WO2023010620A1

WO2023010620A1 - Biological crust restoration material for promoting ecological recovery of ionic rare earth tailings region, application, and restoration method

Info

Publication number: WO2023010620A1
Application number: PCT/CN2021/113744
Authority: WO
Inventors: 张萌; 刘燕; 冯兵; 姚娜; 吴俊伟; 刘足根; 李铭书; 王强; 周慜
Original assignee: 江西省生态环境科学研究与规划院
Priority date: 2021-08-04
Filing date: 2021-08-20
Publication date: 2023-02-09
Also published as: AU2021286451A1; CN113857235A; CN113857235B

Abstract

A biological crust restoration material for promoting the ecological recovery of an ionic rare earth tailings region, an application, and a restoration method, which belong to the technical field of mine ecological restoration. Provided is a biological crust restoration material for promoting an ionic rare earth tailings region, comprising cosmopolitan algae and/or native soil algae, wherein species of the native soil algae comprise cyanobacteria lyngbya and/or green algae Chlamydomonas, and species of the cosmopolitan algae comprise Nostoc and/or Microcoleus vaginatus. Applying a culture solution of cosmopolitan algae and native soil algae to soil in an ionic rare earth tailings region to be restored may significantly increase the content of organic matter in tailings soil, reduce the content of ammonia nitrogen, increase the crust area of the tailings surface, and greatly reduce the exposed surface layer, which may quickly improve extremely degraded ecological environments caused by tailings waste in an ionic rare earth abandoned mine region, and improve soil degradation and environmental pollution in the mining region caused by the destruction and exploitation of rare earth mines.

Description

A kind of biological crust restoration material and its application and restoration method for promoting the ecological restoration of ion-type rare earth tailings area

This application is required to be submitted to the China Patent Office on August 4, 2021. The application number is 202110889960.8, and the invention title is "a biological crust repair material and its application and repair method for promoting the ecological restoration of ionic rare earth tailings areas". priority, the entire contents of which are incorporated in this application by reference.

technical field

The invention belongs to the technical field of mine ecological restoration, and in particular relates to a biocrust restoration material for promoting ecological restoration of an ion-type rare earth tailings area and its application and restoration method.

Background technique

Ionic rare earths are national strategic resources, non-renewable, and are widely used in national defense construction and high-tech fields. The mining of ionic rare earths creates high profits while causing a series of ecological and environmental problems such as the destruction of vegetation and land resources, water and soil pollution, such as: loose soil, severe soil desertification, and the phenomenon of barren grass (known as "Southern Desert"). In the rainy season in the south, water and soil erosion is also prone to occur, resulting in a large number of abandoned slopes, unstable slopes, bare surfaces, and lack of vegetation, causing serious geological disasters such as subsidence, landslides, and landslides, which seriously restrict and hinder the agricultural and social development of the region. develop. Therefore, in view of the damage to the surrounding environment caused by the mining of ion-type rare earth mines, it is imminent to carry out the ecological reconstruction of the ion-type rare earth tailings area in southern Jiangxi.

Contents of the invention

In view of this, the object of the present invention is to provide a biological crust restoration material and its application and repair method for promoting the ecological restoration of ion-type rare earth tailings areas. The biological crust restoration material can increase the content of organic matter in the tailings soil and reduce ammonia nitrogen. content, increase the surface crust area of tailings, greatly reduce the exposed surface layer, and improve the problems of soil degradation and environmental pollution in mining areas.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a biocrust restoration material for promoting the ecological restoration of ion-type rare earth tailings areas, including broadspread algae and/or native soil algae; algae; the preservation number of the cyanophyta Coleurophyta is CCTCC No: M 2021758; the preservation number of the Chlorophyta Chlamydomonas is CCTCC No: M 2021324; Microcoleum.

Preferably, when the biocrust repair material for promoting the ecological restoration of the ion-type rare earth tailings area includes broad-leaved algae and local soil algae, the content ratio of chlorophyll in the described broad-leaved algae and the content ratio of chlorophyll in the local soil algae is ( 0～5): (0～5), and the content of chlorophyll in the broad-leaved algae and the chlorophyll in the local soil algae are different from 0 at the same time;

When the biocrust repair material for promoting the ecological restoration of the ion-type rare earth tailings area includes Cyanophyta and Chlorophyta Chlamydomonas, the content of chlorophyll in the Cyanophyta Chlamydomonas and Chlorophyta Chlamydomonas The content ratio is (0～5):(0～5), and the content of chlorophyll in the cyanophyta Chlamydomonas and the chlorophyll in Chlamydomonas cyanophyta are both 0;

When the biocrust repair material for promoting the ecological restoration of the ion-type rare earth tailings area includes nitrogen-fixing Nostoc and Microcoletheca, the content ratio of chlorophyll in the nitrogen-fixing Nostoc and Microcoletheca is: (0-5): (0-5), and the content of chlorophyll in the nitrogen-fixing Nostoc and the chlorophyll in Microcoletheca are different from 0 at the same time.

Preferably, the application amount of the biological crust restoration material for promoting the ecological restoration of the ion-type rare earth tailings area is 100-1000 μg (chlorophyll)·m ⁻² (soil to be repaired).

The present invention also provides the application of the biological crust repair material for promoting the ecological restoration of the ion-type rare earth tailings area described in the above technical solution in repairing the ion-type rare earth tailings area.

The present invention also provides a method for repairing the ionic rare earth tailings area, comprising the following steps:

The biological crust repair material for promoting the ecological restoration of the ion-type rare earth tailings area described in the above technical solution is applied to the soil of the ion-type rare earth tailings area to be repaired for restoration.

