Chlorohydrocarbon-degrading bacteria Stutzerimonas degradans SH1 strain and its application

Abstract

The invention discloses chlorinated hydrocarbon degrading bacteria Stutzerimonas degradants The invention discloses SH1 strain and application thereof, belonging to the field of environmental microorganisms. Stutzerimonas degradans The SH1 strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20221863 and a deposit date of December 5, 2022. The strain has the function of completely biodechlorinating tetrachloroethylene and trichloroethylene under both aerobic and anaerobic conditions. The growth of the strain has low requirements for environmental conditions and can effectively degrade chlorinated hydrocarbon pollutants under different oxygen content conditions without accumulation of toxic intermediates. The strain or the microbial agent prepared from it can be used for bioremediation of soil or groundwater in industrial contaminated sites contaminated by chlorinated hydrocarbons, and has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of environmental microorganisms, and specifically relates to chlorinated hydrocarbon degrading bacteria Stutzerimonas degradans SH1 strain and its application in dechlorination remediation of chlorinated hydrocarbon pollution. Background Art

[0002] Chlorinated hydrocarbons, as a type of halogenated hydrocarbons formed by the substitution of chlorine atoms, have special physical and chemical properties. As organic solvents and chemical products, they are widely used in the chemical, pharmaceutical, dry cleaning and other industries. However, along with volatilization, leakage and sewage discharge caused by improper storage, chlorinated hydrocarbons are discharged into natural soil and groundwater ecosystems. Chlorinated hydrocarbons such as chloroform (CF), carbon tetrachloride (CT), trichloroethylene (TCE), and tetrachloroethylene (PCE) have become the most common organic pollutants. Studies have shown that chlorinated hydrocarbon pollutants such as trichloroethylene have potential "three-cause" (carcinogenic, teratogenic, and mutagenic) effects. Long-term exposure can cause damage to human health by damaging the liver, kidneys and central nervous system. In addition, their chemical properties make them classified as heavy non-aqueous phase fluids (DNAPLs), which will automatically settle under the aquifer in natural ecosystems to form stubborn and difficult-to-remove pollution sources.

[0003] At present, the remediation of chlorinated hydrocarbon organic pollution includes electrical remediation, physical or chemical remediation, and biological remediation. Among them, biological remediation has attracted widespread attention due to its low energy consumption, economic applicability, and low environmental harm. The current bioremediation methods focus on the reductive dechlorination process under anaerobic conditions. This process removes the chlorine atoms on the molecules of highly chlorinated chlorinated hydrocarbons (tetrachloroethylene, trichloroethylene) one by one to form low-chlorinated chlorinated hydrocarbons (dichloroethylene, vinyl chloride), and finally further dechlorinates to convert vinyl chloride into ethylene to achieve harmlessness. However, since the activity of microorganisms in the natural environment is inhibited by chlorinated hydrocarbons, the remediation process is slow, and the resources of specialized reductive dechlorinating bacteria that can reduce dichloroethylene to ethylene are scarce and limited to... Dehalococcoides Genus and Dehalogenimonas The growth conditions of these microorganisms are extremely harsh. Therefore, in situ pollution often makes it difficult to completely reduce dechlorination, and bioremediation has the risk of causing the accumulation of more toxic intermediates (ethylene dichloride, vinyl chloride).

[0004] In contrast to reductive dechlorination, the oxidative biodegradation pathway of chlorinated hydrocarbons has attracted attention due to its advantages of no accumulation of intermediates, strong adaptability, and fast growth. Currently, the known microbial resources for co-metabolic oxidation and direct oxidation of chlorinated hydrocarbons are concentrated in

[0005] Xanthobacter、Ralstonia、Mycobacterium、Methylobacterium、Pseudomonas

[0006] However, these aerobic microorganisms have extremely low ability to degrade chlorinated hydrocarbons in underground anoxic environments, which limits their application. Summary of the invention

[0007] Based on this, the present invention provides a chlorinated hydrocarbon degrading bacterium, named Stutzerimonas degradans The SH1 strain is deposited in the China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC NO: M 20221863 and the deposit date of December 5, 2022.

[0008] Among them, the Stutzerimonas degradans The SH1 strain is derived from soil from an industrial site co-contaminated with chlorinated hydrocarbons and benzene or benzene-based compounds. The 16S rRNA gene sequence is shown in SEQ ID NO: 1. The strain has the function of dechlorinating tetrachloroethylene and trichloroethylene under both aerobic and anaerobic conditions.

