Method for repairing high-ground-temperature area of strip mine through combination of microbial agent and herbaceous plants

Abstract

The invention discloses a method for restoring a high-ground-temperature area of a strip mine by combining a microbial agent with herbaceous plants, and belongs to the technical field of ecological restoration. According to the method, firstly, the high-temperature area of the strip mine is designed to be physically cooled, vegetation restoration is carried out after the temperature is reduced to 40 DEG C, and the method mainly comprises the steps that physiological and biochemical index data of chlorophyll, malondialdehyde, proline, soluble sugar and the like of herbaceous plants in the high-ground-temperature environment of the mine area, which can reflect damage of the plants when the plants suffer from stress, are compared; herbaceous plants with high heat resistance are screened out, and a microbial agent is applied to increase the heat resistance of the herbaceous plants. It is found that the heat resistance of the zoysia sinensis is high under the high-temperature condition, the fresh weight of the overground part of the zoysia sinensis and osmotic regulation substances can be remarkably increased through application of the microbial agent under high-temperature stress treatment, the malondialdehyde content of the zoysia sinensis is reduced, and therefore the damage degree of plants is relieved, and the yield of the plants is increased. And finally, the heat resistance of the zoysia sinensis is improved, so that the zoysia sinensis grows for a long time in a high-temperature environment to achieve the purpose of repairing the high-ground-temperature area of the strip mine.

Description

Technical Field

[0001] The invention belongs to the field of ecological restoration, and specifically relates to a method for restoring a high geothermal area in an open-pit mine by combining a microbial agent with herbal plants. Technical Background

[0002] After mining, an open-pit coal mine was formed with a length of 3.9 km from east to west, a width of 1.8 km from north to south, a maximum depth of 367 m, and an area of ​​7.02 km. 2 The mine pits. The dumping grounds and waste rock piles around the mining area have caused the land occupation area to be about 15.5km 2 The total area of ​​subsidence caused by coal mining exceeds 20km 2 , and it has not yet completely settled. The spontaneous combustion of the residual coal gangue has caused high temperatures in some areas. The underground temperature in some areas is as high as 60-70°C, and the surface temperature is as high as 43°C, making it difficult for vegetation to grow. Based on the fact that high temperatures in some areas of the Haizhou open-pit mine make it difficult for vegetation to grow, we are considering introducing a herb with extremely strong heat resistance. Zoysia is a plant of the genus Zoysia in the family Gramineae, Zoysiae. Zoysia plants prefer warm and humid climates and have strong heat resistance. Zoysia has a well-developed root system and good creeping ability, so it is often used as a lawn grass. Because it is a warm-season lawn grass, the suitable temperature is 26-32°C, but if the suitable temperature is exceeded, the normal growth, development and health of the lawn grass will be affected.

[0003] At present, a variety of microbial agents have been studied to promote the growth and improve the resistance of different plants, but there are almost no beneficial microbial species suitable for perennial zoysia grass. Therefore, we should actively explore microbial agents that promote the growth of perennial zoysia grass and improve its resistance. Applying suitable microbial agents to the production practice of perennial zoysia grass can reduce the damage caused by high temperature stress to perennial zoysia grass. This can provide a theoretical basis for the screening of heat-resistant plant cultivation in high-temperature areas of mining areas. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art. First, a physical cooling device is designed for the high and low temperature areas of the open-pit mine to reduce the temperature to below 40°C, and then a method for repairing the area using a microbial agent combined with herbal plants is provided. The Bacillus subtilis agent provided in the present invention has a very strong stress resistance. The application of the agent can significantly improve the stress resistance of Zoysia japonica in a high temperature environment, and can provide a theoretical basis and a solid practical foundation for the screening of heat-resistant plant cultivation in high temperature areas of mining areas. The method has good economic and social benefits, no secondary pollution, and is conducive to large-scale promotion and application.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows: a method for repairing high geothermal areas in open-pit mines by combining microbial agents with herbal plants, comprising the following steps:

[0006] 1) Physical cooling devices are buried underground in high geothermal areas of open-pit mines;

[0007] 2) Screening of heat tolerance of herbaceous plants under high temperature stress;

[0008] 3) Apply microbial agents to herbaceous plants to improve their heat resistance;

[0009] 4) Physiological and biochemical indicators of herbaceous plants under each temperature treatment were measured every 2 days;

[0010] 5) The results were obtained by comparing various indicators of herbaceous plants after being treated at different temperatures.

