AU2021286451A1

AU2021286451A1 - Biological crust repairing material and restoration method for promoting ecological restoration of ion-absorbed rare earth tailings area and use thereof

Info

Publication number: AU2021286451A1
Application number: AU2021286451A
Authority: AU
Inventors: Bing Feng; Mingshu Li; Yan Liu; Zugen LIU; Qiang Wang; Junwei WU; Na Yao; Meng Zhang; Min Zhou
Original assignee: Jiangxi Academy Of Eco Env Sciences And Planning
Current assignee: Jiangxi Academy Of Eco Environmental Sciences And Planning
Priority date: 2021-08-04
Filing date: 2021-08-20
Publication date: 2023-02-23
Also published as: WO2023010620A1; CN113857235A; CN113857235B

Abstract

OF THE DISCLOSURE The present disclosure belongs to the technical field of ecological restoration of mines, and specifically relates to a biological crust repairing material and restoration method for promoting ecological restoration of an ion-absorbed rare earth tailings area and use thereof. The biological crust repairing material for promoting ecological restoration of an ion-absorbed rare earth tailings area provided in the present disclosure comprises cosmopolitan algae and/or native soil-dwelling algae; the native soil-dwelling algae include L. sp. and/or C. sp.; and the cosmopolitan algae include N. sp. and/or M. vaginatus. In the present disclosure, applying the culture medium of cosmopolitan algae and native soil-dwelling algae to the soil in the ion-absorbed rare earth tailings area to be repaired may significantly increase the content of organic matter in the tailings soil matrix, reduce the content of ammonia nitrogen, increase the crust area on the surface of the tailings, and greatly reduce the exposed surface, which may quickly improve the extremely degraded ecological environment caused by abandoned tailings in abandoned ion-absorbed rare earth mines, and improve soil degradation and environmental pollution in mining areas caused by the mining of rare earth mines.

Description

BIOLOGICAL CRUST REPAIRING MATERIAL AND RESTORATION METHOD FOR PROMOTING ECOLOGICAL RESTORATION OF ION-ABSORBED RARE EARTH TAILINGS AREA AND USE THEREOF CROSS REFERENCE TO RELATED APPLICATION

[01] This patent application claims the priority of Chinese Patent Application No. 202110889960.8, entitled "biological crust repairing material and restoration method for

promoting ecological restoration of an ion-absorbed rare earth tailings area and use

thereof' filed with the China National Intellectual Property Administration on August

04, 2021, which is incorporated by reference herein in its entirety as part of the present

application.

TECHNICAL FIELD

[02] The present disclosure belongs to the technical field of ecological restoration of

mines, and specifically relates to a biological crust repairing material and a restoration

method for promoting ecological restoration of an ion-absorbed rare earth tailings area

and use thereof.

BACKGROUNDART

[03] Ion-absorbed rare earth is a national strategic resource, which is non-renewable

and is widely used in national defense construction and high-tech fields. The mining of

ion-absorbed rare earths creates high profits while causing a series of ecological and

environmental problems such as destruction of vegetation and land resources, and water

and soil pollution, such as loose soil, serious soil desertification, and the phenomenon of

no grass growing (known as the "southern desert"). During the rainstorm season in

south of China, water and soil erosion is also prone to occur, resulting in a large number

of abandoned slopes, and severe geological disasters such as land subsidence, collapse

and landslide caused by unstable slope, exposed surface and lack of vegetation, which

severely restrict and hinder the agricultural and social development. Therefore, in view of the damage to the surrounding environment caused by the mining of ion-absorbed rare earth mines, it is urgent to carry out ecological reconstruction of the ion-absorbed rare earth tailings area in southern Jiangxi.

SUMMARY

[04] In view of this, the object of the present disclosure is to provide a biological crust repairing material and restoration method for promoting ecological restoration of an ion-absorbed rare earth tailings area and use thereof. The biological crust repairing material may increase the content of organic matter in the tailings soil, reduce the content of ammonia nitrogen, increase the crust area of the tailings surface, greatly reduce the exposed surface, and may improve the problems of soil degradation and environmental pollution in mining areas.

[05] In order to achieve the above objectives, the present disclosure provides the following technical schemes:

[06] The present disclosure provides a biological crust repairing material for promoting ecological restoration of an ion-absorbed rare earth tailings area, comprising cosmopolitan algae species and/or native soil algae; the native soil-dwelling algae species include Lyngbya sp. and/or Chlamydononas sp.; the patent preservation number of the L. sp. is CCTCC No. M 2021758; the patent preservation number of the C. sp. is CCTCC No. M 2021324; the cosmopolitan algae species include Nostoc sp. and/or Microcoleus vaginatus.

[07] In some embodiments, when the biological crust repairing material for promoting ecological restoration of an ion-absorbed rare earth tailings area includes the cosmopolitan algae and native soil-dwelling algae species, a ratio of chlorophyll a content in the cosmopolitan algae to chlorophyll a content in the native soil-dwelling algae is (0-5): (0-5), and chlorophyll a content of the cosmopolitan algae and chlorophyll a of the native soil-dwelling algae are not both 0;

[08] when the biological crust repairing material includes L. sp. and C. sp., a ratio of chlorophyll a content in L. sp. to chlorophyll a content in C. sp. is (0-5): (0-5), and chlorophyll a content of L. sp. and chlorophyll a content of C. sp. are not both 0;

[09] when the biological crust repairing material includes N. sp. and M. vaginatus, a ratio of chlorophyll a content in the N. sp. to chlorophyll a content in the M. vaginatus is

(0-5): (0-5), and chlorophyll a content of N. sp. and chlorophyll a content of M.

vaginatus are not both 0.

[10] In some embodiments, the applied amount of the biological crust repairing material is 100-1000 pg (chlorophyll a)-m-2 (soil under repair).

[11] The present disclosure also provides a use of the biological crust repairing

material for promoting ecological restoration of an ion-absorbed rare earth tailings area

described in the above technical schemes in the repair of ion-absorbed rare earth tailings

areas.

