Method for phytomicrobially remediating heavy metal contaminated soil

Abstract

The application discloses a method for repairing heavy metal contaminated soil by combining plants and microorganisms, and comprises the following steps: planting alfalfa seeds in heavy metal contaminated soil; inoculating lead and zinc copper resistant bacteria L1 into the heavy metal contaminated soil after 30 days when the seeds germinate; and harvesting the alfalfa after the growth period of the alfalfa ends. The application has a very obvious effect on adsorbing lead, zinc and copper heavy metals in the soil. The plant raw material is low in cost, the preparation process is simple and easy to operate, and the bacterial liquid is easy to culture. The application has small investment for removing heavy metals and no secondary pollution.

Description

Technical Field

[0001] This invention belongs to the field of soil heavy metal remediation technology, and relates to a method for the combined remediation of heavy metal contaminated soil by plants and microorganisms. Background Technology

[0002] Industrial activities are a major source of heavy metal pollution in soil, primarily due to waste residues and tailings left over from mining and other production activities. These waste residues and tailings contain large amounts of heavy metals. Long-term open-air dumping, through atmospheric deposition and rainwater runoff, transforms these metals into soluble components that migrate into the soil, causing heavy metal pollution in the waste dumping area and surrounding regions. Under natural conditions, plants often have close contact with soil microorganisms, and some microorganisms can even influence plant metabolism and environmental compatibility. Microbial-phytoremediation is more effective than phytoremediation or microbial remediation in removing heavy metal pollutants. Some microorganisms can directly or indirectly promote plant growth. Microbial remediation technology has advantages over physicochemical methods, such as lower cost and greater safety for the environment and consumers. Summary of the Invention

[0003] The purpose of this invention is to provide a system for adding an external carbon source to a biological treatment tank under low-temperature conditions, thereby solving the problem of reduced total nitrogen removal efficiency.

[0004] The technical solution adopted in this invention is a method for plant-microorganism co-remediation of heavy metal contaminated soil. Alfalfa seeds are planted in heavy metal contaminated soil. After the seeds germinate, lead-zinc-copper tolerant bacteria L1 are inoculated into the heavy metal contaminated soil 30 days later. The alfalfa is harvested after the end of its growth period.

[0005] Furthermore, the lead-, zinc-, and copper-resistant bacterium L1 was deposited on October 22, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 23539.

[0006] Furthermore, the specific steps are as follows:

[0007] Step 1. Pretreatment of alfalfa seeds;

[0008] Step 2. Screening for lead-, zinc-, and copper-resistant bacteria L1;

[0009] Step 3. Pre-treat the heavy metal contaminated soil by irrigating it with 200 mL of water every two days and incubating it for 14 days;

[0010] Step 4. Plant alfalfa in heavy metal contaminated soil and set up a control experimental group;

[0011] Step 5. Add the bacterial solution inoculated with lead-zinc-copper tolerant bacteria L1 to the heavy metal contaminated soil where alfalfa is planted to establish a symbiotic system;

[0012] Step 6. Harvest the alfalfa after its growing season is over.

[0013] Further, step 1 specifically involves taking 25g of alfalfa seeds and placing them in 50mL of 10% hydrogen peroxide solution for disinfection for 10 minutes.

[0014] Further, step 2 specifically involves taking 5g of soil and adding it to 45mL of 0.9% sterile physiological saline, shaking it on a shaker for 1 hour, allowing it to stand, and then taking 5mL of the supernatant and transferring it to Pb. 2+ (The compound is Pb(NO3)2), Zn 2+ (compound ZnCl2) and Cu 2+ The culture was incubated in 45 mL of a medium containing 50 mg / L Cu₂SO₄ at 30 °C with shaking for 48 h; 0.1 mL of the bacterial culture was then serially diluted to 10⁻⁶. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Take 10 of the bacterial suspension. -4 -10 -6 0.2 mL of the bacterial suspension at a certain concentration was spread on a solid culture medium containing heavy metals and incubated in a 30°C incubator. The growth of the bacteria was observed, and lead-zinc-copper resistant bacteria L1 were obtained.

[0015] Furthermore, the control group in step 4 specifically includes:

[0016] 1. Soil free from heavy metal contamination (CK0);

[0017] 2. Heavy metal contaminated soil T0

[0018] 3. Soil free of heavy metal contamination for planting alfalfa (CK1);

[0019] 4. Heavy metal content in soil used for planting alfalfa (T1);

[0020] 5. Soil H0 free of heavy metal pollution, planted with alfalfa and supplemented with lead-, zinc-, and copper-tolerant bacteria L1 solution;

[0021] 6. Heavy metal contaminated soil H1, planted with alfalfa and supplemented with lead-, zinc-, and copper-tolerant bacteria L1 solution.

[0022] Furthermore, step 5 also includes the cultivation of lead-zinc-copper resistant bacteria L1, specifically: the lead-zinc-copper resistant bacteria L1 is inoculated into LB liquid medium and cultured at 30°C and 140 rpm for 8-10 h with shaking. Its OD value is measured at 600 nm to ensure that its OD600 is between 0.5 and 1, thus obtaining the seed culture of lead-zinc-copper resistant bacteria L1; then, the seed culture of lead-zinc-copper resistant bacteria L1 is inoculated into 1 L of medium at an inoculation rate of 2%, and cultured at 30°C and 140 rpm for 48 h to obtain the fermentation broth of lead-zinc-copper resistant bacteria L1.