[0011] In the above method, in step 1), the structure of the physical cooling device is as follows: the water pump 1 pumps the mine water out and stores it in the water reservoir 12, and then the water in the water reservoir 12 is connected to the water heat exchanger 7 through the water pipe 9, and the outlet of the water heat exchanger 7 is connected to the water reservoir 12. The solar panel 2 is connected to the controller 3, the inverter 4, and the transformer 6 in sequence, and is also provided with a temperature sensor and a temperature display screen 11 connected thereto. After the solar panel generates electricity, it supplies power to the water pump and the water heat exchanger through the transformer and the transmission line 10.

[0012] In the above method, in step 2), the herbaceous plant is Zoysia japonica or Zoysia japonica.

[0013] In the above method, in step 2), Chinese Zoysia knotweed and Japanese Zoysia knotweed are transplanted on the ground buried with a physical cooling device, and high temperature stress treatment is carried out at a temperature gradient of 5°C at 30°C-40°C, and the heat resistance of the herbaceous plants is observed under natural light conditions.

[0014] In the above method, in step 3), the microbial agent is Bacillus subtilis, the application concentration is 0.34 g / ml, and 8.9 L / ml of the agent solution is applied on the 10th, 20th and 30th day after the herbaceous plant is transplanted. 2 .

[0015] In the above method, in step 4), the physiological and biochemical measurement indicators are aboveground biomass, malondialdehyde, proline, and soluble sugar content, which are measured five times in total.

[0016] The Bacillus subtilis inoculant provided in the present invention will enter a spore dormancy period in an unsuitable environment such as a harsh growth environment and a lack of nutrients, and form spores with extremely strong stress resistance and can survive in polar environments such as high temperature, acid and alkali, thereby adapting to the environment and surviving.

[0017] The physiological and biochemical indicators measured in the present invention are selected for the following reasons: high temperature stress will inhibit plant growth, which is ultimately reflected in the accumulation of biomass, so the change in plant biomass can be used as one of the criteria for measuring its heat resistance. After plant organs age or suffer damage under adverse conditions, membrane lipid peroxidation often occurs. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of damage suffered by plants under adverse conditions. Proline, soluble sugars and soluble proteins are the osmotic regulating substances that accumulate the most in plants under adverse conditions, which can essentially explore the inherent physiological mechanism of stress resistance of plants themselves.

[0018] Compared with the shortcomings of the prior art, the beneficial results created by the present invention are:

[0019] The present invention designs a method of first physically cooling the temperature and then performing vegetation restoration, and achieves the purpose of repairing the high and low temperature areas of the open-pit mine by means of a microbial agent combined with herbal plants. The present invention draws a conclusion that the Chinese Zoysia is more resistant to high temperatures by comparing the chlorophyll, malondialdehyde, proline, soluble sugar content and other indicators of the herbal plants Chinese Zoysia and Japanese Zoysia under high temperature conditions, and finds that compared with the control group, the Chinese Zoysia treated with the agent has high biomass, low malondialdehyde content, and high content of osmotic regulating substances such as proline, soluble sugar and soluble protein, indicating that the application of the Bacillus subtilis agent not only promotes the growth of Chinese Zoysia, but also reduces the degree of damage to Chinese Zoysia under high temperature conditions, thereby improving the heat resistance of Chinese Zoysia, and can provide a theoretical basis for the screening of heat-resistant plant cultivation in high geothermal areas of mining areas, and lay a solid foundation for the next step of production practice, thereby achieving the purpose of ecological restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the design of a device for physically cooling high and low temperature areas in an open-pit mine in Example 1 of the present invention.

[0021] Figure 2 This is the effect of different temperature treatments on the chlorophyll content of Zoysia japonica and Zoysia japonica along with the high temperature stress time in Example 2 of the present invention.

