Methanol bacterium and application thereof

Abstract

The invention belongs to the technical field of environmental microorganism screening, and discloses a methanol bacterium and application thereof. The method comprises the following steps: adding high-arsenic sediment into an aerobic inorganic salt culture medium taking methanol as a unique carbon source, and placing in a shaking table at 30 DEG C for dark culture at a constant rotating speed; after enrichment and passage, diluting a bacterial solution obtained by culture, uniformly coating the diluted bacterial solution on a solid culture medium taking methanol as a unique carbon source, and placing the solid culture medium in a constant-temperature incubator at 30 DEG C for culturing in a dark place; selecting single colonies from the grown bacterial colonies, selecting the selected single colonies to a new solid culture medium for lineation, placing the new solid culture medium in a constant-temperature incubator for culture, and repeating lineation for more than three times after the single colonies grow to obtain the pure bacterial beads utilizing methanol. The strain can be stored in a liquid inorganic salt culture medium containing 300 mg / L of As < 3 + > and has good arsenic resistance. Gram staining experiment is negative, 16s. RRNA sequence identification shows that the arsenic-resistant strain is methylotrophicus methylobacterium, and strain resources are provided for microbial remediation of arsenic-polluted environment.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganism screening, and specifically relates to an arsenic-resistant methanol-utilizing bacterium and its screening and application in repairing an arsenic-polluted environment. Background Art

[0002] Industrial activities such as metallurgy and mining continuously discharge heavy metal arsenic into the natural environment. Due to its high toxicity and bioaccumulation, the prevention, control and remediation of arsenic pollution has become increasingly urgent. Arsenic and its compounds in the natural environment are eventually enriched in soil, sediments and other environments after migration and transformation. Microbial-mediated organic carbon degradation in sediments is a complex process, which is divided into three stages: (1) hydrolysis and fermentation; (2) hydrogen and acetic acid production; (3) methane production. Sulfate-reducing bacteria, as the dominant group in sediments, can promote the oxidation of nearly 50% of organic matter. However, many sulfate-reducing bacteria do not use methanol. Methanol, as a one-carbon compound, can be used as the sole carbon source to provide energy for the growth and survival of certain microorganisms. From a resource perspective, the development and utilization of methanol has many advantages.

[0003] In summary, the discovery of arsenic-resistant and methanol-utilizing bacteria is of great significance in the existing microbial control of arsenic-contaminated environments. Summary of the invention

[0004] The purpose of the present invention is to screen out a methanol-utilizing bacterium with arsenic resistance and provide application for the restoration of arsenic-contaminated environment.

[0005] The technical solution of the present invention:

[0006] A methanol bacteria (Methylorubrumzatmanii) HLD-8, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 21, 2024, with the deposit number CGMCC 1.62480.

[0007] A method for screening arsenic-resistant methanol-utilizing bacteria comprises the following steps:

[0008] In the first step, the collected high-arsenic sediment samples were added to an aerobic inorganic salt culture medium with methanol as the only carbon source in an ultra-clean bench, and placed in a shaker at 30°C and cultured in the dark at a constant speed;

[0009] In the second step, the cultured bacterial solution was subcultured in an aerobic inorganic salt medium with methanol as the sole carbon source, placed in a shaker at 30°C, and cultured in the dark at a constant speed of 160 rpm / min for 72 hours; this step was repeated three times to obtain a bacterial solution enriched with methanol-utilizing bacteria;

[0010] The third step is to dilute the methanol-utilizing bacteria enriched bacterial solution with 9 g / L sterile physiological saline in a gradient manner, then spread it on a solid culture medium with methanol as the only carbon source, and place it in a constant temperature incubator at 30°C in the dark for 48-72 hours;

[0011] Step 4: Pick a single colony cultured on the solid culture medium in step 3 and place it on a new solid culture medium with methanol as the sole carbon source, streak it on a plate, and place it in a constant temperature incubator at 30°C in the dark for 48-72 hours;

[0012] Step 5, repeating steps 3 and 4 three times to obtain purified pure bacterial bead single colony;

[0013] Step 6. Pick the purified single colony into LB liquid culture medium, place it in a 30℃ shaker, and culture it in the dark at a constant speed to obtain the bacterial solution. Add it to the bacterial preservation tube in a 1:1 ratio with 0.5ml / ml glycerol and freeze it at -80℃.

[0014] Furthermore, in the first step, the constant rotation speed was 160 rpm / min for 72 h in dark culture;

[0015] Furthermore, in the third step, the gradient dilution was performed to obtain 10 -1 -10 -8 Concentration of bacterial solution.

