WO2021114784A1

WO2021114784A1 - Strain capable of tolerating high concentration of chromium, and microbial agent used for hexavalent chromium pollution control

Info

Publication number: WO2021114784A1
Application number: PCT/CN2020/114951
Authority: WO
Inventors: 李琳; 吕宪俊; 胡术刚; 申宪伟; 肖新峰; 薛建良; 高洪阁; 高宇; 尚秀芳
Original assignee: 山东科技大学
Priority date: 2019-12-11
Filing date: 2020-09-14
Publication date: 2021-06-17
Also published as: ZA202201093B

Abstract

Provided is a degrading bacterium which can tolerate a high concentration of chromium, and a bacterial agent prepared therefrom. The degrading bacterium is Stenotrophomonas acidaminiphila 4-1, preserved in the China Center for Type Culture Collection, having the preservation number CCTCC M 2019031. The strain can grow under a high concentration of heavy metal ions, and can absorb Cr(VI) in the environment into the cell body so as to achieve the purpose of enriching chromium.

Description

Strains that can tolerate high concentrations of chromium and microbial agents for hexavalent chromium pollution treatment

Technical field

The invention relates to the technical field of microbial remediation of heavy metal chromium in the environment, in particular to a microbial agent used for the treatment of hexavalent chromium pollution and a method for the treatment of hexavalent chromium pollution.

Background technique

Chromium is a toxic heavy metal widely used in metallurgy, mining, electroplating, printing and dyeing industries. Environmental pollution caused by chromium and its compounds mainly comes from inferior cosmetic raw materials, leather preparations, chrome-plated parts of metal parts, industrial pigments, and raw materials for tanning, rubber and ceramics. Chromium wastewater and chromium slag leachate are two major pollution sources of heavy metal chromium, which have serious impacts on soil, surface water and shallow groundwater. Studies have shown that Cr(VI) is easily absorbed by the human body, causing damage to the skin and mucous membranes, breathing, digestion, immunity, reproduction and other systems. Residents in areas with serious water pollution, frequent contact or excessive intake, are prone to rhinitis, tuberculosis, diarrhea, and bronchial tubes. Inflammation, dermatitis, etc., can cause cancer and induce gene mutations. The search for an economical, effective and non-secondary pollution treatment technology for the heavy metal chromium in the environment has been a hot spot in the environmental engineering community in recent years.

There are many traditional chromium pollution treatment methods, such as physical method, adsorption method, chemical reduction method, ion exchange, electrolysis method, etc., but these methods have disadvantages such as high cost, difficult operation, easy to produce secondary pollution, and it is difficult to deal with large quantities. Harmful pollution with low flow rate. Compared with physical and chemical treatment methods, bioremediation is a treatment method with low energy consumption, high efficiency and environmentally friendly treatment. In recent years, various biological materials (including microorganisms, algae, plants and agricultural wastes) have been developed to treat chromium pollution. At present, the treatment of chromium pollution is mainly focused on microbial treatment. Among them, obtaining strains with high-efficiency removal ability is the most critical and first condition for the application of bioremediation technology.

Summary of the invention

The purpose of the present invention is to provide a microbial agent for the treatment of hexavalent chromium pollution and a method for the treatment of hexavalent chromium pollution.

A strain that can tolerate high concentrations of hexavalent chromium, Stenotrophomonas acidaminiphila 4-1, was deposited in the China Type Culture Collection on January 8, 2019 (Address: China Wuhan. Wuhan University), the deposit number is: CCTCC M 2019031. The strain can grow under the condition of high concentration of heavy metal ions, and can significantly absorb the Cr(VI) in the environment into the cell body to achieve the purpose of enriching chromium. At the same time, the fermentation broth of the bacteria also has the ability to remove chromium ions Cr( VI), therefore, has very important application prospects in the field of heavy metal pollution control.

The invention also discloses a treatment method for hexavalent chromium pollution, which includes the step of inoculating Stenotrophomonas acidaminiphila 4-1 into heavy metal chromium pollutants.

The present invention also discloses a microbial agent for the treatment of chromium pollution, the active ingredient of which is said Stenotrophomonas acidaminiphila 4-1 or Stenotrophomonas acidaminiphila ) 4-1 of fermentation broth.