[12] The present disclosure also provides a method for repairing an ion-absorbed rare earth tailings area, comprising the following steps:

[13] applying the biological crust repairing material for promoting ecological restoration of an ion-absorbed rare earth tailings area described in the above technical

schemes to the soil of the ion-absorbed rare earth tailings area to be repaired for

restoration.

[14] In some embodiments, the biological crust repairing material for promoting

ecological restoration of an ion-absorbed rare earth tailings area is applied to the soil of

the ion-absorbed rare earth tailings area to be repaired in the form of an algal

suspension.

[15] In some embodiments, a method for preparing the algal suspension includes the

following steps:

[16] inoculating the biological crust repairing material for promoting ecological restoration of an ion-absorbed rare earth tailings area in a culture medium and

cultivating to obtain an algal suspension.

[17] In some embodiments, the culture medium is a sterile BG-11 culture medium.

[18] In some embodiments, a light intensity of the cultivating is 2500-3500 lx; a

light-to-dark ratio of the cultivating is 16 h: 8 h or 12 h: 12 h; a method for cultivating is

static cultivation, and a temperature of the cultivating is 25± 5C; and a time of the

cultivating is 3-4 weeks.

[19] In some embodiments, chlorophyll a content in the algal suspension is 100-1000 pg-'.

[20] The present disclosure provides a biological crust repairing material for promoting

ecological restoration of an ion-absorbed rare earth tailings area, comprising

cosmopolitan algae and/or native soil-dwelling algae; the native soil-dwelling algae

include L. sp. and/or C. sp.; the patent preservation number of the L. sp. is CCTCC No.

M 2021758; the patent preservation number of the C. sp. is CCTCC No. M 2021324;

the cosmopolitan algae include N. sp. and/or M. vaginatus. When the repairing material

for promoting ecological restoration of ion-absorbed rare earth tailings area provided by

the present disclosure is used to promote the ecological restoration of the ion-absorbed

rare earth tailings area, the extracellular secretions of algae cells are an important source

of organic matter in the soil, which may significantly increase the content of organic

matter in the soil of the tailings area. The salt ions in the nutrient solution may promote

the leaching of ammonia nitrogen in the soil, thereby reducing the content of ammonia

nitrogen in the soil, increasing the crust area on the surface of the tailings, greatly

reducing the exposed surface, and quickly improving the extremely degraded ecological

environment caused by abandoned tailings in abandoned ion-absorbed rare earth mines,

and solving the problems of soil degradation and environmental pollution in mining

areas caused by leaching mining of rare earth mines.

BRIEF DESCRIPTION OF THE DRAWINGS

[21] FIG. 1 shows the morphology of native soil-dwelling algae under the microscope;

[22] FIG. 2 shows the morphology of the purified and cultivated C. sp. in Example 1 and the morphology of the purified and cultivated L. sp. in Example 2;

[23] FIG. 3 shows the changes of algae crusts on the surface of the soil after

inoculation of algae with different treatments;

[24] FIG. 4 shows the changes in the chlorophyll a content of the native soil-dwelling

algae in Example 1 and Example 2 with the cultivation time;

[25] FIG. 5 shows the changes of algae crust on the surface after 46 d of adding BG-11

culture medium without algae to the rare earth tailings soil;

[26] FIG. 6 shows the SEM microstructure of algae crust on the surface of tailings soil after inoculation with different algae combinations at magnification of 2000 times and

5000 times, respectively.

MATERIALS DEPOSITION

[27] Lyngbya sp. JXSHKY-1 was deposited in China Center for Type Culture Collection on June 24, 2021, the address is Wuhan University Depository Center

(opposite to the First Affiliated Primary School of Wuhan University), No. 299, Bayi

Road, Wuchang District, Wuhan City, Hubei Province, and the deposition number is

CCTCC No. M 2021758;

[28] Chlamydononas sp. JXSHKY-2 was deposited in China Center for Type Culture Collection on April 2, 2021, the address is Wuhan University Depository Center

CCTCC No. M 2021324.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[29] The present disclosure provides a biological crust repairing material for promoting

ecological restoration of an ion-absorbed rare earth tailings area, comprising

cosmopolitan algae and/or native soil-dwelling algae;

[30] the native soil-dwelling algae include L. sp. JXSHKY-1 and/or C. sp. JXSHKY-2;

the deposition number of the L. sp. is CCTCC No. M 2021758; the deposition number

of the C. sp. is CCTCC No. M 2021324.

[31] The cosmopolitan algae include N. sp. and/or M. vaginatus.

[32] In some embodiments, the preferred species of the native soil-dwelling algae are

selected from the abandoned rare earth mining area of Daluoshi, Wenfeng Town,

Xunwu County, Ganzhou City, Jiangxi Province.

[33] In some embodiments, a method for separating and purifying the native

soil-dwelling algae preferably includes the following steps:

[34] collecting biological crust samples formed under natural conditions in the abandoned rare earth mining area of Daloshi, Wenfeng Town, Xunwu County, Ganzhou

City, Jiangxi Province, sterilizing with 75 % alcohol to prevent cross-contamination

during the collection process, placing the sterilized sample in a sterile aluminum box

(specification: 50 mm x 30 mm), transporting the sample back to the laboratory, and

storing at a low temperature of -4°C; taking 0.5 g of biological crust sample into a 150

mL Erlenmeyer flask containing sterile BG-11 culture medium (see Table 1 for the

composition and preparation method of BG-11 culture medium) under sterile

environmental conditions, culturing statically in a light incubator under specific

environmental conditions (light-to-dark ratio = 16 h: 8 h, light intensity of 3000 lx,

temperature of 25°C) for 3-4 weeks after being evenly dispersed in a homogenizer,

taking 2 pL of the algal solution into a 24-well cell culture plate containing 2 mL of

sterile BG-11 culture medium by a capillary tube to for cultivation when a clear algal

solution is formed (cultivation conditions are as described above), and when it is clearly

green, picking up and selecting L. sp. JXSHKY-1 or C. sp. JXSHKY-2 by a capillary

tube under a microscope (optical microscope, CX33RTFS2, OLYMPUS, Japan),

respectively, inoculating the selected L. sp. JXSHKY-1 or C. sp. JXSHKY-2 into sterile

BG-11 culture medium under aseptic conditions for separate cultivation (The cultivation

conditions are as described above), and repeating the separate cultivation for 8 times to

obtain a single purified culture of L. sp. JXSHKY-1 or C. sp. JXSHKY-2.