[0023] Furthermore, in step 5, 200 mL of the lead-, zinc-, and copper-resistant bacteria L1 fermentation broth is added each time.

[0024] Furthermore, in step 4, after the alfalfa has grown for 30 days, 25 vigorous plants that are free from pests and diseases and are similar in size and maturity are selected and retained, while the excess plants are removed.

[0025] The beneficial effects of this invention are:

[0026] This invention exhibits significant effects in adsorbing heavy metals such as lead, zinc, and copper from soil. The plant-based raw materials are inexpensive, the preparation process is simple and easy to operate, and the bacterial culture is readily available. The removal of heavy metals requires minimal investment and produces no secondary pollution. Attached Figure Description

[0027] Figure 1 This is a graph showing the chlorophyll content of alfalfa leaves in each treatment group of this invention.

[0028] Figure 2 This is a graph showing the content of reduced glutathione in alfalfa leaves of each treatment group in this invention.

[0029] Figure 3 This is a graph showing the malondialdehyde content in alfalfa leaves of each treatment group in this invention.

[0030] Figure 4 This is a graph showing the soluble sugar content of alfalfa leaves in each treatment group of the present invention.

[0031] Figure 5 This is a graph showing the peroxidase activity of alfalfa leaves in each treatment group of the present invention;

[0032] Figure 6 This is a graph showing the catalase activity of alfalfa leaves in each treatment group of the present invention.

[0033] Figure 7 This is a graph showing the ascorbate peroxidase activity of alfalfa leaves in each treatment group of the present invention;

[0034] Figure 8 This is a graph showing the superoxide dismutase activity in alfalfa leaves of each treatment group in this invention. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0036] like Figure 1 As shown: A method for plant-microbe co-remediation of heavy metal contaminated soil involves planting alfalfa seeds in heavy metal contaminated soil, and 30 days after seed germination, inoculating the heavy metal contaminated soil with lead-zinc-copper tolerant bacteria L1, and harvesting the alfalfa after the end of its growth period.

[0037] Lead-zinc-copper tolerant bacteria L1 were isolated and purified from non-rhizosphere soil in Bijia Mountain mining area, Cheng County, Longnan City, Gansu Province.

[0038] The lead-zinc-copper resistant bacterium L1 was deposited at the China General Microbiological Culture Collection Center on October 22, 2021, with accession number CGMCC NO: 23539.

[0039] The specific steps are as follows:

[0040] Step 1. Pretreatment of alfalfa seeds;

[0041] Step 2. Screening for lead-, zinc-, and copper-resistant bacteria L1;

[0042] Step 3. Pre-treat the heavy metal contaminated soil by irrigating it with 200 mL of water every two days and incubating it for 14 days;

[0043] Step 4. Plant alfalfa in heavy metal contaminated soil and set up a control experimental group;

[0044] Step 5. Add the bacterial solution inoculated with lead-zinc-copper tolerant bacteria L1 to the heavy metal contaminated soil where alfalfa is planted to establish a symbiotic system;

[0045] Step 6. Harvest the alfalfa after its growing season is over.

[0046] Step 1 specifically involves taking 25g of alfalfa seeds and placing them in 50mL of 10% hydrogen peroxide solution for disinfection for 10 minutes.

[0047] Step 2 specifically involves taking 5g of soil and adding it to 45mL of 0.9% sterile physiological saline solution, placing it on a shaker and shaking for 1 hour, allowing it to stand, and then taking 5mL of the supernatant and transferring it to Pb. 2+ (The compound is Pb(NO3)2), Zn 2+ (compound ZnCl2) and Cu 2+ The culture was incubated in 45 mL of a medium containing 50 mg / L Cu₂SO₄ at 30 °C with shaking for 48 h; 0.1 mL of the bacterial culture was then serially diluted to 10⁻⁶. -1 10 -2 10-3 10 -4 10 -5 10 -6 Take 10 of the bacterial suspension. -4 -10 -6 0.2 mL of the bacterial suspension at a certain concentration was spread on a solid culture medium containing heavy metals and incubated in a 30°C incubator. The growth of the bacteria was observed, and lead-zinc-copper resistant bacteria L1 were obtained.

[0048] The control group in step 4 is specifically as follows:

[0049] 1. Soil free from heavy metal contamination (CK0);

[0050] 2. Heavy metal contaminated soil (T0);

[0051] 3. Soil free of heavy metal contamination for planting alfalfa (CK1);

[0052] 4. Heavy metal content in soil used for planting alfalfa (T1);

[0053] 5. Soil H0 free of heavy metal pollution, planted with alfalfa and supplemented with lead-, zinc-, and copper-tolerant bacteria L1 solution;

[0054] 6. Heavy metal contaminated soil H1, planted with alfalfa and supplemented with lead-, zinc-, and copper-tolerant bacteria L1 solution.

[0055] Step 5 further includes the cultivation of lead-zinc-copper resistant bacteria L1, specifically: L1 is inoculated into LB liquid medium and cultured at 30°C with shaking at 140 rpm for 8–10 h. Its OD value is measured at 600 nm to ensure its OD600 is between 0.5 and 1, thus obtaining the L1 seed culture. Then, the L1 seed culture is inoculated into 1 L of medium at a 2% inoculation rate and cultured at 30°C with shaking at 140 rpm for 48 h to obtain the L1 fermentation broth. The LB liquid medium contains 10 g / L tryptone. -1 5g·L yeast extract -1 10 g·L of sodium chloride -1 15-20 g / L of agar -1 pH 7.2–7.4.