[0016] Furthermore, in the fourth step, a single colony is picked and inoculated into a solid inorganic salt medium with methanol as the sole carbon source in a clean bench, and then placed in a constant temperature incubator at 30°C in the dark for 48-72 hours;

[0017] The components of the aerobic inorganic salt culture medium with methanol as the only carbon source are: 0.2 g / L magnesium sulfate heptahydrate, 3.0 g / L diammonium hydrogen phosphate, 1.0 g / L sodium chloride, 2.0 g / L dipotassium hydrogen phosphate, 10.0 mg / L ferrous sulfate heptahydrate, 3.5 mg / L manganese sulfate monohydrate, 1 L ultrapure water, pH 6.80-7.20. Methanol needs to be filtered through a sterilized 0.22 μm filter membrane. When adding, the culture medium should be cooled to avoid volatilization of overheated methanol. The amount of methanol added is 2.0 ml / L.

[0018] The components of the solid culture medium with methanol as the sole carbon source are: 0.2 g / L magnesium sulfate heptahydrate, 3.0 g / L diammonium hydrogen phosphate, 1.0 g / L sodium chloride, 2.0 g / L dipotassium hydrogen phosphate, 10.0 mg / L ferrous sulfate heptahydrate, 3.5 mg / L manganese sulfate monohydrate, 15.0 g / L agar, 1 L ultrapure water, pH 6.80-7.20. Methanol needs to be filtered through a sterilized 0.22 μm filter membrane. When adding, the culture medium should be cooled to avoid volatilization of overheated methanol. The amount of methanol added is 2.0 ml / L.

[0019] A new method for treating arsenic-contaminated environment with microbial remediation, the application of the above-mentioned methanol bacteria, the arsenic-resistant methanol bacteria has 300 mg / L As 3+ Tolerance, used for microbial remediation of arsenic-contaminated environments.

[0020] Beneficial effects of the present invention: The present invention screens out a microorganism that can metabolize methyl in a high-arsenic environment, providing application for the restoration of arsenic-contaminated environments. Excessive methanol concentration in the environment is toxic to the growth of other microorganisms, while Methylobacterium can metabolize methyl and use methanol as a carbon source to provide energy for its own growth. Arsenic is a toxic metalloid, and a high-arsenic environment will inhibit the growth of microorganisms. In summary, the strain can survive in 300 mg / L of As. 3+ The plant is arsenic-resistant when grown at low concentrations and can use methanol as the sole carbon source for growth metabolism, which is of great significance for bioremediation processes such as arsenic pollution and methanol accumulation in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The invention provides a method and process for screening arsenic-resistant methanol-utilizing bacteria.

[0022] Figure 2 The present invention provides a growth curve of arsenic-resistant methanol-utilizing bacteria using different methanol contents as carbon sources.

[0023] Figure 3 This is a growth curve of arsenic-resistant methanol utilizing bacteria under trivalent arsenic stress implemented by the present invention. DETAILED DESCRIPTION

[0024] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0025]

[0026] Example 1

[0027] The arsenic-resistant methylbacterium of the present invention is isolated and screened from high-arsenic polluted estuary sediments downstream of the sewage outlet of the zinc plant in Jinzhou Bay area of ​​Huludao City. Unless otherwise specified, the screening methods are all well-known methods for those skilled in the art.

[0028] like Figure 1 As shown, the method for screening arsenic-resistant methanol-utilizing bacteria provided by the present invention comprises the following steps:

[0029] Activation of bacterial flora: Take 1.0g sediment sample and inoculate it into 100ml inorganic salt aerobic culture medium with 2.0ml / L methanol as the only carbon source. The components of the culture medium are: 0.2g / L magnesium sulfate heptahydrate, 3.0g / L diammonium hydrogen phosphate, 1.0g / L sodium chloride, 2.0g / L dipotassium hydrogen phosphate, 10.0mg / L ferrous sulfate heptahydrate, 3.5mg / L manganese sulfate monohydrate, 1L ultrapure water, pH 6.80-7.20. After the culture medium is configured, it must be sterilized first. The sterilization conditions are: 121℃, 30 minutes. After sterilization, add 0.2ml methanol in the clean bench. Methanol needs to be filtered through a sterilized 0.22μm filter membrane. When adding, wait for the liquid culture medium to cool down slightly to avoid overheating and methanol volatilization. Finally, place the culture medium inoculated with 1.0g sample in a 30℃ shaker with a constant speed of 160rpm / min for dark culture for 72h;

[0030] Enrichment culture: After 72 hours, take 1.0 ml of the activated bacterial solution on a clean bench and inoculate it into 100 ml of inorganic salt aerobic culture medium with 2.0 ml / L methanol as the only carbon source. Place it in a 30°C shaker with a constant speed of 160 rpm / min and culture it in the dark for 72 hours. Repeat 3 times.