Preferably, the biological crust restoration material for promoting the ecological restoration of the ion-type rare earth tailings area is applied to the soil of the ion-type rare earth tailings area to be repaired in the form of algae suspension.

Preferably, the preparation method of described algae suspension comprises the following steps:

Inoculate the biological crust restoration material in the ion-promoting rare earth tailings area into the culture solution, and cultivate to obtain the algae suspension.

Preferably, the culture medium is sterile BG-11 culture medium.

Preferably, the light intensity of the cultivation is 2500-3500 lx; the light-dark ratio of the cultivation is 16h:8h or 12h:12h; the cultivation method is static cultivation; the cultivation temperature is 25±5°C ; The culture time is 3 to 4 weeks.

Preferably, the chlorophyll content in the algal suspension is 100-1000 μg·L ^-1 .

The invention provides a biocrust restoration material for promoting the ecological restoration of ion-type rare earth tailings areas, including broadspread algae and/or native soil algae; algae; the preservation number of the cyanophyta Coleurophyta is CCTCC No: M 2021758; the preservation number of the Chlorophyta Chlamydomonas is CCTCC No: M 2021324; Microcoleum. When the restoration material for the ion-promoting rare earth tailings area provided by the present invention is used for the ecological restoration of the ion-promoting rare earth tailings area, the extracellular secretions of algae cells are an important source of soil organic matter, which can significantly increase the content of organic matter in the soil of the tailing area. The salt ions in the nutrient solution can promote the leaching of ammonia nitrogen in the soil, thereby reducing the ammonia nitrogen content in the soil, increasing the surface crust area of tailings, and greatly reducing the exposed surface layer, which can quickly improve the ionic rare earth abandoned mining area due to tailings abandonment. The extremely degraded ecological environment caused by the land, and solve the problems of soil degradation and environmental pollution in mining areas caused by the destruction of mountains and leaching of rare earth mines.

Instructions attached

Figure 1 is a morphological diagram of native soil algae under a microscope;

Fig. 2 is the morphological diagram of the Chlamydomonas chlorophyta purified and cultivated in Example 1 and the morphological diagram of the Cyanophyta cyanophyta Purified and cultivated in Embodiment 2;

Fig. 3 is the change chart of soil surface algae crust after different treatment algae after inoculation;

Fig. 4 is the change figure of soil algae chlorophyll content with culture time in embodiment 1 and embodiment 2;

Figure 5 is a graph showing the change of surface algae crusts after adding the BG-11 culture solution without algae to the rare earth tailings soil for 46 days;

Fig. 6 is the SEM microstructure of the algal crust on the surface of the tailings soil after inoculation with different algae combinations at magnifications of 2000 times and 5000 times, respectively.

Biological Deposit Instructions

Cyanophyta sheath filament algae (Lyngbya sp.) JXSHKY-1, was preserved in the China Center for Type Culture Collection on June 24, 2021, and the address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University No. Opposite to First Attached Primary School), Wuhan University Collection Center, the collection number is CCTCC No: M 2021758;

Chlamydononas sp. JXSHKY-2 was preserved in the China Center for Type Culture Collection on April 2, 2021. The address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University No. Opposite to First Attached Primary School), Wuhan University Collection Center, the collection number is CCTCC No: M 2021324.

Detailed ways

The invention provides a biocrust restoration material for promoting the ecological restoration of ion-type rare earth tailings areas, including broadspread algae and/or native soil algae;

The species of the native soil algae include Lyngbya sp. JXSHKY-1 and/or Chlamydononas sp. JXSHKY-2; the preservation number of the Cyanophyta sp. is: CCTCC No: M 2021758; the preservation number of the Chlorophyta Chlamydomonas is: CCTCC No: M 2021324.

The species of Widespread algae include Nostoc sp. and/or Microcoleus vaginatus.

In the present invention, the preferred species of native soil algae are selected from the abandoned rare earth mining area of Daluoshi, Wenfeng Town, Xunwu County, Ganzhou City, Jiangxi Province.

In the present invention, the separation and purification method of the native soil algae preferably includes the following steps:

The biocrust samples formed under natural conditions in the abandoned rare earth mining area of Daluoshi, Wenfeng Town, Xunwu County, Ganzhou City, Jiangxi Province were collected and sterilized with 75% alcohol to prevent cross-contamination of the samples during the collection process. The sterilized samples were Put it in a sterile aluminum box (specification: 50mm×30mm), transport it back to the laboratory, and store it at a low temperature of -4°C; In the 150mL Erlenmeyer flask of -11 culture fluid (composition and preparation method of BG-11 culture fluid are shown in Table 1), after being uniformly dispersed in the homogenizer, it is cultured statically under specific environmental conditions in the light incubator (light and dark Ratio = 16h:8h, light intensity at 3000lx, temperature at 25°C), after 3 to 4 weeks of cultivation, when an obvious algae solution is formed, use a capillary to take 2 μL of the algae solution into 24 wells containing 2mL of sterile BG-11 culture solution In the cell culture plate, carry out culture (cultivation conditions as above), when forming obvious green, use capillary under microscope (optical microscope, CX33RTFS2, OLYMPUS, Japan) respectively draw and select Cyanophyta Sheath filament algae (Lyngbya sp.) JXSHKY- 1 or Chlamydononas sp. JXSHKY-2, and the selected cyanobacteria Lyngbya sp. JXSHKY-1 or Chlamydononas sp. JXSHKY-2 in the absence of Under bacterial conditions, inoculate in aseptic BG-11 culture medium and carry out separate culture (cultivation conditions are as above), repeat the process of separate culture 8 times, obtain the single cyanophyta Sheath filamentous algae (Lyngbya sp.) JXSHKY- 1 or Chlamydononas sp. JXSHKY-2.