[0056] In step 5, add 200 mL of the lead-zinc-copper resistant bacteria L1 fermentation broth each time.

[0057] In step 4, after the alfalfa has grown for 30 days, select 25 vigorous plants that are free from pests and diseases and are similar in size and maturity to keep, and remove the excess plants.

[0058] Studies have found that certain bacteria can produce substances that promote plant growth and development, including indoleacetic acid (IAA), siderophores, ACC deaminase, and phosphate-solubilizing substances. Indoleacetic acid is a signaling substance secreted during plant growth to regulate cell growth, increasing cell volume and weight, promoting cell division and differentiation, and regulating physiological functions such as rooting. ACC deaminase degrades the ethylene precursor ACC, thereby reducing ethylene levels during plant growth and contributing to plant health. Siderophores influence plant health by promoting nutrient absorption, enhancing plant resistance, and inhibiting pathogen growth. Finally, phosphate-solubilizing substances produced by microorganisms effectively increase the utilization of available phosphorus, and high-quality phosphorus is beneficial to plant growth.

[0059] If lead-zinc-copper tolerant bacteria L1 can produce growth-promoting substances, it can promote the growth of alfalfa. However, when measuring physiological indicators, it is impossible to determine whether the changes in the indicators are due to the effect of lead-zinc-copper tolerant bacteria L1 on the plant or the effect of heavy metal soil on the plant. Therefore, the determination of growth-promoting substances was designed.

[0060] Quantitative determination of growth-promoting substances in lead-zinc-copper resistant bacteria L1:

[0061] (1) Determination of iron carrier content

[0062] Lead-, zinc-, and copper-resistant bacteria L1 were inoculated into KMB liquid medium (15 mL glycerol, 5 g / L casein amino acids). -1 K2HPO4 2.5g·L -1 MgSO4·H2O 2.5 g·L -1 The culture was incubated overnight (approximately 18 hours) at 28°C and 100 rpm. The bacterial culture was transferred to centrifuge tubes and centrifuged at 7000 rpm for 10 minutes. The mixture was filtered, and the supernatant was collected. The supernatant was diluted 10-fold with deionized water and mixed with an equal volume of CAS (chromium azure S) detection solution. After reacting for 60 minutes, the absorbance (A) was measured at 630 nm. The absorbance (Ar) obtained by mixing a blank KMB medium diluted 10-fold with deionized water and an equal volume of CAS detection solution served as a control. The A / Ar ratio represents the relative content of siderophores in the sample; a lower value indicates a higher siderophore content.

[0063] Solution A: Dissolve 0.079 g of CAS in 50 mL of deionized water, then add 10 mL of 1 mM FeCl3 solution (containing 10 mM HCl);

[0064] Solution B: Dissolve 0.069g of hexadecyltrimethylammonium bromide (HDTMA) in 40mL of deionized water; Solution C: Gently mix Solution A into Solution B and shake gently to obtain Solution C: CAS blue detection solution, sterilize at 121℃ for 15 minutes.

[0065] (2) Determination of indoleacetic acid (IAA)

[0066] IAA Standard Curve Construction: (IAA concentration from 0 to 35 μg / mL) Construction: Prepare IAA solutions with a concentration gradient from 0 to 35 μg / mL according to the table below, where the concentration of the IAA standard solution is 35 μg / mL (weigh 3.5 mg (0.0035 g) of IAA standard and dissolve it in 1000 mL of distilled water).

[0067] Table 1 Preparation of IAA standard solutions

[0068]

[0069] Add 4 mL of reagent B (Reagent B: Mix 10 mL of 0.5 mol / L FeCl3 solution with 500 mL of 35% perchloric acid, shake well, and store in a brown reagent bottle; 2 mL of this reagent should be added to 1 mL of test solution) to each 2 mL IAA solution prepared above, shake well, and let stand for 30 min at 30℃ in the dark to allow the color reaction to occur. Then measure the absorbance value of each concentration of IAA solution at 530 nm and plot a standard curve.

[0070] Assay for IAA production capacity of the strain: Lead-, zinc-, and copper-resistant bacteria L1 were inoculated onto a substrate supplemented with 0.5 mg / mL... -1 The culture medium was incubated in L-tryptophan liquid medium at 28°C with shaking for 48 h. 2 mL of the culture medium was centrifuged at 10000 r / min for 15 min. For each 1 mL of supernatant after centrifugation, 2 mL of Salkowski's reagent (10 mL of 0.5 mol / L FeCl3 solution and 500 mL of 35% perchloric acid, mixed well before use and stored in the dark) was added. The mixture was then placed in a dark place at room temperature for 30 min for color development, and the absorbance was measured at 530 nm. Uninoculated medium was used as a control group, and absorbance values ​​at corresponding wavelengths were measured using pure IAA (concentration gradients of 0, 7, 14, 21, 28, and 35 μg mL⁻¹) for calculation.