The fermentation broth of Stenotrophomonas acidaminiphila 4-1 is prepared by the following method:

1) First inoculate Stenotrophomonas acidaminiphila 4-1 in the above-mentioned LB medium, and activate it in a constant temperature incubator at 30°C for 24 hours;

2) Pick the single colony activated in step 1) and inoculate it in the LB medium with a bottle volume of 30mL/100mL, 160r·min ^-1 , 30°C constant temperature shaking culture, to obtain a concentration of 4.1×10 ⁷ CFU·mL ^-1 cell culture solution of the strain;

3) Centrifuge the cell culture solution prepared in step 2) at 10000 r·min ^-1 for 20 minutes at 4° C., collect the supernatant and filter it with a 0.22 μm microporous membrane. The supernatant is the sterile fermentation broth.

The Stenotrophomonas acidaminiphila 4-1 provided by the present invention is inoculated with chromium pollutants, which can effectively remove free chromium ions in the environment, and can tolerate Cr(VI) 1000 mg·L ^-1 , It is an excellent microbial material for the treatment of heavy metal chromium Cr(VI) pollution.

Description of the drawings

Figure 1 is a scanning electron micrograph of strain 4-1 in a normal state;

Figure 2 is a scanning transmission electron microscope (TEM) image of strain 4-1 cultured for 7 days under the condition of a Cr(VI) concentration of 90mg·L ^-1;

Figure 3 is an energy spectrum analysis diagram (EDS) of strain 4-1 after 7 days of cultivation under the condition of Cr(VI) concentration of 90mg·L ^-1;

Figure 4 is a surface scan energy spectrum of the Cr element in the selected area of Figure 3;

Figure 5 shows the effect of different Cr(VI) concentrations on the performance of strain 4-1;

Figure 6 shows the effect of different diesel fuel concentrations on the performance of strain 4-1;

Figure 7 shows the effect of contaminated soil with different Cr(VI) concentrations on the performance of strain 4-1;

Figure 8 is a phylogenetic tree of 16S rDNA of strain 4-1;

Figure 9 shows the treatment effect of aseptic fermentation broth on Cr(VI)-containing water bodies.

Detailed ways

The present invention will be described in detail below with reference to the embodiments. It should be pointed out that the embodiments of the present invention are limited to the description of the present invention without limiting the effect. The other various operations involved in the present invention are conventional techniques in the field, and there is no special description in the text. Ordinary technicians can refer to various commonly used reference books, scientific and technological documents or related instructions, manuals, etc. before the filing date of the present invention for implementation.

Example 1 Isolation and identification of strain 4-1

The specific screening plan is as follows:

Take 1g of oilfield contaminated soil (collected in Shengli Oilfield, Dongying City, China in September 2018) and add it to the screening medium. After shaking culture for one week in a shaker with a temperature of 30°C and a rotation speed of 160 rpm, inoculate 5% The culture medium was transferred to the fresh medium to continue the culture, and the transfer was carried out 3 times under the same conditions. Then take the culture solution for gradient dilution, pick out the colonies of different shapes, and use the screening medium to carry out the degradation experiment.

among them:

Screening medium: add 30mg·L ^-1 Cr(VI) on the basis of LB medium.

LB medium: 10g peptone, 5g yeast powder, 5g NaCl, 1000mL distilled water, pH 7.2-7.5.

After isolation and screening, a strain 4-1 with stable resistance to chromium was obtained, and the strain was further identified.

Example 2 Identification of strains

1. Morphological characteristics: the colony is moist, smooth and light yellow.

2. Biological and biochemical characteristics

(1) Gram stain

A: Dye:

1. Crystal violet mixture (liquid A: crystal violet 2g, 95% ethanol 20ml; liquid B: ammonium oxalate 0.8g, distilled water 80ml. Mix liquid A with liquid B, and filter for use at rest for 48 hours);

2. Liquid iodine (1g of iodine, 2.0g of potassium iodide, 300mL of distilled water, first dissolve potassium iodide with 3-5mL of distilled water, then put in the iodine tablets, after dissolving, add water to dilute to 300mL);

3. Decolorizing solution (95% ethanol 70mL);

4. Counterstaining solution (0.5% safranin aqueous solution).

B: Dyeing steps:

1. Use an inoculation needle to pick up a small amount of fungal moss, spread it on a drop of sterile distilled water on a clean glass slide, and fix it by air drying.

2. After dyeing with crystal violet mixture for 1 minute, wash with water.

3. Act on the iodine solution for 1 minute and blot it dry.

4. Decolorize with 95% ethanol or acetone ethanol solution and drip until the eluent is colorless.

5. Use saffron dye solution for 2-3 minutes, wash with water, and air dry.

C: Observed by a phase-contrast microscope, red is Gram-negative bacteria, and dark purple is Gram-positive bacteria.

The experimental results showed that bacteria 4-1 were Gram-negative bacteria.