[0009] Said Stutzerimonas degradans The screening method of SH1 strain comprises the following steps:

[0010] (1) Soil samples were collected from industrial sites contaminated with trichloroethylene and benzene. 5 g of contaminated soil was then inoculated into 50 mL of basic anaerobic culture medium. 500 μM PCE and 5 mM acetate were added as stimulants to enrich the bacterial flora. The enriched culture was transferred to fresh culture medium every 10 days, and the transfer was repeated 10 times.

[0011] (2) Using the gradient dilution method to obtain a diluted bacterial suspension of the enriched culture, and spreading the bacterial suspension on TSA medium, and performing isolation and culture under aerobic and anaerobic conditions, respectively, and separating and purifying the grown single colonies to obtain the strain;

[0012] (3) Amplifying and sequencing the full-length fragment of the 16S rRNA gene of each strain isolated in step (2) to screen out a strain having a 16S rRNA gene sequence as shown in SEQ ID NO: 1.

[0013] The basic inorganic salt culture medium is prepared by dissolving 10 mL of 100× inorganic salt mother solution, 1 mL of trace element solution A, 1 mL of trace element solution B, 5 mL of 200× vitamin mother solution and 50 mL of reducing buffer in 1 L of water.

[0014] The present invention also provides a microbial agent, which comprises the above Stutzerimonas degradans SH1 strain.

[0015] The present invention also provides the above Stutzerimonas degradans Application of SH1 strain or microbial agent in dechlorination bioremediation of chlorinated hydrocarbon pollution, especially including dechlorination bioremediation of soil or groundwater in industrial contaminated sites.

[0016] In addition, the chlorinated hydrocarbon pollution includes the combined pollution of chlorinated hydrocarbons and benzene or benzene compounds.

[0017] More particularly, the Stutzerimonas degradans When the SH1 strain or the microbial agent is used to repair the soil of an industrial contaminated site contaminated by chlorinated hydrocarbons, the Stutzerimonas degradans The amount of SH1 strain used is not less than 10 7 cells / gram of soil.

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

[0019] Stutzerimonas degradans

[0020] The SH1 strain is derived from the original soil environment of a site where chlorinated hydrocarbons and aromatic compounds are co-contaminated. It can achieve efficient degradation of chlorinated hydrocarbons under both aerobic and anaerobic conditions, and there is no accumulation of toxic intermediates. The strain has low requirements for environmental conditions for growth and has high tolerance to multiple complex pollutions. The microbial agent prepared from it can effectively dechlorinate and degrade the vinyl chloride series pollutants in contaminated sites and repair chlorinated hydrocarbon pollution in industrial sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 In the embodiment Stutzerimonas degradans Colony morphology of SH1 strain on TSA medium.

[0022] Figure 2 In the embodiment Stutzerimonas degradans Cell microscopic morphology of SH1 strain (magnification 1000 times).

[0023] Figure 3 In the embodiment Stutzerimonas degradans Phylogenetic tree of SH1 strain and other similar strains in the database.

[0024] Figure 4 In the embodiment Stutzerimonas degradans Changes in the degradation rate of trichloroethylene by strain SH1 in a basal inorganic salt medium supplemented with 100 μM TCE and 5 mM acetate under aerobic and anaerobic conditions.

[0025] Figure 5 In the embodiment Stutzerimonas degradans Community composition of the enrichment culture derived from the SH1 strain at the genus level.

[0026] Figure 6 In the embodiment Stutzerimonas degradans Community composition of the domesticated flora of the SH1 strain at the genus level.

[0027] Figure 7 In the embodiment

[0028] Stutzerimonas degradans

[0029] Changes in tetrachloroethylene degradation rate of the domesticated bacterial flora of SH1 strain under anaerobic conditions in a basic inorganic salt medium supplemented with 100 μM PCE and 5 mM acetate. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described in combination with the embodiment and the accompanying drawings. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0031] The following examples screened Stutzerimonas degradans The SH1 strain is deposited in the China Type Culture Collection at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC NO: M20221863 and the deposit date is December 5, 2022.

[0032] In the following embodiments Stutzerimonas degradans The screening method of SH1 strain comprises the following steps:

[0033] (1) Soil samples were collected from different contaminated sites at an industrial site contaminated with trichloroethylene and benzene. Through correlation analysis between in situ pollutant concentrations and soil microbial community structure, high-throughput sequencing of the 16S rRNA gene V3-V4 region revealed that the second highest average abundance in the entire contaminated site belonged to Pseudomonas stutzeri ( Stutzerimonas ) has a strong correlation with chlorinated hydrocarbon pollution. Then 5 g of contaminated soil was inoculated into 50 mL of basic anaerobic medium, and 500 μM PCE and 5 mM acetate were added as stimulants to enrich the bacterial flora. The enriched culture was transferred to fresh culture medium every 10 days, and the transfer was continued for 10 times. High-throughput sequencing verified that the abundance of ASV1, the most abundant in the enriched culture, reached 97.3% ( Figure 5 ), and its sequence is similar to that of Stutzerimonas The ASV2 nucleotide sequence identity of the samples reached 100%, and it was concluded that the enrichment system with chlorinated hydrocarbon degradation ability was used as Stutzerimonas degradans Source of SH1 strain.

