methanol, ethyl acetate, acetaldehyde or ammonia water; the initial concentration of the organic pollutants in the inorganic salt culture solution is 175-475 mg / L, preferably 400 mg / L.

[0011] The Pseudomonas putida SD-1 is added in the form of resting cells. In the inorganic salt culture solution, the amount of resting cells added is 10-100 mg / L, preferably 50 mg / L, based on the dry weight of the bacteria.

[0012] The inorganic salt culture solution is composed of: K2HPO4·3H2O 0.942g / L, KH2PO4 0.234g / L, NaNO3 1.7g / L, NH4Cl 0.98g / L, MgCl2·6H2O 0.2033g / L, CaCl2·2H2O 0.11g / L, FeCl3 0.0162g / L, trace element mother solution 5ml / L, deionized water, pH 7.0; wherein the trace element mother solution is composed of: CuSO4·5H2O 0.02g / L, FeSO4·7H2O 1.0g / L, MnSO4·4H2O 0.02g / L, CoCl2·6H2O 0.02g / L, H3BO3 0.014g / L, ZnSO4·7H2O 0.10g / L, the solvent is deionized water.

[0013] The Pseudomonas putida SD-1 of the present invention is inoculated in the form of resting cells, and the resting cells are prepared according to the following steps:

[0014] (1) Slant culture: Pseudomonas putida SD-1 was inoculated into a slant LB solid medium and cultured at 30°C for 24-36 h to obtain slant bacteria. The final concentration of the LB solid medium was as follows: NaCl 10 g / L, peptone 10 g / L, yeast powder 5 g / L, agar 18-20 g / L, the solvent was deionized water, and the pH value was natural.

[0015] (2) Expansion culture: Use an inoculation loop to pick the slant bacteria obtained in step (1) and inoculate them into LB liquid culture medium. Cultivate at 30°C and 160 rpm for 24 to 36 h to obtain the OD 600 =0.1-0.2 bacterial liquid, centrifuge, collect wet bacteria, wash with sterile deionized water, and obtain Pseudomonas putida SD-1 resting cells; the final concentration of the LB liquid culture medium is: NaCl 10g / L, peptone 10g / L, yeast powder 5g / L, the solvent is deionized water, and the pH value is natural.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0017] The Pseudomonas putida SD-1 provided by the present invention is taken from the aeration tank of a sewage treatment station of a kitchen waste treatment plant and the biological section circulation section of a kitchen waste odor treatment facility. It has a high efficiency in degrading organic pollutants such as ethanol and can relatively completely convert pollutants into harmless substances such as CO2 and H2O; at the same time, the strain can also degrade other common pollutants in kitchen waste odor such as acetaldehyde, limonene, ammonia water, etc. to varying degrees, and therefore has broad application prospects in the biological purification technology for the treatment of odor generated by kitchen waste composting.

[0018] The Pseudomonas putida SD-1 of the present invention can completely degrade ethanol into inorganic substances (CO2, H2O) and cell biomass, achieve complete mineralization, and the removal rate of ethanol within 400 mg / L is as high as 100%. In addition, the Pseudomonas putida has the ability to degrade common odor components of kitchen waste (such as acetaldehyde, ammonia, etc.) and can withstand higher concentrations of pollutants. (IV) Description of the drawings

[0019] Figure 1 This is a photo of the colony morphology of strain SD-1 on LB medium.

[0020] Figure 2 This is a transmission electron micrograph of strain SD-1.

[0021] Figure 3 This is the phylogenetic tree of strain SD-1.

[0022] Figure 4 This is the degradation curve of Pseudomonas putida SD-1 for different concentrations of ethanol within 48 hours.

[0023] Figure 5 This is the degradation effect of Pseudomonas putida SD-1 on 400 mg / L ethanol at different pH within 48 hours. (V) Specific implementation methods

[0024] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0025] The following examples and drawings are for illustrative purposes only and are not to be construed as limiting the present invention. Unless otherwise specified, the reagent raw materials used in the following examples are conventional commercially available or commercially available biochemical reagent raw materials, and the experimental instruments used are conventional laboratory instruments. Unless otherwise specified, the methods and equipment used in the following examples are conventional methods and equipment used in the art.