[0022] Figure 3 This is the effect of different temperature treatments on the malondialdehyde content of Zoysia japonica and Zoysia japonica along with the high temperature stress time in Example 2 of the present invention.

[0023] Figure 4 This is the effect of different temperature treatments on the proline content of Zoysia japonica and Zoysia japonica along with the high temperature stress time in Example 2 of the present invention.

[0024] Figure 51 is the effect of different temperature treatments on the soluble sugar content of Zoysia japonica and Zoysia japonica along with the high temperature stress time in Example 2 of the present invention.

[0025] Figure 6 This is the effect of the application of Bacillus subtilis inoculant on the fresh weight of aboveground biomass of Zoysia japonica under different temperature treatments along with stress time in Example 3 of the present invention.

[0026] Figure 7 This is the effect of the application of Bacillus subtilis inoculant on the dry weight of aboveground biomass of Zoysia japonica under different temperature treatments along with stress time in Example 3 of the present invention.

[0027] Figure 8 This is the effect of the application of Bacillus subtilis inoculant on the malondialdehyde content of Zoysia japonica under different temperature treatments along with the stress time in Example 4 of the present invention.

[0028] Fig. 9 This is the effect of applying Bacillus subtilis inoculant on the proline content of Zoysia sinensis under different temperature treatments along with stress time in Example 5 of the present invention.

[0029] Fig.10 This is the effect of applying Bacillus subtilis inoculant on the soluble sugar content of Zoysia japonica under different temperature treatments along with stress time in Example 5 of the present invention.

[0030] Fig.11 The present invention is a flow chart of the method of using the microbial agent combined with herbal plants to repair the high geothermal area of ​​an open-pit mine. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods, but the protection scope of the present invention is not limited thereto.

[0032] The method of the present invention firstly physically cools the high and low temperature areas of the mining area, and then plants herbaceous plants for restoration after the temperature drops to 40°C.

[0033] The method of the present invention is used to select herbaceous plants of the genus Zoysia with strong heat resistance, and the heat resistance of Chinese Zoysia and Japanese Zoysia is compared under high temperature stress, so as to screen varieties of Zoysia for planting in high geothermal areas of mining areas.

[0034] The method of the present invention is used to explore the effect of Bacillus subtilis on the growth and heat resistance of Zoysia sinensis. In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods and drawings.

[0035] The Chinese Zoysia and Japanese Zoysia used in the examples of the present invention are both taken from the Ecological Park of Liaoning University. The Bacillus subtilis agent of the present invention can be directly purchased through commercial channels.

[0036] The physiological and biochemical index determination methods in the embodiments of the present invention are all based on national standards.

[0037] A technology and method for repairing high geothermal areas in open-pit mines by combining microbial agents with herbal plants, comprising the following steps:

[0038] Example 1 Physical cooling device for high ground temperature area in open pit mine

[0039] The physical cooling device is a device that buries two physical cooling devices three meters and five meters below the high temperature area. The water pump 1 pumps the mine water out and stores it in the water reservoir 12. The water in the water reservoir 12 is then connected to the water heat exchanger 7 through the water pipe 9. The outlet of the water heat exchanger 7 is connected to the water reservoir 12. The solar panel 2 is connected to the controller 3, the inverter 4, and the transformer 6 in sequence. A temperature sensor and a temperature display screen 11 are also provided underground. After the solar panel generates electricity, it supplies power to the water pump and the water heat exchanger through the transformer and the transmission line 10. A water pump 1 is also provided to extract groundwater to supply water to the water reservoir 12. Copper plates and fans are installed in the water reservoir 12. Figure 1 shown.

[0040] When the temperature exceeds 40°C, the temperature sensor 8 alarms and starts the physical cooling device. The water source is obtained by the water pump 1 to pump water from underground in the mine, and then enters the physical cooling device through the water pipe 9. The heat exchanged water flows back to the water reservoir 12, in which copper plates and fans are installed to accelerate heat dissipation, and exchange resin is added to prevent the reflux water from calcifying and clogging the pipeline. When the physical cooling device does not have enough water, it enters the physical cooling device together with the mine water to realize circulation. The electricity used during this period is supplied by the electricity generated by the solar panels.