In the present invention, the preferred species of the broad-leaved algae are purchased from the National Algae Species Bank of the Institute of Hydrobiology, Chinese Academy of Sciences. Among them, the preservation number of nitrogen-fixing Nostoc sp. is FACHB-119, and the preservation number of filamentous Microcoleus vaginatus is FACHB-253.

In the present invention, when the biocrust repair material for the ecological restoration of the ion-promoting rare earth tailings area includes broad-leaved algae and native soil algae, the ratio of the content of chlorophyll in the broad-spectrum algae to the content of chlorophyll in the native soil algae is It is preferably (0～5): (0～5), and the content of chlorophyll in the broad-leaved algae and the chlorophyll in the local soil algae are different from 0 at the same time; The content ratio of is more preferably (1～4): (1～5);

When the biocrust repair material for promoting the ecological restoration of the ion-type rare earth tailings area includes Cyanophyta and Chlorophyta Chlamydomonas, the content of chlorophyll in the Cyanophyta Chlamydomonas and Chlorophyta Chlamydomonas The content ratio of is preferably (0～5):(0～5), and the content of chlorophyll in the cyanophyta Chlamydomonas and the chlorophyll in the green algae Chlamydomonas are different from 0 at the same time; The ratio of the content of chlorophyll to the content of chlorophyll in Chlamydomonas Chlamydomonas is more preferably (1-4): (1-5);

When the biocrust repair material for promoting the ecological restoration of the ion-type rare earth tailings area includes nitrogen-fixing Nostoc and Microcoletheca, the content ratio of chlorophyll in the nitrogen-fixing Nostoc to the content ratio of chlorophyll in Microcoletheca is preferably It is (0～5): (0～5), and the content of chlorophyll in the nitrogen-fixing Nostoc and the chlorophyll in Microtheca sheaths are different from 0; the content of chlorophyll in the nitrogen-fixing Nostoc and Microsheaths The content ratio of chlorophyll in algae is more preferably (1～4):(1～5);

In the present invention, the application amount of the biological crust restoration material for the ecological restoration of the ion-promoting rare earth tailings area is preferably 100-1000 μg (chlorophyll) m ^-2 (soil to be repaired), more preferably 150-950 μg (chlorophyll )·m ^-2 (soil to be repaired).

The present invention also provides a method for repairing the ion-type rare earth tailings area by utilizing the biological crust that promotes the ecological restoration of the ion-type rare earth tailings area described in the above technical solution, including the following steps:

In the present invention, it is preferable to apply the biological crust restoration material for promoting the ecological restoration of the ion-type rare earth tailings area to the soil of the ion-type rare earth tailings area to be repaired in the form of algae suspension.

In the present invention, the preparation method of the algae suspension preferably comprises the following steps:

In the present invention, the nutrient solution is preferably a sterile BG-11 nutrient solution; the composition of the sterilized BG-11 nutrient solution is as shown in Table 1; For special limitations, it is enough to prepare the mother solution according to the formula in Table 1 according to the process well known in the art and mix to form the BG-11 culture solution.

Table 1 Composition and preparation method of BG-11 culture medium

Note: A5 solution (diluted to 1000mL with water): H ₃ BO ₃ (61.0mg); MnSO ₄ ·H ₂ O (169.0mg); ZnSO ₄ ·7H ₂ O (287.0mg); CuSO ₄ ·5H ₂ O (2.5 mg); (NH ₄ )Mo ₇ O ₂₄ .4H ₂ O (12.5 mg).

Unless otherwise specified, the present invention has no special requirements on the sources of the raw materials used to prepare the BG-11 culture solution, and commercially available products well known to those skilled in the art can be used. In the present invention, there is no special limitation on the sterilization method of the sterile BG-11 culture solution, and a sterilization method well known in the art can be used.

In the present invention, the inoculation is preferably performed under sterile conditions. The present invention has no special limitation on the inoculation method, and methods well known in the art can be used. In the present invention, the cultivation device is preferably a light incubator; the cultivation method is preferably static cultivation; the light-dark ratio of the cultivation is preferably 16h:8h or 12h:12h, and the light intensity of the cultivation Preferably 2500-3500 lx, the temperature of the culture is preferably 25±5°C; the time of the culture is preferably 3-4 weeks.

In the present invention, the chlorophyll content in the algal suspension is preferably 100-1000 μg·L ^-1 , more preferably 150-950 μg·L ^-1 .