[0026] The inorganic salt culture solution is composed of: K2HPO4·3H2O 0.942g / L, KH2PO4 0.234g / L, NaNO3 1.7g / L, NH4Cl 0.98g / L, MgCl2·6H2O 0.2033g / L, CaCl2·2H2O 0.11g / L, FeCl3 0.0162 g / L, trace element mother solution 5ml / L, deionized water, pH 7.0; wherein the trace element mother solution is composed of: CuSO4·5H2O 0.02g / L, FeSO4·7H2O 1.0g / L, MnSO4·4H2O 0.02g / L, CoCl2·6H2O 0.02g / L, H3BO3 0.014g / L, ZnSO4·7H2O 0.10g / L, the solvent is deionized water.

[0027] The final concentration of the LB solid culture medium is as follows: NaCl 10 g / L, tryptone 10 g / L, yeast powder 5 g / L, agar 18 g / L, pH is natural, the solvent is deionized water, and the pH value is natural.

[0028] The final concentration of the LB liquid culture medium is as follows: NaCl 10 g / L, peptone 10 g / L, yeast powder 5 g / L, the solvent is deionized water, and the pH value is natural.

[0029] Example 1: Isolation, purification and identification of Pseudomonas putida SD-1 strain

[0030] 1. Isolation and purification of Pseudomonas putida SD-1

[0031] The strain SD-1 was screened from the activated sludge in the aeration tank of a food waste treatment station and the biological section circulating liquid of the food waste odor treatment facility. The specific steps are as follows:

[0032] Initial screening: Collect 500 ml of water sample containing activated sludge from the site, place the water sample in a 2L measuring cup, add 1.5L of the above-mentioned inorganic salt culture medium, use ethanol as the only carbon source, control the concentration at 100 mg / L, and acclimate at room temperature for 15 days. Add ethanol every day to provide sufficient carbon source, and replace the inorganic salt culture medium every three days to ensure a nitrogen source.

[0033] Rescreening: Add 50mL of inorganic salt culture medium to a 250ml shaking flask, add 5mL of the supernatant water sample from the primary screening and 50mg / L ethanol, and perform enrichment culture at 30℃. When the ethanol concentration is 50% of the initial concentration, take out 5mL of the enrichment solution and add it to 50mL of fresh inorganic salt culture medium, add the same amount of ethanol (to make its concentration 50mg / L), repeat the above enrichment process 5 times, dilute the last enrichment solution 1500 times with sterile water, and then streak it on LB solid medium, culture at 30℃, select a single colony and streak it on LB solid medium, culture at 30℃ for 24h, and the colony morphology photos are shown in the figure. Figure 1 .

[0034] A single colony was added to an inorganic salt culture medium, and ethanol at a final concentration of 100 mg / L was added as the only carbon source and energy source. The degradation effect was verified at 30°C for 24 h, and the target strain SD-1 was obtained. Its morphology was determined by transmission electron microscopy ( Figure 2 ).

[0035] 2. Identification of strain SD-1

[0036] The strain SD-1 was identified as Pseudomonas putida by 16S rRNA sequence analysis and physiological and biochemical experiments. The specific steps are as follows:

[0037] DNA of strain SD-1 was extracted using PrepMan Ultra Kits nucleic acid extractor and stored at 4°C. Universal bacterial primers (forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3'; reverse primer: 5'-GGTTACCTTGTTACGACTT-3') were used for amplification in ABI PCR amplification instrument. Applied Biosystems 3500 Gene Analyzer was used to sequence the PCR purified product. Sequencing was completed by Zhejiang Tianke High-tech Development Co., Ltd. The 16SrDNA sequence of the strain is as follows (Genebank accession number is ON705768), SEQ ID NO.1:

[0038]

[0039] The ability of strain SD-1 to utilize 62 carbon sources on the Mérieux GN card: The strain's metabolism of 46 different carbon sources was investigated using the Mérieux fully automatic identification instrument (entrusted to Zhejiang Tianke High-tech Development Co., Ltd. (formerly Zhejiang Institute of Microbiology)). The identification results are shown in Table 1. According to the biochemical reaction of the Mérieux fully automatic identification instrument VITEK, strain SD-1 can strongly utilize 17 carbon sources, but cannot utilize the other 29 carbon sources.