[0071] (3) Quantitative determination of phosphorus-soluble substances

[0072] Accurately weigh 0.4390 g of potassium dihydrogen phosphate (analytical grade), dried at 105℃ for 2 h, into a phosphorus standard stock solution. Dissolve in water, add 5 mL of concentrated sulfuric acid, and then dilute to 1000 mL with water. This solution contains 100 mg / L of phosphorus and can be used long-term in a refrigerator. Phosphorus standard solution: Pipette 5 mL of the phosphorus standard stock solution into a 100 mL volumetric flask and dilute to volume with water. This solution has a short shelf life and should be prepared fresh before use. Phosphorus working curve preparation: Pipette 0, 1, 2, 3, 4, 5, and 6 mL of the phosphorus standard solution into 50 mL volumetric flasks, dilute to 30 mL with distilled water, add mL of molybdenum antimony colorimetric reagent, shake well, and dilute to volume. This yields a series of standard solutions with concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg / L. Simultaneously compare the absorbance of these solutions with the test solution at 700 nm and plot the standard curve.

[0073] Determination of the strain's ability to produce phosphorus-soluble substances: The test bacteria were cultured in the original culture medium for 18-24 hours, centrifuged to collect the bacterial cells, washed twice with sterile water, and inoculated into inorganic phosphorus liquid culture medium. Inoculation and non-inoculation treatments were included, with three replicates for each treatment. The culture was carried out at 28℃ with shaking for 3 days. The fermentation broth was collected, centrifuged at 12000 rpm for 5 minutes, and the supernatant was used to determine the available phosphorus content.

[0074] Table 2 Inorganic Phosphorus Culture Medium Formulation

[0075]

[0076]

[0077] The preparation method of the above-mentioned antimony-molybdenum sulfate stock solution is as follows: Measure 126 mL of concentrated sulfuric acid and slowly add it to 400 mL of water while stirring continuously and cooling. Separately, dissolve 10 g of finely ground ammonium molybdate (GB657) in 300 mL of water at approximately 60°C and cool. Then, slowly pour the sulfuric acid solution into the ammonium molybdate solution. Add 100 mL of 0.5% potassium antimony tartrate solution, cool, dilute with water to 1000 mL, shake well, and store in a brown reagent bottle. This stock solution also contains 1% ammonium molybdate and 2.25 mol / L sulfuric acid.

[0078] Preparation of molybdenum-antimony anti-color developing agent: Weigh 1.5g of ascorbic acid and dissolve it in 100mL of molybdenum-antimony sulfate stock solution.

[0079] ACC deaminase content determination method

[0080] Standard curve: Prepare 100 mM α-butanone using 0.1 M pH 8.5 Tris-HCl buffer and store at 4 °C. Dilute the α-butanone solution to 10 mM before use. Prepare a gradient dilution of α-butanone (0.1–1.0 μmol). Add 300 μL of 0.2% 2,4-dinitrophenylhydrazine (dissolved in 2 mol / L HCl, with a mass concentration of 2 g / L) to each tube, mix well, and incubate at 30 °C for 30 min. Add 2 mL of 2 M NaOH solution for color development and measure OD540. Plot a standard curve with α-butanone concentration on the x-axis and absorbance on the y-axis.

[0081] ACC deaminase content determination of the strain: The screened strain was inoculated into 30 mL of DF liquid medium and cultured in the dark with shaking at 30℃ and 200 rpm for 12 h. The bacterial suspension was centrifuged at 4℃ and 8000 rpm for 10 min, and the bacterial pellet was collected. The bacterial suspension was resuspended in 30 mL of ADF liquid medium and then cultured with shaking at 30℃ and 200 rpm for 24 h to induce ACC deaminase activity. The bacterial suspension was centrifuged at 4℃ and 8000 rpm for 10 min, the bacterial pellet was collected, resuspended in 5 mL of 0.1 mol / L Tris-HCl buffer (pH 7.6), and centrifuged at 4℃ and 8000 rpm for 10 min before collecting the pellet. Add 1 mL of 0.1 mol / L Tris-HCl buffer (pH 7.6) to the bacterial pellet to suspend the bacterial cells, then transfer to a 1.5 mL centrifuge tube and centrifuge at 16,000 rpm for 5 min. Resuspend the bacterial cells in 600 μL of 0.1 mol / L Tris-HCl buffer (pH 8.5), add 30 μL of toluene and vortex for 30 s to disrupt the bacterial cells.

[0082] Take 200 μL of the lysed bacterial suspension, add 20 μL of 0.5 mol / L ACC solution, mix well, and incubate at 30°C for 15 min. Then add 1 mL of 0.56 mol / L HCl solution, mix well, and centrifuge at 16000 rpm for 5 min. Take 1 mL of the supernatant, add 800 μL of 0.56 mol / L HCl solution, mix well, and then add 300 μL of 2 g / L 2,4-dinitrophenylhydrazine solution. Incubate at 30°C for 30 min. Add 2 mL of 2 mol / L NaOH solution for color development and measure OD540. Use sterile water instead of bacterial suspension as a blank control. Substitute the OD540 value of the sample into the standard curve to calculate the corresponding α-butanone content. The unit enzyme activity of ACC deaminase refers to the activity of forming 1 μmol of α-butanone per minute in the enzyme assay system.

[0083] Table 3 Formulations of Components 1 and 2 in DF Culture Medium

[0084]

[0085] After weighing according to the above formula, dissolve component one in 100 mL of sterile distilled water and component two in 10 mL of sterile distilled water, and store at low temperature for later use. Take 0.5 mL of component one and 0.2 mL of component two and add them to an Erlenmeyer flask, then add the following reagents, adjust the pH to 7.0, and bring the volume to 1000 mL. Sterilize at 121°C for 21 min:

[0086] Table 4 DF Culture Medium Formulation

[0087]

[0088] ADF medium: Replace (NH4)2SO4 in DF medium with ACC. The ACC stock solution concentration is 0.5M, and the addition amount in ADF medium is 6 mL / L. For bacterial nutrient agar medium, DF solid medium, and ADF solid medium, simply add 1.8% (w / w) agar to the above formula.