[35] In some embodiments, the preferred species of the cosmopolitan algae are purchased from the Algae Culture Collection at the Institute of Hydrobiology, Chinese

Academy of Sciences, wherein, the deposition number of N. sp. is FACHB-119, and the

deposition number of the M. vaginatus is FACHB-253.

[36] In some embodiments, when the biological crust repairing material for promoting ecological restoration of an ion-absorbed rare earth tailings area includes the

cosmopolitan algae and the native soil-dwelling algae, a ratio of chlorophyll a content in

the cosmopolitan algae to chlorophyll a content in the native soil-dwelling algae is

preferably (0-5): (0-5), and chlorophyll a content in the cosmopolitan algae and

chlorophyll a in the native soil-dwelling algae are not both 0; and a ratio of chlorophyll

a content in the cosmopolitan algae to chlorophyll a content in the native soil-dwelling algae is more preferably (1-4): (1-5);

[37] when the biological crust repairing material for promoting ecological restoration of an ion-absorbed rare earth tailings area includes Lyngbya sp. and Chlamydononas sp.,

a ratio of chlorophyll a content in the L. sp. to chlorophyll a content in the C. sp. is

preferably (0-5): (0-5), and chlorophyll a content in L. sp. and chlorophyll a content in

C. sp. are not both 0; and a ratio of chlorophyll a content in Lyngbya sp. to chlorophyll a

content in C. sp. is more preferably (1-4): (1-5);

[38] when the biological crust repairing material for promoting ecological restoration

of an ion-absorbed rare earth tailings area includes N. sp. and M. vaginatus, a ratio of

chlorophyll a content in Nostoc sp. to chlorophyll a content of M. vaginatus is

preferably (0-5): (0-5), and chlorophyll a content in Nostoc sp. and chlorophyll content

in M. vaginatus are not both 0; and a ratio of chlorophyll a content in the N. sp. to

chlorophyll a content of the M. vaginatus is more preferably (1-4): (1-5);

[39] In some embodiments, the applied amount of the biological crust repairing

is 100-1000 pg (chlorophyll a)-m-2 (soil to be repaired), and more preferably 150-950

pg (chlorophyll a)-m-2 (soil to be repaired).

[40] The present disclosure also provides a use of the biological crust repairing

areas.

[41] The present disclosure also provides a method for repairing an ion-absorbed rare

earth tailings area, comprising the following steps:

[42] applying the biological crust repairing material for promoting ecological restoration of an ion-absorbed rare earth tailings area described in above technical

restoration.

[43] In some embodiments, the biological crust repairing material for promoting

ecological restoration of an ion-absorbed rare earth tailings area is preferably applied to

the soil of the ion-absorbed rare earth tailings area to be repaired in the form of an algal suspension.

[44] In some embodiments, a method for preparing the algal suspension includes the following steps:

[45] inoculating the biological crust repairing material for promoting ecological restoration of an ion-absorbed rare earth tailings area in a culture medium and

cultivating to obtain an algal suspension.

[46] In some embodiments, the culture medium is preferably a sterile BG-11 culture medium; the composition of the sterile BG-11 culture medium is shown in Table 1; the

present disclosure does not have any special restrictions on the method for preparating

the BG-11 culture medium, and the mother liquor can be prepared according to the

formula in Table 1 according to the well-known process in the art and mixed to form the

BG-11 culture medium.

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

[48]

Type of Culture Medium BG-11

Concentration of Dosage Composition Mother Liquor

NaNO3 15 g/100 ml dH20 10 mL-L-'

K2HPO 4 .3H 20 2 g/100 mL dH20 10 mL-L-'

MgSO4-7H20 3.75 g/100 mL dH20 10 mL-L-'

CaCl2-2H20 1.8 g/100 mL dH20 10 mL-L-'

Citric acid 0.3 g/100 mL dH20 10 mL-L-'

Ferric ammonium citrate 0.3 g/100 mL dH20 10 mL-L-'

EDTA Na 2 0.05 g/100 mL dH 20 10 mL-L-'

Na 2 CO 3 1.0 g/100ml dH 20 10 mL-L-'

A5 solution 1mL-L-1 (Trace mental solution)*

[49] Note: A5 solution (diluted to 1000 mL with water): H 3B0 3 (61.0 mg);

MnSO4 -H20 (169.0 mg); ZnSO 4 -7H 20 (287.0 mg); CuSO4-5H 2 0 (2.5 mg); (NH4 )

Mo 702 4 -4H20 (12.5 mg).

[50] Unless otherwise specified, the present disclosure has no special requirements on the source of the raw materials used to prepare the BG-11 culture medium and

commercially available products well known to those skilled in the art can be used. The

present disclosure does not specifically limit the sterilization method of the sterile

BG-11 culture medium, as long as the sterilization method well known in the art can be

used.

[51] In some embodiments, the inoculation is preferably performed under aseptic conditions. The present disclosure does not specifically limit the inoculation method, as

long as the method well known in the art can be used. In some embodiments, the device

for cultivating is preferably a light incubator; a method for cultivating is preferably

static cultivation; a light-to-dark ratio of the cultivating is preferably 16 h: 8 h or 12 h:

12 h. a light intensity of the cultivating is preferably 2500-3500 lx, a temperature of the

cultivating is preferably 25± 5C; a time of the cultivating is preferably 3-4 weeks.

[52] In some embodiments, chlorophyll a content in the algal suspension is preferably

100-1000 pg-L-, and more preferably 150-950 pg-L-1.