The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

Example 1

The biocrust samples formed under natural conditions in the abandoned rare earth mining area of Daluoshi, Wenfeng Town, Xunwu County, Ganzhou City, Jiangxi Province were collected and sterilized with 75% alcohol to prevent cross-contamination of the samples during the collection process. The sterilized samples were Put it in a sterile aluminum box (specification: 50mm×30mm), transport it back to the laboratory, and store it at a low temperature of -4°C; In the 150mL Erlenmeyer flask of -11 culture fluid (composition and preparation method of BG-11 culture fluid are shown in Table 1), after being uniformly dispersed in the homogenizer, it is cultured statically under specific environmental conditions in the light incubator (light and dark Ratio = 16h: 8h, light intensity at 3000lx, temperature at 25°C), after 4 weeks of cultivation, when an obvious algae solution is formed, use a capillary to take 2 μL of the algae solution into a 24-well cell culture containing 2mL of sterile BG-11 culture solution In the plate, culture was carried out (the culture conditions were as described above), and when a clear green color was formed, it was observed using an optical microscope (model CX33RTFS2, OLYMPUS, Japan). The morphology of native soil algae in the algae solution under the microscope is shown in Figure 1. From the results of microscopic examination in Figure 1, it can be seen that the algae obtained through culture mainly include the filamentous Cyanophyta Lyngbya sp. JXSHKY-1 and the spherical Chlamydononas sp. The algae Chlamydomonas mosaic is distributed among the entangled filamentous cyanophyta sheath filaments; use a capillary to draw and select the cyanobacteria Lyngbya sp. JXSHKY-1 under the microscope, and put the selected Algae (Lyngbya sp.) JXSHKY-1 was inoculated in sterile BG-11 culture medium under aseptic conditions to carry out separate culture (cultivation conditions as mentioned above), and the process of separate culture was repeated 8 times to obtain a single cyanobacterium purified and cultivated Lyngbya sp. JXSHKY-1;

The filamentous cyanobacteria Lyngbya sp. JXSHKY-1 was inoculated in sterile BG-11 culture solution, and cultured statically under specific environmental conditions in a light incubator (light-to-dark ratio=16h:8h, The light intensity was 3000 lx, the temperature was 25°C), and cultured for 4 weeks to obtain a suspension of filamentous cyanobacteria Phylum phylum chlorophyll with a chlorophyll content of 192 μg·L ⁻¹ .

Example 2

The biocrust samples formed under natural conditions in the abandoned rare earth mining area of Daluoshi, Wenfeng Town, Xunwu County, Ganzhou City, Jiangxi Province were collected and sterilized with 75% alcohol to prevent cross-contamination of the samples during the collection process. The sterilized samples were Put it in a sterile aluminum box (specification: 50mm×30mm), transport it back to the laboratory, and store it at a low temperature of -4°C; In the 150mL Erlenmeyer flask of -11 culture fluid (composition and preparation method of BG-11 culture fluid are shown in Table 1), after being uniformly dispersed in the homogenizer, it is cultured statically under specific environmental conditions in the light incubator (light and dark Ratio = 16h: 8h, light intensity at 3000lx, temperature at 25°C), after 4 weeks of cultivation, when an obvious algae solution is formed, use a capillary to take 2 μL of the algae solution into a 24-well cell culture containing 2mL of sterile BG-11 culture solution In the plate, culture was carried out (the culture conditions were as described above), and when a clear green color was formed, it was observed using an optical microscope (model CX33RTFS2, OLYMPUS, Japan). The morphology of native soil algae in the algae solution under the microscope is shown in Figure 1. From the results of microscopic examination in Figure 1, it can be seen that the algae obtained from culture mainly include filamentous cyanophyta Lyngbya sp. JXSHKY-1 and spherical green algae Chlamydononas sp. JXSHKY-2, spherical green algae Chlamydomonas mosaic distribution in the intertwined filamentous cyanophyta sheath filaments; using a capillary tube under the microscope to select the Chlamydononas sp. JXSHKY-2, and the selected Chlamydononas sp. ( Chlamydononas sp.) JXSHKY-2 was inoculated in sterile BG-11 culture medium under aseptic conditions for separate culture (culture conditions as above), and the process of separate culture was repeated 8 times to obtain a single Chlorophyta purified and cultivated Chlamydononas sp. JXSHKY-2;

Chlamydononas sp. JXSHKY-2 was inoculated in sterile BG-11 culture medium, and cultured statically in a light incubator under specific environmental conditions (light-to-dark ratio = 16h: 8h, light intensity At 3000 lx, the temperature is 25°C) and cultivated for 4 weeks to obtain a suspension of Chlorophyta Chlamydomonas with a chlorophyll content of 339 μg·L ⁻¹ .

Example 3

The nitrogen-fixing Nostoc sp. of Cyanophyta (purchased from the National Algae Species Bank of the Institute of Hydrobiology, Chinese Academy of Sciences) was inoculated into sterile BG-11 culture medium, and cultured statically in a light incubator under specific environmental conditions. (Light-to-dark ratio = 16h:8h, light intensity at 3000lx, temperature at 25°C) and cultured for 4 weeks to obtain a nitrogen-fixing Nostoc algae suspension with a chlorophyll content of 441μg·L ^-1 .

Example 4

The difference from Example 3 is that the algae biocrust repair material that promotes the rapid ecological restoration of the ion-type rare earth tailings area is Cyanophyta filamentous Microcoleus vaginatus (purchased from the National Algae Species Bank of the Institute of Hydrobiology, Chinese Academy of Sciences) ), other contents are consistent with Example 3, and the chlorophyll content in the microsheathing algae suspension is 558 μg·L ^-1 .

Application example 1

Evaluation

Location: Donghu District, Nanchang City, Jiangxi Province - in the scientific research glass room of Jiangxi Ecological Environmental Science Research and Planning Institute;

The source of the tested soil: rare earth tailings matrix - the abandoned rare earth mining area of Daluoshi, Wenfeng Town, Xunwu County, Ganzhou City, Jiangxi Province; yellow soil - taken from the vicinity of the abandoned rare earth mining area of Daluoshi, Wenfeng Town, Xunwu County, Ganzhou City, Jiangxi Province Undeveloped natural woodland.