[0089] Table 5. Ability of lead-zinc-copper resistant bacteria L1 to produce plant growth-promoting substances.

[0090]

[0091] Based on the results of testing for four growth-promoting substances (side carrier, indoleacetic acid, phosphate-solubilizing substances, and ACC deaminase), it was found that the lead-zinc-copper-tolerant bacterium L1, apart from producing a trace amount of phosphate-solubilizing substances (0.172 mg / L), lacked the ability to produce the other three growth-promoting substances (side carrier, indoleacetic acid, and ACC deaminase). The results indicate that the lead-zinc-copper-tolerant bacterium L1 cannot synthesize certain substances that can affect plant growth and development. Furthermore, it shows that L1 bacteria have no effect on plants, and the changes in plant physiological indicators only reflect the impact of heavy metal pollution in the soil on plants.

[0092] Changes in plant physiological indicators during restoration experiments

[0093] Eight physiological indicators in the plant were measured during the repair experiment, including chlorophyll, malondialdehyde, soluble sugar, reduced glutathione, peroxidase activity, catalase activity, ascorbate peroxidase, and superoxide dismutase.

[0094] Chlorophyll content determination method: Take 0.2g of sample, add 10mL of 95% ethanol, grind quickly and thoroughly, and measure the absorbance at 663nm and 645nm respectively. Calculate the chlorophyll content according to the formula:

[0095] C a =12.7A663 -2.59A 645

[0096] C b =22.9A 645 -4.67A 663

[0097] C a +Cb=20.3A 645 -8.04A 663

[0098] C a Chlorophyll a content; C b Chlorophyll b content; C a +C b Total chlorophyll

[0099] Method for determining malondialdehyde (MDA) content: Weigh 1g of the material, cut it into small pieces, add 2mL of 10% trichloroacetic acid and a small amount of quartz sand to grind it, then add 8mL of 10% trichloroacetic acid and grind it thoroughly. Centrifuge at 4000r / min for 10min, take 2mL of the extract, add 2mL of 0.6% thiobarbituric acid, mix well, react in a boiling water bath for 15min, cool rapidly, and centrifuge. Use 0.6% TBA solution as a blank control group.

[0100] The OD values ​​at 532 nm, 450 nm and 600 nm were measured using the supernatant.

[0101] Calculation of malondialdehyde content:

[0102]

[0103] MDA content (umol / gFW); A 532 A 600 A 450 V represents the absorbance values ​​of the extract at 532 nm, 600 nm, and 450 nm; V: volume of the extract; W: fresh weight of the plant leaves.

[0104] Determination of soluble sugar content: The anthrone method is used to determine the soluble sugar content in plants.

[0105] (1) Anthrone reagent: Dissolve 0.2g of anthrone in 100mL of 80% (V / V) sulfuric acid. The sulfuric acid should be prepared and used on the same day.

[0106] (2) Standard glucose solution (0.1 mg / mL): A few drops of toluene can be added as a preservative;

[0107] (3) Preparation of the standard curve: Take 5 dry test tubes and add 0, 0.1, 0.2, 0.4, and 0.8 mL of standard sugar solution to each tube respectively, and then bring the volume to 1 mL with distilled water. Add 4 mL of anthrone reagent to each tube and shake to mix. Boil in a water bath for 10 min, let stand at room temperature for 10 min, zero the tube with the solution from test tube No. 1, and measure A620 colorimetrically (simultaneously measure the sample). Construct a standard curve with the concentration of the standard sugar solution as the x-axis and the absorbance as the y-axis.

[0108]

[0109] (4) Determination of soluble sugars in the sample

[0110] Weigh 0.1g of plant sample, cut it into small pieces, and place it in a test tube. Add 5mL of distilled water to each tube, then boil in a water bath for 30 minutes and dilute to a volumetric flask with water to a final volume of 25mL. Take 0.5mL of the extract and place it in a test tube. Add 1.5mL of distilled water, 0.5mL of ethyl anthrone acetate, and 5mL of concentrated sulfuric acid. Boil in a water bath for 1 minute and then cool. Use the standard curve blank solution as the blank control.

[0111] calculate:

[0112] Sample sugar content (%) = (c × v1 × D / W × v2 × 10) 6 )×100

[0113] Sugar content (ug) obtained from the C-standard curve

[0114] V1 - Total extraction volume (mL)

[0115] V2 - Total volume measured (mL)

[0116] D - Dilution factor

[0117] W - Sample mass (g)

[0118] Extract 0.2-0.3g of the leaves with enzyme solution, add a small amount of 5mL of pH7.0 phosphate buffer, grind into a homogenate, add buffer and transfer to a 10mL centrifuge tube, centrifuge at 4000rpm for 15min.

[0119] Determination of plant catalase activity

[0120] Take two 10mL test tubes, one for testing and the other for blank.