[53] The technical schemes of the present disclosure will be clearly and completely

described below in conjunction with the embodiments of the present disclosure.

[54] Example 1

[55] The biological crust samples formed under natural conditions in the abandoned rare earth mining area of Daloshi, Wenfeng Town, Xunwu County, Ganzhou City,

Jiangxi Province were collected, sterilized with 75% alcohol to prevent

cross-contamination during the collection process, the sterilized samples were placed in

a sterile aluminum box (specification: 50 mm x 30 mm), transported back to the

laboratory, and stored at a low temperature of -4°C. 0.5 g of biological crust sample was

taken into a 150 mL Erlenmeyer flask containing sterile BG-11 culture medium (see

Table 1 for the composition and preparation method of BG-11 culture medium) under

sterile environmental conditions. The sample was cultured statically in a light incubator

under specific environmental conditions (light-to-dark ratio = 16 h: 8 h, light intensity

of 3000 lx, temperature of 25°C) for 4 weeks after being evenly dispersed in a homogenizer. 2 pL of the algal solution was taken into a 24-well cell culture plate containing 2 mL of sterile BG-11 culture medium by a capillary tube for cultivation when a clear algal solution was formed (cultivation conditions were as described above), and when it was clearly green, the sample was observed under an optical microscope

(CX33RTFS2, OLYMPUS, Japan). The morphology of the native soil-dwelling algae in

the algal solution under the microscope was shown in FIG. 1. From the microscopic

results in FIG 1, it could be seen that the cultured algae mainly included the filamentous

L. sp. JXSHKY-1 and the spherical C. sp. JXSHKY-2. Spherical C. sp. was embedded

and distributed among the entwined filamentous L. sp.. The L. sp. JXSHKY-1 was

picked up and selected using a capillary tube under the microscope, inoculated into

sterile BG-11 culture medium under aseptic conditions for separate cultivation (the

cultivation conditions were as described above), and the separate cultivation was

repeated for 8 times to obtain a single purified culture of L. sp. JXSHKY-1;

[56] The filamentous L. sp. JXSHKY-1 was inoculated into sterile BG-11 culture

medium, and cultured statically under specific environmental conditions in a light

incubator (light-to-dark ratio = 16 h: 8 h, the light intensity was 3000 lx, the temperature

was 25°C) for 4 weeks to obtain an algal suspension of filamentous L. sp., with a

chlorophyll a content of 192 jig-L-1.

[57] Example 2

[58] The biological crust samples formed under natural conditions in the abandoned rare earth mining area of Daloshi, Wenfeng Town, Xunwu County, Ganzhou City,

Jiangxi Province were collected, sterilized with 75 % alcohol to prevent

a sterile aluminum box (specification: 50 mm x 30 mm), transported back to the

taken into a 150 mL Erlenmeyer flask containing sterile BG-11 culture medium (seen as

Lyngbya sp. JXSHKY-1 and the spherical C. sp. JXSHKY-2. Spherical C. sp. was

embedded and distributed among the entwined filamentous L. sp. The C. sp. JXSHKY-2

was picked up and selected using a capillary tube under the microscope, inoculated into

sterile BG-11 culture medium under aseptic conditions for separate cultivation

(cultivation conditions were as described above), and the separate cultivation was

repeated for 8 times to obtain a single purified culture of C. sp. JXSHKY-2.

[59] The C. sp. JXSHKY-2 was inoculated into sterile BG-11 culture medium, and

cultured statically under specific environmental conditions in a light incubator

(light-to-dark ratio=16 h: 8 h, the light intensity was 3000 lx, and the temperature was

°C) for 4 weeks to obtain an algal suspension of C. sp., with the chlorophyll a content

of 339 ig-L-1.

[60] Example 3

[61] The Nostoc sp. (purchased from the Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences) was inoculated in the sterile BG-11

culture medium, and placed in a light incubator under specific environmental conditions.

(the light-to-dark ratio = 16 h: 8 h, the light intensity was 3000 lx, and the temperature

was 25°C). After culturing for 4 weeks, an algal suspension of N. sp. was obtained, with

the chlorophyll a content of 441 pg-L- 1 .

[62] Example 4

[63] The difference from Example 3 was that the biological crust repairing material for

promoting ecological restoration of an ion-absorbed rare earth tailings area was the

filamentous Microcoleus vaginatus (purchased from the Algae Culture Collection at the

Institute of Hydrobiology, Chinese Academy of Sciences), the others were the same as in Example 3. The chlorophyll a content in the algal suspension of Microcoleus vaginatus was 558 pg-L-1.

[64] Application Example 1

[65] Effect evaluation:

[66] Location: Donghu District, Nanchang City, Jiangxi Province-in the research glass room of Jiangxi Eco-Environmental Science Research and Planning Institute;

[67] The source of the tested soil: Rare earth tailings matrix-abandoned rare earth mining area of Daloshi, Wenfeng Town, Xunwu County, Ganzhou City, Jiangxi

Province; Yellow soil-taken from the undeveloped natural forest land near the

abandoned rare earth mining area of Daloshi, Wenfeng Town, Xunwu County, Ganzhou

City, Jiangxi Province.

[68] Time: November 2020-March 2021.

[69] Cultivation conditions: Effective photosynthetic radiation was about 3.5

pmol-m-2-s-', average temperature was 10°C, and average air humidity was 70% RH.

[70] The basic physical and chemical index of rare earth tailings matrix was as follows:

pH of 4.36, the content of ammonia nitrogen of 173.89 mg-kg- , and the content of

organic matter of 0.236 g-kg- .

[71] Test scheme:

[72] A phytoplankton classification fluorimeter PHYTO-PAM-II was used to

determine the chlorophyll a content of the four algal suspensions obtained in Examples

1-4 (L. sp. A, C. sp. B, N. sp. C, and M. vaginatus D) and draw the growth curve. The

chlorophyll a content in the algal suspension obtained in Examples 1-2 was shown in

FIG. 2.