[0034] (2) A diluted bacterial suspension of the enriched culture was obtained by the gradient dilution method, and the bacterial suspension was spread on TSA medium. The culture was separated and cultured under aerobic and anaerobic conditions, respectively, and the grown single colonies were separated and purified to obtain purified strains.

[0035] (3) Performing ERIC-PCR amplification on each strain isolated in step (2), typing the strain based on the gel electrophoresis pattern, and then amplifying and sequencing the full-length fragment of the 16S rRNA gene to obtain a strain with a 16S rRNA gene sequence as shown in SEQ ID NO: 1, which is Stutzerimonas degradans SH1 strain.

[0036] The above-screened Stutzerimonas degradans The SH1 strain was inoculated in TSB medium and cultured at 180 r / min and 30°C for 3 days. The cells were collected by centrifugation at 8000×g and suspended in 5 mL of liquid basic inorganic salt medium. The suspended bacteria were inoculated into 45 mL of sterile basic anaerobic medium. Under aerobic conditions, 100 μM trichloroethylene and 5 mM acetate were directly added exogenously. Under anaerobic conditions, the gas in the serum bottle was replaced with helium three times before adding chlorinated hydrocarbons and acetate. Then, 1 mL samples were collected on the 3rd and 5th days and detected by headspace gas chromatography (HS-GC) under aerobic and anaerobic conditions. Stutzerimonas degradans The results showed that the SH1 strain had the ability to remove chlorinated hydrocarbons. Stutzerimonas degradans The SH1 strain was able to rapidly remove trichloroethylene from the culture medium without accumulating the common low-chlorinated toxic intermediates dichloroethylene and vinyl chloride.

[0037] The technical solution of the present invention is further explained below through specific embodiments. Example 1

[0038] This example screens Stutzerimonas degradans SH1 strain, the specific steps are as follows:

[0039] 1. Culture medium formula

[0040] (1) TSA medium: Dissolve 15 g casein peptone, 5 g soy peptone, 5 g NaCl, and 15 g agar in 1 L water and sterilize at 121°C for 20 min.

[0041] (2) TSB medium: Dissolve 15 g casein peptone, 5 g soy peptone, and 5 g NaCl in 1 L of water and sterilize at 121°C for 20 min.

[0042] (3) Aerobic basal inorganic salt medium: Dissolve 10 mL of 100× inorganic salt stock solution, 1 mL of trace element solution A, and 1 mL of trace element solution B in 1 L of water. Sterilize at 121°C for 20 min, filter using a 22 μm filter membrane, and add 5 mL of 200× vitamin stock solution and 50 mL of reducing buffer.

[0043] (4) Anaerobic basal inorganic salt medium: Dissolve 10 mL of 100× inorganic salt stock solution, 1 mL of trace element solution A, and 1 mL of trace element solution B in 1 L of water. Sterilize at 121°C for 20 min, filter using a 22 μm filter membrane, and add 5 mL of 200× vitamin stock solution and 50 mL of reducing buffer.

[0044] (5) Ringer solution: Dissolve 9 g of sodium chloride, 0.4 g of potassium chloride, and 0.25 g of anhydrous calcium chloride in 1 L of pure water and sterilize at 121°C for 20 min.

[0045] (6) 100× inorganic salt stock solution: 100 g NaCl, 50 g MgCl2∙6H2O, 20 g KH2PO4, 30 g NH3Cl, 30 g KCl, 1.5 g CaCl2∙2H2O dissolved in 1 L pure water and sterilized at 121°C for 20 min.

[0046] (7) Trace element solution A: 10 mL HCl (25%, w / w), 1.5 g FeCl2∙4H2O, 0.19 g CoCl2∙6H2O, 0.1 g MnCl2∙4H2O, 70 mg ZnCl2, 6 mg H3BO3, 36 mg Na2MoO4∙2H2O, 24 mg NiCl2∙6H2O, and 2 mg CuCl2∙2H2O were dissolved in 1 L of pure water and sterilized at 121°C for 20 min.

[0047] (8) Trace element solution B: 6 mg Na2SeO3∙5H2O, 8 mg Na2WO4∙2H2O, and 0.5 g NaOH were dissolved in 1 L pure water and sterilized at 121°C for 20 min.