Time: November 2020 to March 2021.

Culture conditions: the effective photosynthetic radiation is about 3.5 μmol·m ^-2 ·s ^-1 , the average temperature is 10°C, and the average air humidity is 70% RH.

The basic physical and chemical index contents of rare earth tailings matrix are: pH value 4.36, ammonia nitrogen content 173.89mg·kg ^-1 , organic matter content 0.236g·kg ^-1 .

Test plan:

Measure the chlorophyll content of 4 kinds of algae suspensions (sheath filaments A, Chlamydomonas B, nitrogen-fixing Nostoc C, microsheath D) that embodiment 1～4 obtains by phytoplankton classification fluorescence instrument PHYTO-PAM-II and Draw a growth curve. The chlorophyll content in the algal suspension obtained in Examples 1 and 2 is shown in Figure 2.

Treatment group 1:

The Cyanophyta phylloxera suspension A of Example 1 and the Chlorophyta Chlamydomonas suspension B of Example 2 were mixed according to the chlorophyll content of 2:1, and 0.03% agar, 3g yellow soil and no addition were added as 3 Each treatment was supplemented with sterile BG-11 culture solution to 45mL, and three parallel samples were set for each treatment. After shaking for 12h, 45mL treatment solution was evenly applied to the white plastic basin ( Each pot contains 2.5kg of test soil), 4 glass beads are put into the solution, and it is sealed with air-permeable sealing film. The water temperature setting and oscillation frequency are respectively set at 29°C and 100 rpm min ^-1 , and the oscillation time is 24h. Mix the solution and the rare earth tailings matrix evenly. In order to prevent the surrounding litter from entering the potted plants and reduce the rate of water loss in the potted plants, a transparent film was covered after the test was inoculated. In order to ensure water supply, within 2 weeks after the inoculation of algae solution, continue to apply 370mL·m ^-2 ·d ^-1 /2.5kg of soil BG-11 culture solution to supplement the water and nutrients in the rare earth tailings matrix for the growth of algae .

Treatment group 2:

The difference with treatment group 1 is that the inoculated algae suspension is the mixed algae suspension mixed according to the chlorophyll content of 2:1 of the Chlamydomonas algae suspension B of embodiment 2 and the nitrogen-fixing Nostoc algae suspension C of embodiment 3, The rest were the same as treatment group 1.

Treatment group 3:

The difference from treatment group 1 is that the inoculated algae suspension is the mixed algae suspension mixed with the microcoleopsis algae suspension D of embodiment 4 and the nitrogen-fixing nostoc algae suspension C of embodiment 3 according to the chlorophyll content of 2:1 , and the rest are the same as treatment group 1.

Treatment group 4:

The difference from treatment group 1 is that the inoculated algae suspension is the mixed algae suspension mixed with the Cyanophyta phylum suspension A of Example 1 and the Chlorophyta Chlamydomonas suspension B of Example 2 according to the chlorophyll content of 2:5. solution, and the rest were the same as treatment group 1.

Control group:

The difference from treatment group 1 is that algae are not inoculated, and the rest are the same as treatment group 1.

See Table 2 for the specific combinations of the control group and treatment groups 1-4.

The combination mode of the algae suspension prepared in table 2 embodiment 1～4

The specific detection content is as follows:

(1) Identification and determination of dominant species: Direct observation with an optical microscope is used to draw 200 μL of culture solution inoculum with a sampling gun to make temporary water-mounted slices. Take 3 temporary slices for each sample, and observe at least 10 fields of view, use an optical microscope with an imaging system to observe the shape of algae under a 40x objective lens and a 100x oil lens, take pictures of individual algae, and count and count different types. According to their shape, structure, size and other characteristics, refer to " "Freshwater Algae in China" and others compared and identified them, and finally determined the dominant species according to the frequency of occurrence.

Fig. 2 is the morphological figure of the Chlamydomonas purified and cultivated in Example 1 and the morphological figure of the Sheath filamentous algae purified and cultivated in Example 2, wherein (a) and (b) are Chlamydomonas Chlamydomonas, (c) and (d ) is Cyanophyta sheath filaments.

As can be seen from Figure 2, the present invention purifies and cultivates two kinds of algae from the biocrust tissue of the abandoned rare earth mining area in Daluoshi, Wenfeng Town, Xunwu County, Ganzhou City, Jiangxi Province, respectively, which are spherical Chlorophyta Chlamydomonas and filamentous cyanobacteria Phyllanthus phylum.

(2) Coverage: Measured by point needle method, record the number of occurrences of algae crusts under the focal point of a 15cm×15cm grid.

It can be seen from Figure 3 that after the surface layer of rare earth tailings is inoculated with native soil algae and/or widespread algae, the coverage of algal crusts is 100%, indicating that the inoculation of native soil algae and/or widespread algae significantly improves the surface soil algae. For the covered area of the crust, Figure 4b shows that when the Chlamydomonas Chlamydomonas is more than that of the Cyanophyta, the two grow together, and the ratio of chlorophyll content is relatively stable.