[73] Treatment Group 1:

[74] The algal suspension A of L. sp. in Example 1 and the algal suspension B of C. sp.

in Example 2 were mixed according to the chlorophyll a content of 2: 1, and 0.03 % of

agar, 3 g of yellow soil and no addition were added as 3 treatments. Sterile BG-11

culture medium was used to make up the volume of each treatment to 45 mL, and each

treatment was set three parallel samples. After shaking for 12 h, 45 mL of the treatment

solution was evenly applied to white plastic pots (each pot containing 2.5 kg of test soil) filled with rare earth tailings matrix. The solution was filled with 4 glass beads and sealed with a breathable sealing film. The water temperature and oscillation frequency were set to 29°C and 100 rpm-min- , respectively, and the shaking time was 24 h to make the solution and the rare earth tailings matrix uniformly mixed. In order to prevent the surrounding leaf-litter from entering the pot and reduce the rate of water loss in the pot, a transparent film was covered after the inoculation test. In order to ensure water supply, within 2 weeks after the inoculation of the algal solution, BG-11 culture medium with a dosage of 370 mL-m-2-d-1/2.5 kg soil was continued to apply to supplement the water and nutrients in the rare earth tailings matrix for the growth of algae.

[75] Treatment Group 2:

[76] The difference from Treatment Group 1 was that the inoculated algal suspension was a mixed algal suspension of suspension B of C. sp. in Example 2 and suspension C of N. sp. in Example 3 which were mixed according to the chlorophyll a content of 2: 1. The rest was the same as Treatment Group 1.

[77] Treatment Group 3:

[78] The difference from Treatment Group 1 was that the inoculated algal suspension was a mixed algal suspension of suspension D of M. vaginatus in Example 4 and suspension C of N. sp. in Example 3 which were mixed according to the chlorophyll a content of 2: 1. The rest was the same as Treatment Group 1.

[79] Treatment Group 4:

[80] The difference from Treatment Group 1 was that the inoculated algal suspension was a mixed algal suspension of suspension A of L. sp. in Example 1 and suspension B of C. sp. in Example 2 which were mixed according to the chlorophyll a content of 2: 5. The rest was the same as Treatment Group 1.

[81] Control Group:

[82] The difference from Treatment Group 1 was that no algae was inoculated, and the rest was the same as Treatment Group 1.

[83] The specific combinations of the Control Group and Treatment Groups 1-4 were shown in Table 2.

[84] Table 2 Combinations of algal suspensions prepared in Examples 1-4

[851 No. Number Combination

Control Group (no algae + BG-11 solution containing 1 CK1 0.03 % agar)

Control Group (no algae + BG-11 solution containing 0.3 g 2 CK2 yellow soil)

3 CK3 Control Group (no algae + BG-11 solution)

Treatment Group 1 (A + B = 2: 1 + BG-11 solution 4 TQl containing 0.03% agar)

Treatment Group 1 (A + B = 2: 1 + BG-11 solution 5 THI containing 0.3 g of yellow soil)

6 TB1 Treatment Group 1 (A + B = 2: 1+ BG-11 solution)

Treatment Group 2 (C + B = 2: 1 + BG-11 solution 7 TQ2 containing 0.03% agar)

Treatment Group 2 (C + B = 2: 1 + BG-11 solution 8 TH2 containing 0.3 g of yellow soil)

9 TB2 Treatment Group 2 (C + B = 2: 1 + BG-11 solution)

Treatment Group 3 (D + B = 2:1 + BG-11 solution 10 TQ3 containing 0.03% agar)

Treatment Group 3 (D + B = 2:1 + BG-11 solution 11 TH3 containing 0.3 g of yellow soil)

12 TB3 Treatment Group 3 (D + B = 2: 1 + BG-11 solution)

Treatment Group 4 (A + B=2: 5 + BG-11 solution 13 TQ4 containing 0.03% agar)

Treatment Group 4 (A + B=2: 5 + BG-11 solution 14 TH4 containing 0.3 g yellow soil)

15 TB4 Treatment Group 4 (A+ B = 2: 5 + BG-11 solution)

In the early stage of inoculation, the algal solution was Inoculation Amount added intermittently according to the above chlorophyll a

No. Number Combination concentration, 45 mL/pot, and the amount of BG-11 culture medium added was about 10 mL/pot; In the later stage of inoculation, 500-600 mL of BG-11 culture medium was continuously added (calculated according to the area of the surface tailings in the culture pots, the inoculation amount was about 370 mL-m-2-d-1, which was equivalent to 10 mL/pot)

[86] The specific testing content was as follows:

[87] (1) Identification and determination of dominant species: method of direct observation under optical microscope was used. 200 pL of culture medium inoculum was drawn using a sample gun to make temporary water-loaded slices. Totally 3 temporary slices were taken for each sample, and at least 10 fields of view were observed for each slice. The algae morphology under a 40 x objective lens and a 100 x oil lens was observed using an optical microscope with an imaging system, pictures of individual algae were taken, and different species were counted. According to the morphology, structure, size and other characteristics, the algae was compared and identified by referring to "The Freshwater Algae of China", etc. Finally, the dominant species were determined according to the frequency of occurrence.

[88] FIG. 2 showed the morphology of the purified and cultivated C. sp. in Example 1 and the morphology of the purified and cultivated L. sp. in Example 2, wherein (a) and (b) represented the C. sp., (c) and (d) represented the L. sp.

[89] It could be seen from FIG. 2 that two kinds of algae purified and cultivated from the biological crusts of the abandoned rare earth mining area in Daluoshi, Wenfeng Town, Xunwu County, Ganzhou City, Jiangxi Province are spherical C. sp. and filamentous L. sp.

[90] (2) Coverage: Measured by the point-needle method, and the number of occurrences of algal crusts under the focal point of a 15 cm x 15 cm grid was recorded.