[0048] (9) 200× vitamin stock solution: 20 mg biotin, 20 mg folic acid, 100 mg pyridoxine hydrochloride, 50 mg riboflavin, 50 mg thiamine, 50 mg niacin, 50 mg pantothenic acid, 1 mg vitamin B12, 50 mg p-aminobenzoic acid, 50 mg lipoic acid are dissolved in 1 L of pure water and the pH is adjusted to 7.5 with NaOH. The suspension is stirred vigorously and aliquots are frozen and stored in the dark. A 200× working solution is prepared by diluting the 1000× stock solution in a ratio of 1:5 in distilled water.

[0049] (10) Reducing buffer: 0.035 g L-cysteine ​​salt, 0.048 g Na2S∙9H2O, and 2.52 g NaHCO3 were dissolved in 50 mL pure water.

[0050] 2. Preparation of soil bacterial suspension

[0051] (1) Prepare 0.85% NaCl solution and sterilize it at 121°C for 20 min for the preparation of soil bacterial suspension.

[0052] (2) Weigh 50 g of contaminated soil sample and place it in a 500 mL blue-capped bottle.

[0053] (3) Add 500 mL of 0.85% NaCl solution at a ratio of soil: water = 1:10 and add a sterilized magnetic rotor.

[0054] (4) Vortex the mixture three times and mix at 600 rpm for 30 min.

[0055] 3. Enrichment and cultivation of soil microorganisms under anaerobic conditions

[0056] Without standing, in the anaerobic workstation, take 1 mL of the stirred bacterial suspension and add it to 50 mL of sterile anaerobic basic inorganic salt medium. Replace the gas in the serum bottle with helium three times, add 500 μM PCE and 1 mM sodium acetate, and culture at 30°C in the dark. Take 5 mL of culture every 10 days using a disposable sampler and inject it into fresh sterile basic inorganic salt medium, and continue to transfer 10 times.

[0057] 4. Cultivation of soil microorganisms under aerobic conditions

[0058] (1) In a clean bench, take 1 mL of the enriched culture and add it to 9 mL of sterile 0.85% NaCl. Pipet several times to mix thoroughly to obtain 10 -1 Dilution, from 10 -1 1 mL of soil suspension was taken from the diluent and added to 9 mL of sterilized 0.85% NaCl to dilute to 10-2 Then, dilute to 10 -3 , 10 -4 , 10 -5 .

[0059] (2) Incubate under aerobic conditions. Take 100 μL of 10 -2 , 10 -3 , 10 -4 , 10 -5 The bacterial suspension was spread on TSA culture medium in sequence, with 5 replicates for each dilution. The plate was placed upright for 20-30 minutes and then inverted in a 30°C incubator for about 2 weeks.

[0060] 5. Purification of strains

[0061] Observe and record the newly grown single colonies on the gradient dilution plates, and select as many different bacterial isolates as possible based on the size, color, and morphology of the colonies. Streak the purified strains on TSA medium 2-3 times until a single purified colony is obtained.

[0062] 6. Preservation of strains

[0063] After the bacteria purified 2-3 times by streaking grow on the plate, pick a single colony with a sterile inoculation loop and transfer it to TSB liquid culture medium. Culture it in a shaking incubator at 30°C for 1-2 days. Pipette 500 μL of the bacterial solution and add it to a cryovial containing 500 μL of 50% glycerol. Store it in a -80°C refrigerator.

[0064] 7. Morphological and molecular biological identification of strains

[0065] Through high-throughput sequencing analysis, the culture system was enriched Stutzerimonas degradans The relative abundance of SH1 strains reached 97.3% ( Figure 5 ), the 16S rRNA gene full-length sequence analysis was performed on the 27 isolated strains, and the 27 isolates contained 16 Stutzerimonas 16 bacteria were identified as Stutzerimonas The bacteria of the genus were divided into 6 different strains, among which the V3-V4 region sequence of the SH1 strain was 100% similar to the V3-V4 region sequence of the 16S rRNA gene of the second most abundant ASV (ASV2) in the in situ indigenous contaminated soil community, and was also 100% similar to the V3-V4 region sequence of the 16S rRNA gene of the most abundant ASV (ASV1) in the enrichment culture system, so it was identified as Stutzerimonas The SH1 strain of the genus is a representative strain with the function of degrading chlorinated hydrocarbons in in situ soil microbial communities and enrichment culture systems.