[0121] Table 6 Sample Solution Preparation Table for H2O2 Determination by Ultraviolet Absorption Method

[0122]

[0123] After preheating at 25℃, add 0.5 mL of 0.1 mol / L H2O2 to each tube. After each tube is added, quickly pour the solution into a quartz cuvette and measure the absorbance at 240 nm. Take a reading every 20 seconds for a total of 3 minutes. Each 0.01 μL is considered one unit of enzyme activity (u).

[0124]

[0125] Among them: A 240 V represents the decrease in absorbance. t V represents the total volume of the enzyme extract (mL); s The volume of supernatant was drawn to determine absorbance; FW represents the fresh weight of the plant leaves.

[0126] Determine the activity of plant peroxidase POD.

[0127] Enzyme extraction: Weigh 0.2–0.3 g of leaves, place them in a mortar, add an appropriate amount of quartz sand and 2 mL of 50 mmol pH 7.0 phosphate buffer, grind, then add 8 mL of phosphate buffer and transfer to a centrifuge tube. Centrifuge at 4000 rpm for 15 minutes. The 3 mL reaction solution system includes: 50 mmol pH 7.0 phosphate buffer, 2% hydrogen peroxide, and 1% guaiacol.

[0128] Table 7. Reaction system for determining CAT activity

[0129]

[0130] Absorbance was measured at 470 nm, and one unit of enzyme activity (u / g·min) was defined as a change in absorbance of 0.01 μL per minute.

[0131]

[0132] Where: (x-x0) is the decrease in absorbance; V t V represents the total volume of the enzyme extract (mL); s The volume of supernatant was drawn to determine absorbance; FW represents the fresh weight of the plant leaves.

[0133] Determine the activity of plant peroxidase SOD.

[0134] The SOD activity of plants under different treatments was determined using the nitroblue tetrazolium reduction method. 0.2 g of fresh leaves were weighed and placed in a pre-cooled mortar, and 4 mL of pre-cooled 50 mmol / L phosphate buffer (pH 7.8) was added and the mixture was ground. After centrifugation at 1000 r / min for 20 minutes at 4℃, the supernatant was collected to obtain the crude enzyme extract.

[0135] Using the NBT method, reagents were added to the test tubes sequentially:

[0136] Table 8 Reaction system for NBT determination of SOD activity

[0137]

[0138]

[0139] After adding the reagents, place test tube No. 7 in the dark. Place the test tubes (1, 2, 3) and the light control tubes (4, 5, 6) in a light incubator with a light intensity of 4000 lx and react accurately for 20 min. After the reaction is completed, immediately remove them and treat them with a black plastic bag to terminate the reaction. Use the dark control tube as a blank control. Use an ultraviolet spectrophotometer to measure the absorbance of each test tube at 560 nm compared to the light control tube, and calculate the SOD enzyme activity.

[0140]

[0141] Among them: SOD activity (U / g); A c A represents the absorbance value of the control group with light-induced reaction. s To process the absorbance values ​​of the photoreactive group; V T Vs is the total volume of the extract (mL); Vs is the volume of the crude enzyme solution during the determination (mL); W is the fresh weight of the leaf sample (g).

[0142] Determine the activity of plant ascorbate peroxidase APX.

[0143] Weigh 0.5g of leaf material, add extraction solution (50mmol / L pH7.0 phosphate buffer with 1mmol / L EDTA-Na2), grind into a homogenate, centrifuge at 150000r / min for 15min, and bring the supernatant to a final volume of 5mL. Take a portion of the supernatant, dilute it appropriately, and use it for enzyme activity assay.

[0144] To a 3 mL reaction system, add 1.8 mL of 50 mmol / L pH 7.0 phosphate buffer, 0.1 mL of 15 mmol / L AsA, 0.1 mL of enzyme solution, and 1 mL of 0.3 mmol / L H₂O₂. Using phosphate buffer (without enzyme solution) as a blank, measure the change in OD₂₉₀ over a specific time period (3 min). A decrease of 0.01 oz in OD₂₉₀ within 1 min is defined as one unit of enzyme activity (1 U).

[0145] Result calculation:

[0146] APX activity (U·min) -1 ·g -1 FW)=ΔA 290 ×V1 / (0.01×V2×t×W)

[0147] In the formula: ΔA290 - the change in OD290 during the reaction time; V1 - the total volume of the extracted enzyme solution (mL); V2 - the volume of the enzyme solution used for determination (mL); t - the reaction time (min); W - the fresh weight of the sample (g). Determination of reduced glutathione (GSH) content.

[0148] Creating the GSH standard curve

[0149] Standard GSH solutions with concentrations of 0, 0.02, 0.04, 0.06, 0.08, 0.1, and 0.12 mmol / L were prepared. 0.25 mL of each standard solution was pipetted into each tube, and 2.6 mL of 150 mmol / L NaH₂PO₄ (pH 7.7) was added. After mixing thoroughly, 0.15 mL of DTNB reagent was added to each tube, and the mixture was shaken well. The reaction was incubated at 30°C for 5 min, and the absorbance at OD₄¹² was measured. A standard curve was constructed by plotting the results with GSH concentration on the x-axis and optical density on the y-axis.

[0150] Weigh 0.5g of leaf material, add extraction solution (50mmol / L pH7.0 phosphate buffer with 1mmol / L EDTA-Na2), grind into a homogenate, centrifuge at 150000r / min for 15min, and bring the supernatant to a final volume of 5mL. Take a portion of the supernatant, dilute it appropriately, and use it for enzyme activity assay.