[91] It can be seen from FIG. 3 that after the surface of the rare earth tailings is inoculated with native soil-dwelling algae and/or cosmopolitan algae, the coverage of algal crust is 100 %, indicating that the inoculation of native soil-dwelling algae and/or cosmopolitan algae significantly improves the coverage of algae crusts on the soil surface, FIG. 4b shows that when C. sp. is more than L. sp., the two grow together, and the ratio of chlorophyll a content is relatively stable.

[92] (3) Determination of biomass of the algal crusts

[93] Table 3 showed the development characteristics of algal crusts after different treatments, using biomass and thickness to characterize the growth status of the crusts.

Determination of biomass of the algal crust: the biomass of the algal crust was

expressed by chlorophyll a content (Chl-a). 2.0 g of the freeze-dried crust sample was

weighed into a 10 mL centrifuge tube, 5 mL of 95 % ethanol solvent was added. The

mixture was kept away from light overnight at 4°C, centrifuged at 8000 rpm for 5 min.

The supernatant was taken, and the absorbance values at wavelengths of 665 nm and

750 nm, were measured respectively using an ultraviolet spectrophotometer, then 5

drops of 1 mol-L-1 hydrochloric acid was added to acidify, and the absorbance values at

wavelengths of 665 nm and 750 nm after 90 s were measured. The formula for

calculating the content of chlorophyll a was as follows:

[94] Chl-a (mg-g- 1)= 27.9 X [(Efsa - E7 50 ) - (A, - A 7 )] X V/M

[95] E665 and E750 were the absorbance values of the extract before acidification,

A665 and A750 were the absorbance values of the extract after acidification, and V and

M represented the volume of the extract (mL) and the weight of the crust sample (g),

respectively. The results were shown in Table 3.

[96] Table 3 Development characteristics of algal crusts after different treatments

(mean standard deviation)

[97]

Chlorophyll Chlorophyll Treatment Thickness Thickness a Content Composition a Content Grup mm /mm Groups /mg-g- 1 /mg-g-1

Treatment 2.00 1.03a 1.08 Algae 2.14 0.10 1.06

Chlorophyll Chlorophyll Treatment Thickness Thickness a Content Composition a Content Grus/mm /mm /mg-g- 1 /mg-g-1

Group with 0.29 Combination 1 0.27

Agar (TQ1-3) (TQl, THI,

TB1)

Treatment Algae

Group with 0.98 Combination 2 1.02 2.34 1.26a 2.13 1.55 Yellow Soil 0.20 (TQ2, TH2, 0.24

(TH1-3) TB2)

Treatment Algae

Group with 1.02 Combination 3 0.97 2.62+ 2 .7 7b 2.75+3.00 BG-11 0.24 (TQ3, TH3, 0.18

(TB1-3) TB3)

Algae

Control Group Combination 4 1.07 0.31 0.36a - 2.28 1.32 (CK1-3) (TQ4, TH4, 0.30

TB4)

[98] Noted: Lowercase letters with different superscripts indicated that there was a

significant difference between the mean values in the same column (p < 0.05).

[99] It can be seen from Table 3 that the crust thickness increases with the increase of inoculation time. There is a significant difference between the treatment group with

BG-11 and the control group (p < 0.05, p = 0.026), while after adding agar and yellow

soil, there is no obvious difference with the control group, indicating that the main

factor restricting the ecological restoration and succession of rare earth tailings is the

lack of nutrient elements, but human disturbance can shorten the time of fixing and

multiplying of algae. After 30 d of inoculation with algae, the average thickness of algae

crust is 1.22 mm. Early field investigations found that the average thickness of

biological crust naturally formed in rare earth mining areas is 2.14 mm, indicating that artificial inoculation can accelerate the adhesion of algae and tailings particles.

Compared with the control group with BG-11, the control group with agar has no

significant difference in crust thickness between the two, indicating that the addition of

agar also has a cementing effect on soil particles. The thickness of algal crust is in the

order of treatment group with agar > treatment group with BG-11 > treatment group

with yellow soil, the values are 1.08, 1.02 and 0.98 mm, respectively.

[100] It can be seen from Table 3 that the order of the chlorophyll a content of each algae combination added with each additive after inoculation for 60 d is treatment group

with BG-11 > treatment group with yellow soil > treatment group with agar, and the

average concentration of chlorophyll are 2.63, 2.34, and 2.00 mg-g- , respectively.

Under 4 different algae treatment combinations (seen as Table 2 for treatment methods),

when the inoculation ratio of C. sp. and L. sp. is 2: 1, the average chlorophyll a content

is the largest, which is 2.75 mg-g-1, the order of size is algae combination 3 > algae

combination 4 > algae combination 1 > algae combination 2, but the thickness of algae

crust is the lowest. It shows that using agar according to the ratio of N. sp. to spherical C.

sp. of 2: 1 is the best inoculation method, which is more conducive to the rapid

development of algae, but the entanglement ability of N. sp. on soil particles is

relatively weak, resulting in the lowest thickness of algal crust.

[101] (4) Growth curve determination: A certain amount of each component solution

was taken and sterilized in an autoclave (121°C, 30 min), and mixed after cooling to

obtain BG-11 culture medium (Table 1). The two indigenous algae L. sp. and C. sp.

from Example 1 and Example 2 were inoculated in a 1 L Erlenmeyer flask containing

400 mL of the above BG-11, and cultured statically (light-to-dark ratio = 12 h: 8 h, light

intensity was 2500-3500 lx, temperature was 25± 5C), The two indigenous algae were

shaken manually for 3 times every day, and the chlorophyll a content was measured

using a phytoplankton classification fluorimeter (PHYTO-PAM-II, Germany). After 10

days of cultivation, the algae cells began to clump, and the measurement of chlorophyll

a content was stopped. The variation characteristics of the chlorophyll a content of the

two indigenous algae L. sp. and C. sp. in Examples 1 and 2 with the cultivation time

were shown in FIG. 4.