(3) Biomass determination of algal crusts

Table 3 shows the development characteristics of algae crusts after different treatments, using biomass and thickness to characterize the growth state of the crusts. Determination of algal crust biomass: the biomass of algal crust is expressed by chlorophyll content (Chl-a). Weigh 2.0g of the freeze-dried crust sample into a 10mL centrifuge tube, add 5mL of 95% ethanol solvent, overnight at 4°C in the dark, centrifuge at 8000rpm for 5min, take the supernatant, and use a UV spectrophotometer at wavelengths of 665 and 750nm respectively Measure the absorbance value at the place, then add 5 drops of 1mol·L ^-1 hydrochloric acid to acidify, set it at wavelength 665 and 750nm respectively after 90s, and measure the absorbance value again. The formula for calculating the chlorophyll a content of algae is:

Chl-a(mg·g ^-1 )＝27.9×[(E ₆₆₅ -E ₇₅₀ )-(A ₆₆₅ -A ₇₅₀ )]×V/M

E665 and E750 are the absorbance values of the extract before acidification, A665 and A750 are the absorbance values of the extract after acidification, respectively, V and M represent the volume of the extract (mL) and the mass of the crust sample (g), respectively. The results are shown in Table 3.

Table 3 Development characteristics of algal crusts after different treatments (mean ± standard deviation)

Note: Lowercase letters with different superscripts indicate significant differences among the means in the same column (p<0.05).

It can be seen from Table 3 that the crust thickness increases with the increase of inoculation time, and the difference between the BG-11 treatment group and the control group is significant (p<0.05, p=0.026), but after adding agar and yellow soil, there is no obvious difference with the control group. It shows that the main factor limiting the ecological restoration and succession of rare earth tailings is the lack of nutrients, but human disturbance can shorten the time for algae colonization. After 30 days of algae inoculation, the average thickness of the algal crust was 1.22 mm. The previous field survey found that the average thickness of the naturally formed biological crust in the rare earth mining area was 2.14 mm, indicating that artificial inoculation can accelerate the bonding of algae and tailings particles. Compared with the BG-11 control group, there was no significant difference in crust thickness between the agar control group and the BG-11 control group, indicating that the addition of agar also had a cementing effect on soil particles. The order of thickness of algae crust was agar treatment group>BG-11 treatment group>yellow soil treatment group, which were 1.08, 1.02 and 0.98mm respectively.

It can be seen from Table 3 that the order of chlorophyll content of each algae combination after inoculation 60 days after adding each additive is BG-11 treatment group > yellow soil treatment group > agar treatment group, and the average chlorophyll concentrations are 2.63, 2.34, and 2.00 mg·g, respectively ^-1 . However, under the combination of 4 different algae treatments (see Table 2 for treatment methods), when the inoculation ratio of Chlamydomonas chlorophyta and Coleurophyta cyanophyta is 2:1, the average chlorophyll content is the largest, which is 2.75 mg·g ^-1 , the order of size is algae combination 3 > algal combination 4 > algal combination 1 > algal combination 2, but the thickness of algal crust is the lowest. It shows that the use of agar according to nitrogen-fixing Nostoc: Chlamydomonas spheroides = 2:1 is the best inoculation method, which is more conducive to the rapid development of algae, but the ability of nitrogen-fixing Nostoc to soil particles is relatively weak, resulting in algal knots. The skin thickness is the lowest.

(4) Growth curve measurement: A certain amount of each component solution was sterilized in an autoclave (121°C, 30min), cooled and mixed to obtain BG-11 culture solution (Table 1). Inoculate the two kinds of indigenous algae Coleoptera and Chlamydomonas Chlamydomonas of Example 1 and Example 2 into a 1L Erlenmeyer flask containing 400mL of the above-mentioned BG-11, and culture them statically (light-to-dark ratio=12h: 8h, The light intensity is 2500-3500lx, the temperature is 25±5°C), shake manually 3 times a day, and measure the chlorophyll content with a phytoplankton classification fluorescence instrument (PHYTO-PAM-II, Germany). After culturing for 10 days, the algae cells began to agglomerate, and the determination of the chlorophyll content was stopped. The variation characteristics of the chlorophyll content of the two indigenous algae Cyanophyta cyanophyta and the Chlorophyta Chlamydomonas with culture time in Examples 1 and 2 are shown in FIG. 4 .

It can be seen from Fig. 4 that when the biomass of Chlamydomonas cyanophyta is lower than that of Cyanophyta cyanophyta, the biomass increase rate of Cyanophyta cyanophyta increases with the inoculation time at the initial stage of inoculation, and the growth rate is about 48.95 μg L ^{- 1} ·d ^-1 , 7 days after inoculation, the growth rate of Cyanophyta cyanophyta gradually decreased to a stable level, and the growth rate was about 18.2μg·L ^-1 ·d ^-1 . Chlamydomonas chlorophyta grows fast, the growth rate is about 1.4μg·L ^-1 ·d ^-1 , while the growth rate of cyanophyta is about 1.2μg·L ^-1 ·d ^-1 , both of them show growth The amount decreased with the increase of culture time. However, when the biomass of Chlorophyta Chlamydomonas is higher than that of Cyanophyta Chlamydomonas, Chlamydomonas Chlorophyta grows slowly at the initial stage of inoculation, and the growth rate of Chlamydomonas Chlorophyta is much faster than that of Cyanophyta, and Cyanophyta Chlamydomonas grows much faster than that of Cyanophyta. Restricted, after 9 days of culture, the chlorophyll content of Chlamydomonas chlorophyll rose rapidly from 20μg·L ^-1 to 934.9μg·L ^-1 . The total chlorophyll content reached 1076.2μg·L ^-1 , the green algae Chlamydomonas accounted for 87.16%, and the cyanophyta only accounted for 13.20%. The content ratio is maintained between 2:1 and 4:1.