(2) Sugar alcohol fermentation

A: Hugh-Leifson Medium: peptone 5g, NaCl 5g, K ₂ HPO ₄ 0.2g, sugar alcohol (glucose or other sugar, alcohol) 10g, agar 5-6g, 1% bromine 3mL of cresyl violet, 1000mL of distilled water, pH 7.0-7.2, separate into test tubes, the height of the medium is about 4-5cm, and sterilize at 115℃ for 20 minutes.

B: Vaccination and observation. The young slant culture was punctured and inoculated in the above medium, and observed after 3 days of moderate temperature cultivation. If the indicator turns yellow, it means acid production, which is positive; if it does not change or turns blue, it is negative.

The experimental results showed that the sugar alcohol fermentation test of bacteria 4-1 was positive.

(3) Methyl red test

A: Medium: peptone 5g, glucose 5g, K ₂ HPO ₄ 5g, water 1000mL, adjust the pH to 7.0-7.2.

B: Methyl red reagent: 0.1 g of methyl red, 300 mL of 95% ethanol, and 200 mL of distilled water.

C: Observation of inoculation: Inoculate bacteria 4-1 in the above medium and cultivate for 3 days at room temperature. Add 2-3 drops of methyl red reagent to the culture solution. Red is a positive reaction in the methyl red test, and yellow is a negative reaction.

The experimental results showed that bacteria 4-1 was a negative reaction in the methyl red test.

(4) V-P test

B: Creatine, 40% NaOH.

C: Observation of inoculation: Inoculate bacteria 4-1 in the above medium and cultivate for 3 days at room temperature. Take the culture solution and mix the same amount of 40% NaOH. Add a little creatine, if the culture medium appears red for 10 minutes, it is a positive reaction.

In the experimental results, the 4-1 culture medium did not appear red.

(5) Gelatin liquefaction

A: Medium: peptone 5g, gelatin 100-150g, water 1000mL, pH 7.2-7.4, divided into test tubes, the height of the medium is about 4-5cm, and steam sterilized at 115°C for 20 minutes.

B: Vaccination and observation. Take 18-24h slant culture puncture inoculation and have two uninoculated blank controls. Incubate at room temperature for 3 days. Observe the growth and whether the gelatin is liquefied at room temperature. If the bacteria 4-1 has grown, the gelatin surface has no depressions and is a stable clot, it is negative. If part or all of the gelatin is a flowable liquid below 20°C, the gelatin hydrolysis is positive.

The experimental results showed that all gelatin was a flowable liquid below 20°C, indicating that the gelatin was hydrolyzed positively.

(6) Bacterial motility test

Inorganic salt medium is prepared, 0.3%-0.6% agar is added to it to prepare a semi-solid medium. The test tube does not flow when it is placed down, and it will break when it is gently tapped on the hand. Use a straight needle to puncture the inoculation bacteria 4-1 on the semi-solid medium, and cultivate at an appropriate temperature. If the growth only grows on the puncture line and the edge is very clear, it means that the test bacteria has no motility. If the growth spreads from the puncture line to the surroundings in a misty shape and the edges are cloudy, it means that the test bacterium has motility.

The experimental results showed that the growth spread from the puncture line to the surroundings in a cloud shape, and its edge was clouded, indicating that the bacteria 4-1 had mobility.

(7) Contact enzyme experiment

A: Reagent 3% hydrogen peroxide.

B: Inoculation and result observation: Use a platinum wire inoculation loop to take a small loop of slant bacteria 4-1 and smear it on a glass slide with 3% hydrogen peroxide. If there is air bubbles, it is positive, and if there is no air bubbles, it is positive. Is negative.

The result of the experiment showed that there were bubbles, indicating that the bacteria 4-1 was positive.

3. 16S rDNA sequence analysis:

Use TIANGEN (Tiangen Biochemical Technology (Beijing) Co., Ltd.) bacterial genomic DNA extraction kit for bacterial genomic DNA extraction, with bacterial 16S rDNA universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-GGTTACCTTGTTACGACTT-3) '), PCR amplification is performed on the DNA template. The sequence is determined, and the analysis result of the determined sequence shows that the length of the amplified 16S rDNA fragment is 1012, and the sequence is shown in SED ID NO.1.

The phylogenetic tree of Bacteria 4-1 constructed by MEGA7 software is shown in Fig. 8. From Fig. 8 it can be seen that this bacterium is the closest to Stenotrophomonas acidaminiphila strain YFMCD4.4.

After morphological observation, physiological and biochemical reactions and 16S rDNA molecular identification, the strain 4-1 was determined to be Stenotrophomonas acidaminiphila, named Stenotrophomonas acidaminiphila 4-1.