[102] It can be seen from FIG. 4 that when the biomass of C. sp. is lower than that of L. sp., the increase rate of biomass of the L. sp. increases with the inoculation time at the

initial stage of inoculation, and the growth amount is about 48.95 pg -L-I-d-1. After 7 d of

inoculation, the growth rate of L. sp. gradually decreases to a stable level, and the

growth amount is about 18.2 pg'-L--d-1. C. sp. grows fast, the growth rate is about 1.4

pg-L- -d-1, while the growth rate of L. sp. is about 1.2 pg-L- -d-1, and both show the

trend that the growth rate decreases with the increase of cultivation time. When the

biomass of C. sp. is higher than that of L. sp., C. sp. grows slowly at the initial stage of

inoculation, and the growth rate of C. sp. is much faster than that of L. sp., which is

restricted in growth. However, after 9 d of cultivation, the chlorophyll a content of C. sp.

rises rapidly from 20 pg-L-1 to 934.9 Ig-L-1. The total chlorophyll a content reaches

1076.2 pg-L-1, the C. sp. accounts for 87.16 %, and the L. sp. accounts for only 13.20

% of the total. However, the ratio of chlorophyll a content of L. sp. and C. sp. maintains

between 2: 1 and 4: 1 during the cultivation process.

[103] (5) The effect of BG-11 culture medium on the crust tissues in rare earth tailings

[104] FIG. 5 shows the change of rare earth tailings after adding BG-11 culture medium without algae for 46 d. It can be seen from FIG. 5 that through direct observation and

microscopic observation, it's found that the direct addition of BG-11 nutrient solution

is conducive to the fixing and multiplying of air-borne microorganisms (such as spores

of bryophytes) on the surface of tailings sand.

[105] (6) Scanning Electron Microscope (SEM): A small piece of upper algae crust

sample was taken, freeze-dried and then coated with gold, a scanning electron

microscope was used to observe and take pictures of the ultrastructure characteristics of

the upper surface of the sample. FIG. 6 showed the SEM microstructure of the surface

layer of the tailings soil after inoculation with different algae combinations at 2000

times and 5000 times, respectively. The magnifications of (a), (c), (e), (h), () and (1) in

the figure were 2000 times. The magnifications of (b), (d), (f), (i), (k) and (m) were

5000 times, i.e. the image on the right (x 5000 times) was the enlarged part in the red

circle on the left (x 2000 times).

[106] As shown in FIG. 6, a large number of needle-like minerals are present in rare earth tailings. After adding agar, a film is formed on the surface of the rare earth tailings, which has a protective effect on the surface particles. After 30 d of the tailings inoculation, in the treatment group with agar, the algae are attached or embedded on the agar film, and the surface is loose and porous with a large specific surface area. After the blank group is inoculated for the same time, no algae appear on the surface or soil particles form agglomerates, indicating that it takes longer for microorganisms or particles in the air to attach to on the surface of the rare earth tailings compared with the inoculated treatment group. After adding the yellow soil solution containing algae, the algae and the yellow soil particles form agglomerates, and the particles are tightly bound. The rare earth tailings directly added with the yellow soil solution form a particle-particle contact particle matrix, indicating that algae cells can cement fine particles to form aggregates. The microstructure of algae crust on the surface of tailings soil after inoculation with BG-11 nutrient solution containing algae is obviously different from that of algae crust on the surface of tailings soil after 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 body

of the spherical C. sp. is clearly observed, and the indigenous C. sp. with strong

tolerance is dominant. After 30 d of inoculation, the adhesion between soil particles is

still dominated by the adhesion of the extracellular sheath.

[107] (7) Determination of physical and chemical properties of the soil:

[108] A. pH: Measured by a pH meter. The sample was taken back to the room,

subjected to air drying, crushing, and removing impurities, then the soil and distilled

water were mixed according to the water-to-soil ratio of 2.5: 1, stirred and stood for 1 h,

and then a pH meter (Raymagnet) was used to sequentially determine the pH of the

supernatant solution, and 3 replicate values were determined for each sample.

[109] B. Determination of organic matter of the soil: the organic carbon in the soil was

oxidized with dichromate at a certain temperature (100°C, 90 min), and part of the

hexavalent chromium (Cr 6 ) was reduced to green trivalent chromium (Cr3 ), the

absorbance of trivalent chromium was measured 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 the content of organic matter. The reagents and preparation methods involved were referred to "Analytical Methods of Soil Agricultural Chemistry" by Lu Rukun (2000). The calculation method was as follows:

OM, %= x 100

[110] m x 11

[111] mi-the carbon content of the soil sample from the standard curve, mg;

[112] 1.724-the coefficient of conversion of organic carbon to organic matter;

[113] 1.08-oxidation correction coefficient;

[114] m-the quality of the soil sample, g.

[115] C. Determination of ammonia nitrogen (NH 4+-N) in the soil: The NH 4 ' in the soil leaching solution reacts with hypochlorite and phenol in the strong alkaline medium to produce water-soluble fuel of indophenol blue. The blue color of the solution is very stable, and the shade of the color is proportional to the concentration of NH 4 + in the range of 0.05-0.5 mg-L- 1. The reagents and preparation methods involved were referred to "Analytical Methods of Soil Agricultural Chemistry" by Lu Rukun (2000). The calculation method was as follows: p x V x ts x 10-- 1 w(N)-=x1000

[116] m

[117] w(N) - the mass fraction of ammonia nitrogen in the soil, mg -kg-1;

[118] P - the concentration of nitrogen in the developing liquid from the working curve, mg-L- 1;

[119] V - volume of the developing solution, mL;

[120] Ts - dividing ratio, total volume of leaching solution (mL)/volume of pipetted leaching solution (mL);

[121] 10-3, 1000 - respectively means converting mL into L; converting into soil content per kg;

[122] m - the quality of the soil sample.

[123] The results of the basic physical and chemical properties of rare earth tailings soil in different treatment groups were shown in Table 4.