[0041] Example 2 Comparative screening of heat resistance of Chinese Zoysia grass and Japanese Zoysia grass under high temperature stress

[0042] Chinese Zoysia and Japanese Zoysia were transplanted on the ground above the physical cooling device. The land temperature in the transplanting area was controlled at 30℃, 35℃, and 40℃.

[0043] The chlorophyll content, malondialdehyde, proline and soluble sugar content of Zoysia japonica and Zoysia japonica were measured on the 1st, 3rd, 5th, 7th and 9th days after high temperature stress to reflect which herbaceous plant, Zoysia japonica or Zoysia japonica, has stronger heat resistance under heat stress.

[0044] ① Determination of aboveground biomass: First, take the Chinese Zoysia knotweed and Japanese Zoysia knotweed of each treatment, rinse the surface dust of the plants with clean water, absorb the moisture with filter paper, and then place them on an analytical balance to weigh the fresh weight of the aboveground part; then, fix the aboveground part at 105℃ for 1h, dry it at 80℃ to constant weight, and place it on an analytical balance to weigh the dry weight of the aboveground part.

[0045] ② Determination of chlorophyll content: Take 0.2g of fully expanded functional leaves after treatment, add a small amount of quartz sand and calcium carbonate and 2-3ml95% ethanol, and then add 10ml ethanol solution until the tissue turns white, and let it stand for 3-5 minutes. Place the filter paper in a funnel and rinse it into a volumetric flask (25ml) with ethanol solution. Use 96% ethanol as blank to adjust to zero, measure the absorbance A665 and A649 of the extract at 665nm and 649nm respectively, and calculate the chlorophyll a concentration (Ca) and chlorophyll b concentration (Cb) according to the following formula.

[0046] Ca(mg·L-1)=13.95A665 -6.88A649;

[0047] Cb(mg·L-1)=24.96A649-7.32A665;

[0048] Chlorophyll a content (mg / gFW) = (Ca×Vt) / (FW×1000);

[0049] Chlorophyll b content (mg / gFW) = (Cb×Vt) / (FW×1000);

[0050] Total chlorophyll content (Chl) = chlorophyll a content (Chla) + chlorophyll b content (Chlb);

[0051] Wherein, Ca: chlorophyll a concentration; Cb: chlorophyll a concentration; Vt: total volume of extract; FW: fresh weight of sample.

[0052] ② Determination of malondialdehyde content: Take 0.2g of fresh leaves, add 5mL 5% TCA (trichloroacetic acid) in a mortar and grind together, transfer the extract into a centrifuge tube, and centrifuge at 4000r / min for 10min. Take 2mL of the supernatant obtained by centrifugation, add 2mL of 0.67% thiobarbituric acid, mix well and boil in a water bath for 30min, wait for it to cool and centrifuge again at 4000r / min for 10min. Take the centrifuged supernatant and use a UV-1900i visible spectrophotometer to detect its absorbance at 450nm, 532nm and 600nm respectively, recorded as A450, A532 and A600. The MDA concentration can be calculated, and then the MDA content per unit fresh weight sample can be calculated.

[0053] MDA concentration (nmol / L) = [6.45 × (A532-A600)-0.56 × A450] × 1000

[0054] MDA content (nmol / g) = MDA concentration (nmol / L) × extract volume (mL) / plant fresh weight (g)

[0055] ③ Determination of proline content: Use the acid ninhydrin method. Take 0.2g of fresh leaves, cut them into pieces and place them in a large test tube. Add 5ml of 3% sulfosalicylic acid to the test tube, extract in a boiling water bath for 10min (shake frequently during the extraction process), and centrifuge at 4000r / min-1 for 10min after cooling. Pipette 2ml of supernatant and put it in another test tube with a glass stopper, add 2ml of glacial acetic acid and 2ml of acid ninhydrin, heat in a boiling water bath for 30min, and the solution will turn red. After cooling, add 4ml of toluene, shake well and let it stand for a while. Use a pipette to draw the upper proline-toluene red solution into a colorimetric dish, use toluene as a blank, and compare the color at 520nm. Calculate the proline content in 2ml of the test solution based on the standard curve.