[0031] Gradient dilution plate coating: prepare solid culture medium in advance, components: 0.2g / L magnesium sulfate heptahydrate, 3.0g / L diammonium hydrogen phosphate, 1.0g / L sodium chloride, 2.0g / L dipotassium hydrogen phosphate, 10.0mg / L ferrous sulfate heptahydrate, 3.5mg / L manganese sulfate monohydrate, 15.0g / L agar, 1L ultrapure water, pH 6.80-7.20, methanol needs to be filtered through a 0.22μm filter membrane after sterilization on a clean bench, and the culture medium should be cooled slightly when added to avoid overheating and methanol volatilization. The amount of methanol added is 2.0ml / L. Pour the culture medium after adding methanol evenly into sterile culture dishes and let it cool for use.

[0032] In the clean bench, take 1 ml of bacterial solution and 9 ml of 9g / L sterile saline in a 15 ml sterile centrifuge tube for gradient dilution to obtain 10 -1 -10 -8concentration of bacterial solution, and the diluted concentration was 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Place 100 μl of bacterial solution on the culture dish, use a sterile coating stick to evenly spread the bacterial solution over the entire culture dish in the same direction until a slight pause occurs. Repeat the process, seal the culture dish with a sealing film, mark it, and place it in a 30°C constant temperature incubator for 72 hours until colonies grow.

[0033] Strain selection and rescreening: Select plates with clear boundaries of each colony, use a sterile inoculation loop to select strains with larger diameters in a clean bench, and make three-zone lines on a new culture dish. Place it in a 30°C constant temperature incubator for culture, and re-select after a single pure colony grows. Repeat the streaking three times to obtain pure bacterial beads.

[0034] The obtained pure bacterial strain was re-picked into 100 ml inorganic salt aerobic culture medium with 2.0 ml / L methanol as the sole carbon source and placed in a 30°C shaker with a constant speed of 160 rpm / min for dark culture for activation. The culture was carried out for 48 hours, and the bacterial genomic DNA was extracted.

[0035] DNA sequencing and sequence alignment.

[0036] Example 2

[0037] Methanol concentration gradient growth experiment: The arsenic-resistant Methylobacterium obtained by screening was activated with 100 ml of liquid inorganic salt medium with 2.0 ml / L methanol as the sole carbon source, and 1.0 ml was inoculated into 100 ml of fresh inorganic salt liquid medium with methanol as the sole carbon source at 2.0 ml / L, 5.0 ml / L, 10.0 ml / L, and 15.0 ml / L. The absorbance value OD was measured. 600 The growth curve was obtained (see Figure 2 ), it can be seen that Methylobacterium can grow normally in the inorganic salt medium with a methanol concentration of 5.0 ml / L.

[0038] Example 3

[0039] The application of the arsenic-resistant methanol utilizing bacteria provided by the present invention in a high arsenic pollution environment is described in detail with reference to the embodiments.

[0040] Trivalent arsenic gradient stress experiment: The arsenic-resistant Methylobacterium screened was activated for 12 h in 100 ml of liquid inorganic salt medium with 10.0 ml / L methanol as the sole carbon source. Then 1.0 ml of the activated bacterial solution was inoculated into 100 ml of fresh inorganic salt liquid medium with 10.0 ml / L methanol as the sole carbon source and trivalent arsenic concentrations of 50, 100, 150, 200, 250, and 300 mg / L. The absorbance value OD was measured. 600 The growth curve was obtained (see Figure 3 ), it can be seen that Methylobacterium can grow in inorganic salt culture medium with a trivalent arsenic concentration of 300 mg / L.

Claims

1. A methanol bacteria (Methylorubrum zatmanii) HLD-8, characterized in that The methanol bacteria was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 21, 2024, with the deposit number CGMCC1.62480.

2. The use of methanol bacteria according to claim 1 in the microbial remediation of arsenic-contaminated environment, wherein the arsenic-resistant methanol bacteria has an As content of 300 mg / L. 3+ Tolerance, used for microbial remediation of arsenic-contaminated environments.

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