Example 3: Enrichment capacity of Stenotrophomonas acidaminiphila 4-1 on hexavalent chromium in wastewater containing hexavalent chromium

The concentration of Cr(VI) prepared with K ₂ Cr ₂ O ₇ ^{is 15 mg·L -1} , 50 mg·L ^-1 , 100 mg·L ^-1 , 120 mg·L ^-1 , 150 mg·L ^-1 , 180 mg·L ^-1 , 200mg·L ^-1 , 500mg·L ^-1 , 1000mg·L ^-1 , the strain 4-1 inoculation amount is 10% (v/v, the bacterial density is 4.1×10 ⁷ CFU/mL), the shaker speed is 160 rpm at 30 Incubate at ℃ for 7 days, and measure the absorbance at 540nm with a visible spectrophotometer.

The specific determination method is as follows: the culture medium is centrifuged at 4000r/min for 20min, 100μL of the supernatant is taken with a pipette and diluted with water in a 50mL colorimetric tube to the mark, and 0.5mL of 1+1 sulfuric acid solution is added, 0.5mL of 1+ 1 Phosphoric acid solution, and 2mL of color reagent (weigh 0.2g of diphenylcarbazide, dissolve it in 50mL of acetone, dilute to 100mL with water), measure the absorbance at 540nm after 10min.

Figure 2 shows the TEM image of anti-chromium bacteria 4-1 ^{cultured for 7 days under the condition of 90mg·L -1 of} Cr(VI). It can be seen from the figure that there is a layer of extracellular polymer on the outside of the cells of strain 4-1. There are several black dot-like substances evenly dispersed inside the cells. Figure 3 shows the energy spectrum analysis graph (EDS) of strain 4-1 cultured for 7 days under the condition of Cr(VI) concentration of 90mg·L ^-1 ; Figure 4 is the surface scan energy spectrum graph of the selected area in Figure 3 It is obvious from Figure 4 that chromium was detected in both the inside of the cell and the extracellular polymer, indicating that the strain has an enrichment effect on chromium.

Strain 4-1 has a Cr(VI) concentration of 15 mg·L ^-1 , 50 mg·L ^-1 , 100 mg·L ^-1 , 120 mg·L ^-1 , 150 mg·L ^-1 , 180 mg·L ^-1 , 200 mg·L ^{Under the conditions of -1} , 500mg·L ^-1 and 1000mg·L ^-1 , the removal of Cr(VI) in 7 days is shown in Figure 5. Strain 4-1 has a 75.7% removal rate of 15 mg·L ^-1 and can tolerate up to 1000 mg·L ^-1 Cr(VI).

Example 4: Application in diesel

Accurately weigh 0.1g, 0.3g, 0.5g, 0.7g, 1.0g, and 1.5g of diesel oil used in the experiment and add them to 30mL of LB medium. The resulting diesel oil concentration is 3.33g·L ^-1 , 10g·L ^-1 , 16.67g·L ^-1 , 23.33g·L ^-1 , 33.33g·L ^-1 , 50g·L ^-1 , strain 4-1 inoculation amount is 10% (v/v, bacterial density is 4.1×10 ⁷ CFU· mL ^-1 ), incubate at 30°C for 7 days at a shaker rotation speed of 160 rpm, and measure the absorbance at 540 nm with a visible spectrophotometer. The specific measurement method is as described in Example 3.

The degradation of strain 4-1 at the diesel concentration of 3.33g·L ^-1 , 10g·L ^-1 , 16.67g·L ^-1 , 23.33g·L ^-1 , 33.33g·L ^-1 , and 50g·L ^-1 Figure 6 shows that the degradation rate of diesel at 3.33 g·L ^{-1 can reach 68.47%.}