[0056] ④Determination of soluble sugar content: using anthrone colorimetric method.

[0057] Sample extraction: Weigh 0.20-0.30g of fresh leaves, use tweezers to clamp the sample into a graduated test tube, add 10mL of distilled water, seal with plastic film, extract in boiling water for 30min (extract twice), filter the extract into a 25ml volumetric flask, rinse the test tube and residue repeatedly, and make up to the scale.

[0058] Colorimetric determination: Pipette 0.5 ml of sample extract into a 20 ml graduated test tube (repeat twice), add 1.5 ml of distilled water, then add 0.5 ml of anthracene ethyl acetate reagent and 5 ml of concentrated sulfuric acid, shake thoroughly, immediately put the test tube into a boiling water bath, keep each tube at temperature for 1 minute, take it out and naturally cool it to room temperature, use the blank as a reference (do not forget the control). Measure its optical density at a wavelength of 630 nm to determine the optical density of the sample. Calculate the content of soluble sugars based on the standard curve.

[0059] Results Figure 2 , 3 , 4, 5, Figure 2 It shows that with the increase of temperature, the chlorophyll content of the two species of Zoysia grasses decreased in different trends, but the chlorophyll content of Zoysia japonica was always higher than that of Zoysia japonica. Figure 3 The results showed that the MDA content of the two species of Zoysia grass decreased with the increase of temperature, but the MDA content of Zoysia chinensis was lower than that of Zoysia japonica in general. Figure 4The results showed that the proline content of the two zoysias did not increase significantly with stress time and temperature, but overall the proline content of Chinese zoysia was higher than that of Japanese zoysia. Figure 5 The results showed that the soluble sugar content of the two zoysia grasses showed an overall upward trend with the increase of stress time and temperature, and the overall performance was that the soluble sugar content of Chinese zoysia grass was higher than that of Japanese zoysia grass. The results showed that Chinese zoysia grass had stronger stress resistance than Japanese zoysia grass under high temperature conditions.

[0060] Example 3 Effect of application of Bacillus subtilis inoculant on growth and development of Zoysia japonica

[0061] Control group: directly subjected to high temperature stress without adding bacterial agents, but in order to reduce the experimental error, the plants treated with bacterial agents were cultured in batches at the same time.

[0062] Experimental group: Bacillus subtilis was applied on the 10th, 20th and 30th days of growth of Zoysia japonica.

[0063] Results Figure 6 (CK is the control group, BS is the experimental group). The results showed that compared with the suitable temperature treatment, the aboveground fresh weight of Zoysia knotweed under high temperature stress decreased significantly, among which the decrease rate of the inoculant treatment was lower than that of the blank treatment, which were 21.9%, 30.6% and 14%, 16.9% respectively. Compared with the blank treatment, the aboveground fresh weight of the inoculant treatment was significantly increased, which were 52.1%, 57.2% and 60.0% respectively. The aboveground dry weight of Zoysia knotweed showed a downward trend under heat stress treatment. Among them, the decrease rate of the inoculant treatment was lower than that of the blank treatment, which were 22.8%, 39.2% and 17.3%, 25.6% respectively. Compared with the blank treatment, the aboveground fresh weight of the inoculant treatment was significantly increased, which were 51.5%, 52.6% and 67.3% respectively. (Results see Figure 7 ), it was concluded that the fresh and dry weight of the aboveground part of Zoysia japonica decreased significantly after high temperature treatment, indicating that high temperature stress would seriously affect the normal growth of Zoysia japonica. However, after applying Bacillus subtilis to Zoysia japonica, the fresh and dry weight of its aboveground part increased significantly, and the degree of decrease was small under high temperature treatment. This shows that Bacillus subtilis can promote the growth of Zoysia japonica under high temperature stress conditions.