[0040] Table 1 Biochemical reaction results of strain SD-1 using the Mérieux automatic identification instrument VITEK (GN card)

[0041]

[0042]

[0043] Table Notes: +, positive reaction: -: negative reaction

[0044] The morphological and physiological and biochemical characteristics of strain SD-1 were studied and analyzed. The strain sequence was compared with the gene sequence in the NCBI database by Blast, and a phylogenetic tree was constructed by combining 16S RNA homology analysis (such as Figure 3 ), thus determining that the strain SD-1 was Pseudomonas putida, named Pseudomonas putida SD-1, and deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M2022949, the deposit date: June 23, 2022, and the address: Wuhan University, Wuhan, China, 430072.

[0045] Example 2. Obtaining resting cells of Pseudomonas putida SD-1

[0046] 1. Slant culture:

[0047] Pseudomonas putida SD-1 was inoculated into LB liquid culture medium and cultured at 30°C, 160rpm for 24-36h. The activated bacteria were then streaked onto solid LB plates and cultured in a 30°C incubator. Single colonies were taken and continued to be streaked on the plates to detect the purity of the bacteria. The LB test tube slant was stored in a conventional manner (4°C).

[0048] 2. Expand training

[0049] Inoculate the slant cells in step 1 into LB liquid medium and culture at 30°C, 160 rpm for 24-36 h to obtain the OD 600 = 0.1 of the expanded culture medium, centrifuged, collected the wet cells, washed with sterile deionized water, and obtained Pseudomonas putida SD-1 resting cells.

[0050] Example 3: Detection of the degradation performance of Pseudomonas putida SD-1 on different concentrations of ethanol

[0051] The inorganic salt culture solution was divided into 250 ml shake flasks, 50 ml in each bottle, and sterilized at 110°C for 40 min. After sterilization, it was placed at room temperature for 2 days to ensure that no foreign bacteria grew. The resting cells obtained by the method of Example 2 were added to reach a final concentration of 50 mg / L (measured as cell dry weight), and then ethanol was added as the only carbon source to make the final concentrations of 175.6, 272.2, 333.3, 399.2, and 472.4 mg / L, respectively. After the shake flasks were sealed, they were cultured in a shaker at 30°C and 160 rpm, and a blank control without adding bacteria was performed. The residual ethanol concentration in the shake flask was measured regularly, and a curve of the removal rate of the strain at different initial ethanol concentrations over time was plotted. The results are shown in the table below. Figure 4 The results showed that when the ethanol concentration was lower than 400 mg / L, strain SD-1 could quickly degrade all added substrates.

[0052] The concentration of gaseous ethanol was detected by Fuli 9790II gas chromatograph. The chromatographic column was KB-5 (30m×0.32mm×0.5μm). The injection port, detector (FID) and column temperature were 120, 200 and 80°C, respectively, the auxiliary furnace temperature was 100°C, and the split ratio was 100:1. The hydrogen flow rate was 30mL / min, the air flow was 300mL / min, the carrier gas nitrogen flow rate was 30mL / min, and the gas injection volume was 1mL.

[0053] Example 4: Degradation performance test of Pseudomonas putida SD-1 for 400 mg / L ethanol under different initial pH environments

[0054] The inorganic salt culture medium was adjusted to different pH values ​​(5.0, 6.0, 7.0, 8.0, 9.0, 10.0) with 1 mol / L NaOH aqueous solution or 1 mol / L HCl aqueous solution, and the resting cells of Pseudomonas putida SD-1 prepared by the method of Example 2 were inoculated under the condition of an initial ethanol concentration of 400 mg / L, so that the initial cell dry weight in each parallel sample was 50 mg / L. The samples were cultured in a constant temperature shaker at 30°C and 160 rpm, and a blank control without adding bacteria was performed. The residual ethanol concentration in the shake flask was measured regularly, and the removal rate curve of ethanol under different pH environments of the strain over time and the degradation rate of 400 mg / L ethanol at different pH within 48 hours were plotted. The results are shown in Table 1. Figure 5 The results showed that Pseudomonas putida SD-1 could degrade ethanol at all pH values, and the degradation effect of ethanol was the best at pH 7-9.