Example 5: Treatment of soil contaminated by hexavalent chromium

Prepare chromium-contaminated soil and control the concentration of hexavalent chromium ions in the soil samples to 0.2mg·g ^-1 , 0.3mg·g ^-1 , 0.7mg·g ^-1 , 1.0mg·g ^-1 , 3.0mg·g ^{-1 respectively} , 10.0mg·g ^-1 , 15.0mg·g ^-1 , stir evenly and put them in a drying oven (75℃) to dry, so that Cr(VI) can be fully absorbed by the soil. Then, the amount of bacterial solution of the inoculated strain 4-1 was 2.5mL (the bacterial density was 4.1×10 ⁷ CFU·mL ^-1 ), and the control group was not added with bacterial solution. After stirring, it was placed in an incubator (30℃) for cultivation During the cultivation period, water is regularly replenished, and the soil moisture content is controlled at 15% to 20%. Take out after 7 days of cultivation, weigh 0.1000g soil sample into a 100mL small beaker, add 5mL extract (0.4mol·L ^-1 KCl) into the beaker, stir for 5min with an electromagnetic stirrer, and transfer the soil suspension to the centrifuge tube , Centrifuge at 4000r·min ^-1 for 20min, pour the supernatant into a 100mL volumetric flask, add 2mL of deionized water to the residue, stir with a glass rod for 2min, centrifuge for 5min, pour out the supernatant, repeat the washing once, and combine the supernatant Dilute the solution to a constant volume, transfer the solution after the constant volume to a 50mL colorimetric tube, dilute a certain multiple according to the actual situation, add 0.5mL 1+1 sulfuric acid solution, 0.5mL 1+1 phosphoric acid solution, 2mL color reagent, shake well After that, let it stand for 5 minutes, and measure the absorbance at a wavelength of 540nm with a zero-concentration solution as a reference.

Strain 4-1 has a Cr(VI) concentration of 0.2mg·g ^-1 , 0.3mg·g ^-1 , 0.7mg·g ^-1 , 1.0mg·g ^-1 , 3.0mg·g ^-1 , 10.0mg·g ^{Under the conditions of -1} and 15.0 mg·g ^-1 , the treatment situation of 7d is shown in Figure 7. The removal rate of strain 4-1 to 1 mg·g ^{-1 was} as high as 45.8%.

Example 6: Activation and cultivation of strains

1) Preparation of culture medium:

LB medium: 10g peptone, 5g yeast powder, 5g NaCl, 1000mL distilled water, pH 7.2～7.5;

2) Strain activation: first inoculate the microacidophilus Stenotrophomonas 4-1 in the above-mentioned LB medium, and activate it in a constant temperature incubator at 30°C for 24 hours;

3) Liquid culture of the strain: pick the single colony of the micro-acidophilic Stenotrophomonas strain activated in step 2) and inoculate it in the LB medium with a bottle volume of 30mL/100mL, 160r·min ^-1 , 30 Incubate with shaking at constant temperature for 24 hours to obtain a cell culture solution of the strain with a ^{concentration of 4.1×10 7} CFU·mL ^-1;

4) Preparation of the preparation: Centrifuge the cell culture solution prepared in step 3) at 10000 r·min ^-1 for 20 min at 4°C, collect the supernatant and filter it with a 0.22 μm microporous membrane. The supernatant is no Bacterial fermentation filtrate;

The sterile fermentation filtrate prepared by the above method is subjected to degradation test in actual polluted water body, and 6 samples are made respectively. The content (V/V) of the fermentation filtrate in each sample is 5%, 10%, 20%, 30%, 40%, respectively. 50%, the Cr(VI) concentration in each sample is 15mg·L ^-1 before treatment. The treatment effect of 7d and 7d is shown in Figure 9. It can be seen from Figure 9 that the amount of fermentation filtrate (V/V) is 50 %, the removal rate of 7d reaches 53.78%, the effect is excellent.

Claims

A strain that can tolerate high concentrations of chromium, characterized in that the strain is Stenotrophomonas acidaminiphila 4-1, deposited in the China Type Culture Collection, and the deposit number is CCTCC M 2019031.
The microbial agent used for the treatment of hexavalent chromium pollution is characterized in that the active ingredient is Stenotrophomonas acidaminiphila 4-1 or Stenotrophomonas acidaminiphila 4 -1 fermentation broth, the Stenotrophomonas acidaminiphila 4-1, deposited in the China Type Culture Collection, and the deposit number is CCTCC M 2019031.
The microbial agent according to claim 2, wherein the fermentation broth of Stenotrophomonas acidaminiphila 4-1 is prepared by the following method:

1) First inoculate Stenotrophomonas acidaminiphila 4-1 in the above-mentioned LB medium, and activate it in a constant temperature incubator at 30°C for 24 hours;

2) Pick the single colony activated in step 1) and inoculate it in the LB medium with a bottle volume of 30mL/100mL, 160r·min -1 , 30°C constant temperature shaking culture, to obtain a concentration of 4.1×10 7 CFU·mL -1 cell culture solution of the strain;

3) Centrifuge the cell culture solution prepared in step 2) at 10000 r·min -1 for 20 minutes at 4° C., collect the supernatant and filter it with a 0.22 μm microporous membrane. The supernatant is the sterile fermentation broth.
The strain capable of tolerating high concentrations of chromium according to claim 1, characterized in that it is used for the treatment of chromium pollution in chromium-containing wastewater, diesel fuel or combined pollution of diesel fuel and chromium.