[0064] Example 4 Effect of Bacillus subtilis inoculant application on malondialdehyde in Zoysia sinensis under high temperature stress

[0065] Results Figure 8(CK is the control group, BS is the experimental group). The results showed that the blank treatment showed a gradual upward trend under high temperature stress, while the bacterial agent treatment showed a trend of first increasing and then decreasing, and the malondialdehyde content of the bacterial agent treatment was lower than that of the blank treatment. This shows that the application of Bacillus subtilis bacterial agent effectively reduced the malondialdehyde content of the plant under high temperature stress, thereby reducing the degree of cell membrane peroxidation of Zoysia japonica under high temperature stress conditions.

[0066] Example 5 Effect of Bacillus subtilis inoculant application on osmotic regulating substances of Zoysia japonica under high temperature stress

[0067] (1) Proline content

[0068] Results Fig. 9 (CK is the control group, BS is the experimental group). The results showed that compared with the appropriate temperature treatment, the proline content of the bacterial agent treatment under high temperature treatment was higher than that of the blank treatment, and showed an overall upward trend with the increase in temperature, and the peak value appeared at the 9th day of high temperature treatment. This shows that the application of Bacillus subtilis to Zoysia japonica increased the proline content of the plant under high temperature stress, and effectively reduced the degree of damage to Zoysia japonica under high temperature stress.

[0069] (2) Soluble sugar content

[0070] Results Fig.10 (CK is the control group, BS is the experimental group). The results show that compared with the appropriate temperature treatment, the blank treatment did not show significant changes under high temperature stress, and the soluble sugar content of the bacterial agent treatment showed a gradual upward trend. There was no significant difference between the two treatments under the appropriate temperature treatment, but the soluble sugar content of the high temperature treatment bacterial agent treatment was significantly higher than that of the blank treatment, and the peak of the soluble sugar content appeared on the 9th day compared with the control. This shows that the application of Bacillus subtilis to Zoysia japonica increased the soluble content of the plant under high temperature stress, increased the osmotic regulating substances that the plant accumulated the most when suffering from adversity, and thus reduced the damage of Zoysia japonica to Zoysia japonica due to high temperature stress.

Claims

1. A method for remediating high geothermal areas in open-pit mines by combining microbial agents with herbal plants, which is characterized by: The following steps are involved: 1) Physical cooling devices are buried underground in high geothermal areas of open-pit mines; 2) Screening of heat tolerance of herbaceous plants under high temperature stress; 3) Apply microbial agents to herbaceous plants to improve their heat resistance; 4) Physiological and biochemical indicators of herbaceous plants under each temperature treatment were measured every 2 days; 5) The results were obtained by comparing various indicators of herbaceous plants after being treated at different temperatures.

2. The method according to claim 1, characterized in that In step 1), the structure of the physical cooling device is as follows: a water pump (1) pumps out the mine water and stores it in a water reservoir (12), and then the water in the water reservoir (12) is connected to the water heat exchanger (7) through a water pipe (9), and the outlet of the water heat exchanger (7) is connected to the water reservoir (12). The solar panel (2) is connected to the controller (3), the inverter (4), and the transformer (6) in sequence, and is also provided with a temperature sensor and a temperature display screen (11) connected thereto. After the solar panel generates electricity, it supplies power to the water pump and the water heat exchanger through the transformer and the transmission line (10).

3. The method according to claim 1, characterized in that In step 2), the herbaceous plants are Zoysia japonica and Zoysia japonica.

4. The method according to claim 1, characterized in that In step 2), Chinese Zoysia knotweed and Japanese Zoysia knotweed are transplanted on the ground buried with a physical cooling device, and high temperature stress treatment is carried out at a temperature gradient of 5°C at intervals of 30°C-40°C. The heat resistance of the herbaceous plants is observed under natural light conditions.

5. The method according to claim 1, characterized in that In step 3), the microbial agent is Bacillus subtilis, the application concentration is 0.34 g / ml, and 8.9 L / ml of the agent solution is applied on the 10th, 20th and 30th day after the herbaceous plant is transplanted. 2 .

6. The method according to claim 1, characterized in that In step 4), the physiological and biochemical determination indicators are aboveground biomass, malondialdehyde, proline, and soluble sugar content, which are determined five times in total.

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