(5) Effect of BG-11 culture medium on crust structure in rare earth tailings

Fig. 5 is a graph showing the change of rare earth tailings after adding BG-11 culture solution without algae for 46 days. It can be seen from Fig. 5 that direct addition of BG-11 nutrient solution is conducive to the colonization of surface tailings by airborne microorganisms (such as bryophyte spores, etc.) through direct apparent observation and microscope observation.

(6) Scanning electron microscope (SEM): Take a small sample of the upper layer of algae crust, freeze-dry it and spray it with gold coating, use a scanning electron microscope to observe and take pictures of the ultrastructural features of the upper layer of the sample. Figure 6 is the SEM microstructure diagram of the algal crust on the surface of the tailings soil after inoculation of different algae combinations with magnifications of 2000 times and 5000 times respectively, and the magnifications of numbers a, c, e, h, j and l in the figure are 2000 times, The magnifications of b, d, f, i, k and m are 5000 times, that is, the picture on the right (×5000 times) is the magnified part inside the red circle of the picture on the left (×2000 times).

As shown in Figure 6, a large number of needle-like minerals exist in rare earth tailings. After adding agar, a layer of film was formed on the surface of rare earth tailings, which has a protective effect on the surface particles. 30 days after tailings inoculation, add algae to the agar treatment group, the algae attached or embedded on the agar film, the surface is loose and porous, with a larger specific surface area. After the blank group was inoculated for the same time, no algae appeared on the surface or soil particles aggregated, indicating that compared with the inoculated test group, it took longer for microorganisms or particles in the air to attach to the rare earth tailings on the surface. After adding the algae-containing yellow soil solution, the algae and the yellow soil particles formed aggregates, and the aggregates were closely combined. However, the rare earth tailings directly added to the yellow soil solution formed a granular matrix with particle-particle contact, indicating that algal cells can cement fine particles to form aggregates. The microstructure of adding BG-11 nutrient solution containing algae is obviously different from that of directly adding BG-11 nutrient solution. The content of coarse particles is obviously more than that of fine particles, and the algae cells are embedded in the soil particles. In Fig. 6, the spherical green algae Chlamydomonas were observed clearly, and the native Chlorophyta Chlamydomonas with strong tolerance were dominant. After 30 days of inoculation, the cohesion between soil particles was still dominated by the extracellular gelatin sheath.

(7) Soil physical and chemical properties test:

A. pH: measured by pH meter method. After taking the sample back to the room, air-drying, pulverizing, and removing impurities, mix the soil and distilled water according to the water-to-soil ratio of 2.5:1, stir and let it stand for 1 hour, and measure the pH of the supernatant solution sequentially with an acidity meter (Lemag). Three replicates were determined.

B. Determination of soil organic matter: at a certain temperature (100°C, 90min) oxidize the organic carbon in the soil with heavy chromium, part of the hexavalent chromium (Cr ⁶⁺ ) is reduced to green trivalent chromium (Cr ³⁺ ), and use The absorbance of trivalent chromium was determined by colorimetry, and the carbon oxidation solution in the glucose standard solution was used as the standard color scale to calculate the organic carbon in the soil and convert it into organic matter content. For the reagents and preparation methods involved, refer to "Soil Agricultural Chemical Analysis Methods" written by Lu Rukun (2000). The calculation method is as follows:

m ₁ - the carbon content of the soil sample detected by the standard curve, mg;

1.724—the conversion coefficient of organic carbon into organic matter;

1.08—Oxidation correction coefficient;

m—mass of soil sample, g.

C. Determination of soil ammonia nitrogen (NH ₄ ⁺ -N): the NH ₄ ⁺ strong alkaline medium in the soil leachate reacts with hypochlorite and phenol to generate water-soluble fuel indophenol blue, and the blue color of the solution is very stable. When the concentration of NH ₄ ⁺ is in the range of 0.05-0.5 mg·L ^-1 , its depth is directly proportional to the content. For the reagents and preparation methods involved, refer to "Soil Agricultural Chemical Analysis Methods" written by Lu Rukun (2000). The calculation method is as follows:

w(N)—mass fraction of ammoniacal nitrogen in the soil, mg kg ^-1 ;

ρ—the concentration of nitrogen in the chromogenic solution obtained from the working curve, mg·L ^-1 ;

V—the volume of the chromogenic solution, mL;

ts—multiple of points,

10 ^-3 , 1000—represents the conversion of mL into L; conversion into soil content per kg;

m—the mass of the soil sample.

The basic physical and chemical properties of rare earth tailings soil in different treatment groups are shown in Table 4.

Table 4 Basic physical and chemical properties of rare earth tailings soil in different treatment groups

Table 4 shows the basic physical and chemical properties of rare earth tailings in different treatment groups. It can be seen from Table 4 that compared with the rare earth tailings soil without algae, the ammonia nitrogen content in the yellow soil, agar, BG-11 treatment group and control group decreased by 93.51%, 94%, 91.75% and 91.66%, respectively. The soil pH was between 4.5 and 5.5, the organic matter content increased by 85.26%, and the organic matter content in the rare earth tailings soil increased from 0.23g·kg ^-1 to 1.56g·kg ^-1 .

From the results of the foregoing examples, it can be seen that when the biological crust restoration material for the ion-promoting rare earth tailings area provided by the present invention is used for rapid ecological restoration of the ion-promoting rare earth tailings area, it can significantly increase the content of organic matter in the tailings soil matrix, Reducing the content of ammonia nitrogen, increasing the surface crust area of tailings, and greatly reducing the exposed surface can quickly improve the extremely degraded ecological environment caused by the abandoned tailings in ion-type rare earth abandoned mines, and improve the mining area caused by the destruction of mountains and mining of rare earth mines. Soil degradation and environmental pollution.