[0066] Stutzerimonas degradans After 3 days of culture of SH1 strain on TSA solid medium, the colonies were translucent white, with smooth surface and round protrusions ( Figure 1 ), the cells are short rod-shaped ( Figure 2 ). FastDNA Spin DNAKit for Soil was used for extraction and purification. Stutzerimonas degradans The DNA of SH1 strain was stored at 4°C. Stutzerimonas degradans The full-length base sequence of the 16S rRNA gene of the SH1 strain is as follows:

[0067]

[0068] Compared with similar strains in the GenBank database, based on Stutzerimonas degradans The 16S rRNA gene sequence homology of the SH1 strain was used to construct a phylogenetic tree using MEGAX software, such as Figure 3 shown.

[0069] Application Example 1

[0070] The screened samples obtained in Example 1 Stutzerimonas degradans SH1 strain was streaked on TSA medium and cultured at 30°C for 2-3 days. Stutzerimonas degradans SH1 single colony was inoculated into 50 mL TSB liquid medium and cultured at 30℃ 180 r / min for 1.5 days until the bacterial solution became turbid. Centrifuge the bacteria at 4000 × g for 5 minutes, discard the supernatant, and collect Stutzerimonas degradans SH1 bacteria, collect Stutzerimonas degradans SH1 strain cells were inoculated in TSB medium, cultured at 180 r / min and 30°C for 3 days, and then centrifuged at 8000 × g to collect the cells, suspend the cells with 5 mL of liquid basic inorganic salt medium, and inoculate the suspended bacteria into 45 mL of sterile basic anaerobic medium. For the test of aerobic conditions, 100 μM trichloroethylene and 5 mM acetate were directly added exogenously, while for the test of anaerobic conditions, the gas in the serum bottle was replaced three times with helium before adding chlorinated hydrocarbons and acetate. The inoculated culture solution was cultured at 30°C, and 1 mL samples were collected on the 3rd and 5th days, and the HS-GC was used to detect the aerobic and anaerobic conditions. Stutzerimonas degradans The results showed that the SH1 strain had the ability to remove chlorinated hydrocarbons. Stutzerimonas degradant SH1 strain can quickly remove trichloroethylene from the culture medium ( Figure 4 ), and there is no accumulation of common low-chlorinated toxic intermediates such as dichloroethylene and vinyl chloride, which reduces the pollution of chlorinated hydrocarbons in industrial sites and has important application value.

[0071] Application Example 2

[0072] Inoculate 5 g containing the Stutzerimonas degradans The soil sample of the site contaminated by chlorinated hydrocarbons and benzene series was added to 50 ml of basic anaerobic medium, and the gas in the serum bottle was replaced with helium three times. 500 μM tetrachloroethylene and 5 mM acetate were added as stimulants to acclimate the bacterial flora. The enriched culture was transferred to fresh medium every 10 days, and the transfer was continued for 10 times. Stutzerimonas degradans Domesticated flora of SH1 strain ( Figure 6) was inoculated into 50 mL of sterile basic anaerobic medium at a volume ratio of 10%. The gas in the serum bottle was replaced with helium three times before adding chlorinated hydrocarbons and acetate. 500 μM tetrachloroethylene and 5 mM acetate were added exogenously, and the inoculated culture medium was cultured anaerobically at 30 °C. Then 1 mL of culture samples were collected on the 5th and 10th days, and the content of anaerobic bacteria under anaerobic conditions was detected by HS-GC. Stutzerimonas degradans The results showed that the composite bacterial community of SH1 strain could quickly remove tetrachloroethylene in the system. Figure 7 ), and there is no accumulation of common low-chlorinated toxic intermediates such as dichloroethylene and vinyl chloride. The bacterial agent can effectively remove tetrachloroethylene pollutants in contaminated sites and repair chlorinated hydrocarbon pollution in industrial sites, and has important application value.

Claims

1. A chlorinated hydrocarbon degrading bacteria strain SH1, named Stutzerimonas degradans SH1, the deposit number is CCTCC NO: M 20221863, and the deposit date is December 5, 2022.

2. A microbial agent comprising the SH1 strain according to claim 1.

3. Use of the SH1 strain according to claim 1 or the microbial agent according to claim 2 in dechlorination bioremediation of tetrachloroethylene and trichloroethylene pollution.

4. The use according to claim 3, characterized in that: The dechlorination bioremediation includes the dechlorination bioremediation of soil or groundwater in industrial pollution sites polluted by tetrachloroethylene and trichloroethylene.

5. The use according to claim 4, characterized in that: When the SH1 strain or the microbial agent is used to repair the soil of industrial contaminated sites contaminated by tetrachloroethylene and trichloroethylene, the amount of the SH1 strain used is not less than 10 7 cells / gram of soil.

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