[0151] Take 0.25 mL of the extract, add 2.6 mL of 150 mmol / L Na₂HPO₄ (pH 7.7) and 0.15 mL of DTNB reagent to each, and use phosphate buffer instead of DTNB reagent as a blank. After shaking well, incubate at 30 °C for 5 min, and measure the absorbance at 412 nm. Calculate the GSH content (ug / gFW) of the sample according to the standard curve.

[0152] Depend on Figure 1 It was found that, compared with the control soil free of heavy metals (CK1), the chlorophyll content of the heavy metal-contaminated soil planted with alfalfa (T1), the heavy metal-free soil planted with alfalfa and supplemented with L1 (H0), and the heavy metal-contaminated soil planted with alfalfa and supplemented with L1 (H1) decreased by 4.51%, 20.62%, and 18.03%, respectively. Figure 2 It can be seen that, compared with CK1, the GSH content of T1 and H1 decreased by 13.49% and 23.38% respectively; while the GSH content of H0 increased by 65.37% compared with CK1.

[0153] Depend on Figure 3 It can be seen that the malondialdehyde (MDA) contents of T1, H0, and H1 all decreased compared to CK1, decreasing by 28.03%, 23.21%, and 0.11%, respectively. Figure 4It can be seen that the soluble sugar content of T1 and H0 is higher than that of CK1, increasing by 8.16% and 7.13% respectively, while the soluble sugar content of H1 increases by 25.53% compared to CK1. Figure 5 It can be seen that the peroxidase (POD) activities of T1 and H0 increased by 64.64% and 53.58% respectively compared with CK1, while the POD activity of H1 decreased by 22.82% compared with CK1. Figure 6 It can be seen that the changes in catalase activity are opposite to those in peroxidase activity. The catalase (CAT) activities of T1 and H0 decreased by 39.03% and 55.60% respectively compared to CK1, while the POD activity of H1 increased by 5.35% compared to CK1. Figure 7 It can be seen that the ascorbate peroxidase (APX) levels in T1 and H1 decreased by 26.78% and 67.14% respectively compared to CK1, while H0 increased by 32.10%. Figure 8 It can be seen that the superoxide dismutase (SOD) levels of T1, H0, and H1 were all higher than those of CK1, increasing by 9.36%, 34.84%, and 31%, respectively.

[0154] Depend on Figures 1 to 8 It can be seen that, in the heavy metal treatment group, compared with the sterile group (T0), the levels of chlorophyll, GSH, soluble sugar, POD, and APX decreased by 14.16%, 11.43%, 31.15%, 53.12%, and 55.12%, respectively, when bacterial L1 was added (H1). Conversely, in the heavy metal treatment group, compared with the sterile group (T0), the levels of malondialdehyde (MDA), CAT, and SOD activity increased by 38.78%, 72.78%, and 19.78%, respectively, when bacterial L1 was added (H1).

[0155] Changes in plant physiological indicators indicate that, in the process of using plant microorganisms to remediate heavy metal-contaminated soil, the addition of microorganisms also has a certain influence and effect on the physiology of plants under heavy metal pollution stress.

[0156] The removal of heavy metals by plants and lead-, zinc-, and copper-tolerant bacteria L1

[0157] After the remediation experiment, the heavy metal content in various parts of the plants and the soil was measured, and the plant enrichment coefficient and translocation coefficient were calculated. The accumulation and transport capacity of heavy metals in plants directly affects the plant's absorption and tolerance to heavy metals. The enrichment coefficient and translocation coefficient are important indicators for evaluating the absorption and accumulation of heavy metal pollutants by plants.

[0158] The enrichment coefficient = heavy metal content in the plant (mg / kg) / heavy metal content in the soil (mg / kg) reflects the degree of migration of heavy metal elements and the plant's ability to enrich heavy metal elements.

[0159] The translocation coefficient = heavy metal content in the aboveground parts of the plant (mg / kg) / heavy metal content in the underground parts of the plant (mg / kg) reflects the ability of plant roots to transport heavy metal ions from underground to aboveground.

[0160] In this experiment, the heavy metal content of each part of the plant (roots, stems, and leaves) and the soil were all tested at the Institute of Soil and Fertilizer, Fujian Academy of Agricultural Sciences.

[0161] Table 9 Heavy metal content in plants and soil

[0162]

[0163] According to Table 9, the Pb content in alfalfa stems and leaves ranged from 27.93 to 78.68 mg / kg; the Pb content in alfalfa roots ranged from 201.80 to 214.66 mg / kg; the Zn content in alfalfa stems and leaves ranged from 178.31 to 244.27 mg / kg; the Zn content in alfalfa roots ranged from 142.48 to 155.46 mg / kg; the Cu content in alfalfa stems and leaves ranged from 8.34 to 10.17 mg / kg; the Cu content in alfalfa roots ranged from 26.69 to 32.19 mg / kg. In alfalfa, heavy metals Pb and Cu are mainly concentrated in the roots, while heavy metal Zn is mainly concentrated in the leaves.

[0164] Compared with the aseptic addition (T1), the Pb content in the roots increased by 6.37%; the Pb, Zn, and Cu contents in the stems increased by 33.85%, 28.39%, and 20.33%, respectively; and the Cu content in the leaves increased by 22.00%. Based on the heavy metal content in the soil, it can be seen that, compared with the aseptic group (T1), the addition of lead-zinc-copper-tolerant bacteria L1 effectively reduced the lead and zinc content in the soil, with Pb and Zn decreasing by 15.22% and 21.80%, respectively.