Although the foregoing embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention rather than all embodiments, and people can also obtain other embodiments according to the present embodiment without inventive step, and these embodiments are all Belong to the protection scope of the present invention.

Claims

A biological crust restoration material for promoting the ecological restoration of ionic rare earth tailings, characterized in that it includes broad-leaved algae and/or native soil algae;

The native soil algae include Cyanophyta Chlamydomonas and/or Chlorophyta Chlamydomonas; the preservation number of the Cyanophyta Chlamydomonas is CCTCC No: M 2021758; the preservation number of the Chlorophyta Chlamydomonas is CCTCC No. : M 2021324;

The broad-leaved algae include Nostoc nitrogen-fixing and/or Microcoletheca.
The biological crust restoration material for promoting the ecological restoration of the ion-type rare earth tailings area according to claim 1, characterized in that, when the biological crust restoration material for the ecological restoration of the ion-promoting rare earth tailings area includes broad-leaved algae and native soil When algae, the ratio of the content of chlorophyll in the broad-leaved algae to the content of chlorophyll in the local soil algae is (0-5): (0-5), and the content of the chlorophyll in the said broad-spread algae and the chlorophyll in the native soil algae not at the same time is 0;

When the biocrust repair material for promoting the ecological restoration of the ion-type rare earth tailings area includes Cyanophyta and Chlorophyta Chlamydomonas, the content of chlorophyll in the Cyanophyta Chlamydomonas and Chlorophyta Chlamydomonas The content ratio is (0～5):(0～5), and the content of chlorophyll in the cyanophyta Chlamydomonas and the chlorophyll in Chlamydomonas cyanophyta are both 0;

When the biocrust repair material for promoting the ecological restoration of the ion-type rare earth tailings area includes nitrogen-fixing Nostoc and Microcoletheca, the content ratio of chlorophyll in the nitrogen-fixing Nostoc and Microcoletheca is: (0-5): (0-5), and the content of chlorophyll in the nitrogen-fixing Nostoc and the chlorophyll in Microcoletheca are different from 0 at the same time.
The biological crust restoration material for the ecological restoration of the ion-promoting rare earth tailings area according to claim 2, wherein the biological crust restoration material for the ecological restoration of the ion-promoting rare earth tailings area includes broad-leaved algae and native soil algae, the ratio of the content of chlorophyll in the broad-leaved algae to the content ratio of the chlorophyll in the native soil algae is (1～4):(1～5);

When the biocrust repair material for promoting the ecological restoration of the ion-type rare earth tailings area includes Cyanophyta and Chlorophyta Chlamydomonas, the content of chlorophyll in the Cyanophyta Chlamydomonas and Chlorophyta Chlamydomonas The content ratio is (1～4): (1～5);

When the biocrust repair material for promoting the ecological restoration of the ion-type rare earth tailings area includes nitrogen-fixing Nostoc and Microcoletheca, the content ratio of chlorophyll in the nitrogen-fixing Nostoc and Microcoletheca is: (1~4): (1~5).
The biological crust repair material for ecological restoration of ion-promoting rare earth tailings area according to claim 1, characterized in that the application amount of the bio-skin repair material for ecological restoration of ion-promoting rare earth tailing area is 150-950 μg ( chlorophyll) m -2 (soil to be repaired).
The application of the biological crust restoration material for promoting the ecological restoration of the ion-type rare earth tailings area according to any one of claims 1 to 4 in repairing the ion-type rare earth tailings area.
A method for repairing ionic rare earth tailings area, characterized in that it comprises the following steps:

Applying the biological crust restoration material for promoting the ecological restoration of the ion-type rare earth tailings area according to any one of claims 1 to 4 to the soil of the ion-type rare earth tailings area to be repaired for restoration.
The method according to claim 6, characterized in that, the biological crust repair material for promoting the ecological restoration of the ion-type rare earth tailings area is applied to the soil of the ion-type rare earth tailings area to be repaired in the form of algae suspension.
The method according to claim 6, characterized in that the application amount of the biological crust restoration material for the ecological restoration of the ion-promoting rare earth tailings area is 100-1000 μg (chlorophyll)·m −2 (soil to be repaired).
The method according to claim 7, characterized in that, the preparation method of the algae suspension of the biological crust repair material for the ecological restoration of the ion-promoting rare earth tailings area comprises the following steps:

Inoculate the biological crust restoration material in the ion-promoting rare earth tailings area into the culture solution, and cultivate to obtain the algae suspension.
The method according to claim 9, characterized in that the culture solution is a sterile BG-11 culture solution.
The method according to claim 9, characterized in that the light intensity of the cultivation is 2500-3500 lx.
The method according to claim 9 or 11, characterized in that the light-dark ratio of the culture is 16h:8h or 12h:12h.
The method according to claim 9, characterized in that, the cultivation method is static cultivation, and the cultivation temperature is 25±5°C.
The method according to claim 9 or 13, characterized in that the culture period is 3-4 weeks.
The method according to claim 7, characterized in that the content of chlorophyll in the algae suspension of the biological crust restoration material for the ecological restoration of the ion-promoting rare earth tailings area is 100-1000 μg·L -1 .
The method according to claim 7, characterized in that the content of chlorophyll in the algae suspension of the biological crust restoration material for the ecological restoration of the ion-promoting rare earth tailings area is 150-950 μg·L -1 .