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

[125]

Content of Content of Treatment Methods pH Ammonia Nitrogen Organic Matter

(mg -kg-1) (g-kg-')

Treatment Group with 4.93 0.88 11.23 5.27 1.56 0.29 Yellow Soil (TH1-3)

Different Treatment Group with 4.93 0.16 10.94 2.88 1.56 0.43 Additive Agar (TQ1-3)

s Treatment Group with 4.89 0.20 14.28 6.15 1.60 0.48 BG-11 (TB1-3)

Control Group (CK1-3) 5.25 0.17 10.91 2.07 1.22 0.18

Algae Combination 1 4.94 ±0.14 12.49 4.38 1.65 0.53 (TQl, THI, TB1)

Different Algae Combination 2 4.86 0.13 9.95 2.97 1.39 0.22 Algae (TQ2, TH2, TB2) Combina Algae Combination 3 4.89 0.13 11.58 5.84 1.67 0.18 tions (TQ3,TH3,TB3)

Algae Combination 4 4.97 0.23 14.42 5.95 1.58 0.54 (TQ4, TH4, TB4)

[126] 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 adding algae, the content of ammonia nitrogen in the rare earth tailings soil of the treatment group with yellow soil, treatment group with agar, treatment group with BG-11 and the control group decreases by 93.51%, 94%, 91.75%

and 91.66%, respectively. The pH of the soil is 4.5-5.5, the content of organic matter increases by 85.26%, and the content of organic matter in rare earth tailings soil increases from 0.23 g-kg-' to 1.56 g-kg-1.

[127] It can be seen from the results of the above examples that the biological crust repairing material for promoting ecological restoration of ion-absorbed rare earth tailings area provided by the present disclosure can significantly increase the content of organic matter in the tailings soil matrix when it is used to promote the rapid ecological restoration of the ion-absorbed rare earth tailings area, reduce the content of ammonia nitrogen, increase the crust area on the surface of the tailings, and greatly reduce the exposed surface, which can quickly improve the extremely degraded ecological environment caused by abandoned tailings in abandoned ion-absorbed rare earth mines, and improve soil degradation and environmental pollution in mining areas caused by the mining of rare earth mines.

[128] Although the above embodiments give a detailed description of the present

disclosure, they are only a part of the embodiments of the present disclosure and not all

of them. People can also obtain other embodiments based on this embodiment without

creativity. These embodiments are all belong to the protection scope of the present

disclosure.

Claims

WHAT IS CLAIMED IS:

1. A biological crust repairing material for promoting ecological restoration of an

ion-absorbed rare earth tailings area, comprising cosmopolitan algae and/or native

soil-dwelling algae;

the native soil-dwelling algae comprise Lyngbya sp. and/or Chlamydononas sp.; a

patent preservation number of the L. sp. is CCTCC No. M 2021758; a patent

preservation number of the C. sp. is CCTCC No. M 2021324;

the cosmopolitan algae comprise Nostoc sp. and/or Microcoleus vaginatus.

2. The biological crust repairing material according to claim 1, wherein when the

biological crust repairing material comprises the cosmopolitan algae and the native

soil-dwelling algae, a ratio of chlorophyll a content in the cosmopolitan algae to

chlorophyll a content in the native soil-dwelling algae is (0-5): (0-5), and chlorophyll a

content in the cosmopolitan algae and chlorophyll a in the native soil-dwelling algae are

not both 0;

when the biological crust repairing material comprises L. sp. and C. sp., a ratio of

chlorophyll a content in L. sp. to chlorophyll a content in C. sp. is (0-5): (0-5), and the

chlorophyll a content of L. sp. and chlorophyll a content of C. sp. are not both 0;

when the biological crust repairing material comprises N. sp. and M. vaginatus, a

ratio of chlorophyll a content in N. sp. to chlorophyll a content in M. vaginatus is (0-5):

(0-5), and the chlorophyll a content of N. sp. and chlorophyll a content of M. vaginatus

are not both 0.

3. The biological crust repairing material according to claim 2, wherein when the

chlorophyll content in the native soil-dwelling algae is (1-4): (1-5);

chlorophyll a content in L. sp. to chlorophyll a content in Chlamydononas sp. is (1-4):

(1-5);

when the biological crust repairing material comprises N. sp. and M. vaginatus, a ratio of chlorophyll a content in N. sp. to chlorophyll a content of M. vaginatus is (1-4):

(1-5).

4. The biological crust repairing material according to claim 1, wherein a applied

amount of the biological crust repairing material is 150-950 pg (chlorophyll a)-m-2 (soil

under repair).

5. Use of the biological crust repairing material according to any one of claims 1-4

in the repair of ion-absorbed rare earth tailings areas.

6. A method for repairing an ion-absorbed rare earth tailings area, comprising the

following steps:

applying the biological crust repairing material according to any one of claims 1-4

to the soil of the ion-absorbed rare earth tailings area to be repaired for restoration.

7. The method according to claim 6, wherein the biological crust repairing material

is applied to the soil of the ion-absorbed rare earth tailings area to be repaired in the

form of an algal suspension.

8. The method according to claim 6, wherein a applied amount of the biological

crust repairing material is 100-1000 pg (chlorophyll a)-m-2 (soil under repair).

9. The method according to claim 7, wherein a method for preparing the algal

suspension of the biological crust repairing material comprises the following steps:

inoculating the biological crust repairing material in a culture medium and

cultivating to obtain an algal suspension.

10. The method according to claim 9, wherein the culture medium is a sterile

BG-11 culture medium.

11. The method according to claim 9, wherein a light intensity of the cultivating is

2500-3500 lx.

12. The method according to claim 9 or 11, wherein a light-to-dark ratio of the

cultivating is 16 h: 8 h or 12 h: 12 h.

13. The method according to claim 9, wherein a method for cultivating is static

cultivation, and a temperature of the cultivating is 25± 5C.

14. The method according to claim 9 or 13, wherein a time of the cultivating is 3-4

weeks.

15. The method according to claim 7, wherein the chlorophyll a content in the algal

suspension of the biological crust repairing material is 100-1000 pg -L-'.

16. The method according to claim 7, wherein the chlorophyll a content in the algal

suspension of the biological crust repairing material is 150-950 g-L-1.