[0165] Table 10. Accumulation coefficients and biotransfer coefficients of different heavy metals after 5 months of planting on heavy metal-contaminated plants.

[0166]

[0167] According to Table 10, in the experiment of remediating heavy metal contaminated soil, the addition of bacteria L1 significantly improved the enrichment coefficients of Pb and Zn in alfalfa. The Pb enrichment coefficient increased from 2.826 to 3.360, and the Zn enrichment coefficient increased from 2.817 to 3.431. This indicates that the addition of lead-, zinc-, and copper-tolerant bacteria L1 greatly improved the enrichment capacity of alfalfa for Zn and Cu. In addition, lead-, zinc-, and copper-tolerant bacteria L1 also increased the translocation coefficients of Zn and Cu (from 1.176 and 0.548 to 1.352 and 0.569, respectively), indicating that the addition of lead-, zinc-, and copper-tolerant bacteria L1 greatly improved the absorption and translocation capacity of alfalfa for Zn and Cu.

Claims

1. A method for plant-microorganism co-remediation of heavy metal contaminated soil, characterized in that, Alfalfa seeds were planted in heavy metal-contaminated soil. Thirty days after germination, lead-, zinc-, and copper-tolerant bacteria L1 were inoculated into the heavy metal-contaminated soil. The alfalfa was harvested after the end of its growth period. The heavy metal-contaminated soil refers to soil contaminated with Pb and / or Zn. The lead-, zinc-, and copper-tolerant bacteria L1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO: 23539.

2. The method for plant-microorganism co-remediation of heavy metal contaminated soil according to claim 1, characterized in that, The specific steps are as follows: Step 1. Pretreatment of alfalfa seeds; Step 2. Screening for lead-, zinc-, and copper-resistant bacteria L1; Step 3. Pre-treat the heavy metal contaminated soil by irrigating it with 200 mL of water every two days and incubating it for 14 days; Step 4. Plant alfalfa in heavy metal contaminated soil and set up a control experimental group; Step 5. Add the bacterial solution inoculated with lead-zinc-copper tolerant bacteria L1 to the heavy metal contaminated soil where alfalfa is planted to establish a symbiotic system; Step 6. Harvest the alfalfa after its growing season is over.

3. The method for plant-microorganism co-remediation of heavy metal contaminated soil according to claim 2, characterized in that, Step 1 specifically involves taking 25g of alfalfa seeds and placing them in 50mL of 10% hydrogen peroxide solution for disinfection for 10 minutes.

4. The method for plant-microorganism co-remediation of heavy metal contaminated soil according to claim 2, characterized in that, Step 2 specifically involves taking 5g of soil and adding it to 45mL of 0.9% sterile physiological saline solution, placing it on a shaker and shaking for 1 hour, allowing it to stand, and then taking 5mL of the supernatant and transferring it to Pb. 2+ Zn 2+ and Cu 2+ The Pb was cultured in 45 mL of a 50 mg / L medium at 30 °C with shaking for 48 h. 2+ The Zn from compound Pb(NO3)2 2+ From compound ZnCl2, the Cu 2+ Derived from compound Cu2SO4; 0.1 mL of bacterial culture was serially diluted to 10⁻⁶. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Take 10 of the bacterial suspension. -4 -10 -6 0.2 mL of bacterial suspension at a certain concentration was spread onto a solid culture medium containing heavy metals and incubated in a 30°C incubator. The growth of the bacteria was observed, and lead-zinc-copper resistant bacteria L1 were obtained.

5. The method for plant-microorganism co-remediation of heavy metal contaminated soil according to claim 2, characterized in that, The control group in step 4 is specifically as follows: CK0: Soil free from heavy metal contamination; Heavy metal contaminated soil T0; CK1, a soil free of heavy metal contamination used for planting alfalfa; T1, a soil containing heavy metals in alfalfa cultivation; Soil H0 free of heavy metal pollution, planted with alfalfa and supplemented with lead-zinc-copper resistant bacteria L1 solution; Soil H1 contaminated with heavy metals, planted with alfalfa and supplemented with lead-, zinc-, and copper-tolerant bacteria L1.

6. The method for plant-microorganism co-remediation of heavy metal contaminated soil according to claim 2, characterized in that, Step 5 also includes the cultivation of lead-zinc-copper resistant bacteria L1, specifically: the lead-zinc-copper resistant bacteria L1 is inoculated into LB liquid medium and cultured at 30°C and 140 rpm for 8-10 h with shaking. Its OD value is measured at 600 nm to ensure that its OD600 is between 0.5 and 1, thus obtaining the seed culture of lead-zinc-copper resistant bacteria L1; then, the seed culture of lead-zinc-copper resistant bacteria L1 is inoculated into 1 L of medium at an inoculation rate of 2% and cultured at 30°C and 140 rpm for 48 h to obtain the fermentation broth of lead-zinc-copper resistant bacteria L1.

7. The method for plant-microorganism co-remediation of heavy metal contaminated soil according to claim 6, characterized in that, In step 5, add 200 mL of the lead-zinc-copper resistant bacteria L1 fermentation broth each time.

8. The method for plant-microorganism co-remediation of heavy metal contaminated soil according to claim 2, characterized in that, In step 4, after the alfalfa has grown for 30 days, select 25 vigorous plants that are free from pests and diseases and are similar in size and maturity to keep, and remove the excess plants.

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