[0055] Example 5: Degradation ability of Pseudomonas putida SD-1 on different carbon source substrates

[0056] In practical applications, there is not only ethanol as an organic pollutant, but kitchen waste odor generally contains a variety of volatile organic waste gases. Therefore, it is necessary to study the degradation effect of Pseudomonas putida SD-1 on other substrates. The experimental operation of Example 3 was adopted, the initial cell dry weight was 50 mg / L, the pH value was 7.0, and the substrate was changed to toluene, methanol, ethyl acetate, acetaldehyde, carbon disulfide, and ammonia water with an initial concentration of 100 mg / L. Other operations were the same as in Example 3, and the degradation effect is shown in Table 2.

[0057] Table 2. Degradation ability of Pseudomonas putida SD-1 on different substrates

[0058]

[0059]

[0060] Note: “+” means degradation ability; “-” means no degradation ability

Claims

1. A Pseudomonas putida ( Pseudomonas putida )SD-1, deposited in China Center for Type Culture Collection, deposit number: CCTCC NO: M2022949, deposit date: June 23, 2022, address: Wuhan University, Wuhan, 430072, China.

2. A use of Pseudomonas putida SD-1 according to claim 1 in degrading organic pollutants, characterized in that: The application is to inoculate Pseudomonas putida SD-1 into an inorganic salt culture solution with a pH of 5-10 containing organic pollutants, and culture at 25-35°C and 100-200rpm to achieve degradation of organic pollutants; The organic pollutant is ethanol, methanol, ethyl acetate or acetaldehyde.

3. The use according to claim 2, characterized in that The initial concentration of ethanol in the inorganic salt culture solution is 175-475 mg / L.

4. The use according to claim 2, characterized in that The Pseudomonas putida SD-1 is added to the inorganic salt culture solution in the form of resting cells, and the amount of resting cells added is 10-50 mg / L based on the dry weight of the bacteria.

5. The use according to claim 2, characterized in that The inorganic salt culture solution is composed of: K2HPO4·3H2O 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl2·6H2O 0.2033 g / L, CaCl2·2H2O 0.11 g / L, FeCl3 0.0162 g / L, trace element mother solution 5 ml / L, solution deionized water, pH 7.0; wherein the trace element mother solution Composition: CuSO4·5H2O 0.02 g / L, FeSO4·7H2O 1.0g / L, MnSO4·4H2O 0.02 g / L, CoCl2·6H2O 0.02g / L, H3BO3 0.014g / L, ZnSO4·7H2O 0.10 g / L, the solvent is deionized water.

6. The use according to claim 4, characterized in that The resting cells are prepared according to the following steps: (1) Slant culture: Pseudomonas putida SD-1 was inoculated into a slant LB solid medium and cultured at 30°C for 24-36 hours to obtain slant bacteria. The final concentration of the LB solid medium was as follows: NaCl 10 g / L, peptone 10 g / L, yeast powder 5 g / L, agar 18-20 g / L, the solvent was deionized water, and the pH value was natural. (2) Expansion culture: Use an inoculation loop to pick the slant bacteria obtained in step (1) and inoculate them into LB liquid culture medium. Cultivate at 30°C and 160 rpm for 24 to 36 hours to obtain the OD 600 =0.1-0.2 bacterial liquid, centrifuge, collect wet bacteria, wash with sterile deionized water, and obtain Pseudomonas putida SD-1 resting cells; the final concentration of the LB liquid culture medium is: NaCl 10g / L, peptone 10g / L, yeast powder 5g / L, the solvent is deionized water, and the pH value is natural.

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