CN-121988603-A - Method for restoring gangue soil based on CUE (compute unified element) synthetic microbial community

CN121988603ACN 121988603 ACN121988603 ACN 121988603ACN-121988603-A

Abstract

The invention discloses a method for repairing gangue soil based on CUE synthetic microbial communities, and belongs to the technical field of microorganisms. Adding biochar or glucose into the coal gangue polluted soil and inoculating and restoring the synthetic flora, wherein the addition amount of the biochar or glucose added into the coal gangue polluted soil is that 0.5-1.5% (m/m) of biochar or 15-25 ppm% of glucose is added into the coal gangue polluted soil containing 85-95% (m/m), the inoculation amount of the synthetic flora is that 40-60 mL bacterial liquid is applied to each kilogram of polluted soil, and the bacterial liquid content is 1X 10 7 ~3×10 7 CFU/mL. The invention is based on CUE synthetic flora, maximally utilizes modifier (glucose/biochar), realizes rapid establishment and metabolism and mutual culture of the flora, ensures that strains can survive in a carbon-limited environment, drives downstream ecological functions such as nutrient circulation, community stress resistance and the like by the modifier, realizes integration of microorganisms and physicochemical means, remarkably improves the colonization capacity and nutrient circulation efficiency of microorganisms, has repair efficiency superior to that of a single modifier, and solves the bottleneck of carbon deficiency of gangue tailings.

Inventors

CHEN QUAN
DONG JIHONG
LIN RONGRONG
XIONG YANQING
HUANG YU
WU MIN
YI PENG
PAN BO

Assignees

昆明理工大学

Dates

Publication Date: 20260508
Application Date: 20260120

Claims (10)

1. A method for restoring coal gangue soil based on CUE synthetic microflora is characterized in that biochar or glucose is added into coal gangue contaminated soil and a synthetic flora is inoculated for restoration, wherein the synthetic flora comprises one or more of Arthrobacter (Arthrobacter), nocardioides (Nocardioides), arthrobacter (Pseudarthrobacter), paramethomyces (Pararhizobium), new grass spirochete (Noviherbaspirillum), bacillus (Cytobacillus), paenibacillus (Paenibacillus) and Bacillus (Bacillus).
2. The method for restoring coal gangue soil based on CUE synthetic microorganism community as claimed in claim 1, wherein the adding amount of biochar or glucose in the coal gangue polluted soil is that 0.5-1.5% (m/m) of biochar or 15-25 ppm glucose is added to the polluted soil containing 85-95% (m/m), the inoculating amount of the synthetic flora is that 40-60 mL bacterial liquid is applied to each kilogram of polluted soil, and the bacterial liquid content is 1X 10 7 ~ 3×10 7 CFU/mL.
3. The method for restoring coal gangue soil based on CUE synthetic microorganism community as claimed in claim 1, wherein the synthetic flora is obtained by the following method: (1) Performing gradient dilution in buffer solution by a limiting dilution method after adding carbon sources into coal gangue polluted soil for improvement to form bacterial solutions with different concentrations, respectively separating and culturing the diluted bacterial solutions with different gradients in a culture medium, purifying bacterial strains according to colony morphology, and screening local core strains; (2) Selecting core strains representing different species from the native core strains and the non-native core strains in the step (1), inoculating and culturing, and screening core strains with a relatively high growth rate and a relatively short doubling time after culturing, namely screening out high-carbon utilization-efficiency CUE core strains as high CUE candidate strains for constructing synthetic flora; (3) Inoculating and culturing candidate strains, culturing each strain and the supernatant of another candidate strain in a pairwise co-culture mode, quantifying the interaction strength between the strains, selecting high CUE strains with strong forward interaction, randomly assembling into different flora, culturing the different flora respectively, re-suspending, and mixing the bacterial suspensions of the agreed flora in equal volumes to form different synthetic flora.
4. The method for restoring gangue soil based on CUE synthetic microorganism consortium as claimed in claim 3, wherein in the step (1), the gradient dilution of 10 - to 10 -7 is performed in buffer; the culture mediums are TSB agar culture medium and LB agar culture medium, diluted bacterial solutions with different gradients are respectively cultured in the TSB agar culture medium and the LB agar culture medium, the culture environment in the culture medium is 25-30 ℃, and the culture time is 5-7 days.
5. The method for restoring gangue soil by using a CUE-based synthetic microbial community according to claim 3, wherein in the step (1), the screening of the core strain is to analyze the width of the ecological niche of each amplicon sequence variant ASV in the ecological environment of gangue-polluted soil and healthy soil respectively, the screening calculation is carried out by adopting a Levins ecological niche width index of B=1/Σpi2, the B values of each ASV are arranged in a descending order, the first 10% is taken as a generalized seed, the last 10% is taken as a specialized seed, the classification unit of the generalized seed is compared with the strains which are separately cultured through cluster analysis and MEGA software, and if the similarity of the strains and the generalized seed ASV corresponds to the same classification level, the strains are regarded as the core strain.
6. The method for restoring gangue soil based on CUE synthetic microbial communities, as claimed in claim 3, wherein in the step (2), the process of selecting the core strains representing different species is that the native core strains and the non-native core strains separated in the step (1) are subjected to 16S rRNA gene sequencing, the species are annotated to a species level, if a plurality of strains are annotated as the same species, the strain with the fastest growth rate is reserved, the core strains which are obtained by different species and have the optimal reserved growth rate of the same species are screened, and the screened core strains are inoculated and cultured.
7. The method for restoring gangue soil based on CUE synthetic microorganism community as claimed in claim 3, wherein in the step (2), the condition of inoculation culture is that each strain is inoculated into a 96-well plate according to the volume ratio of 1:98-1:99, specifically, 200-400 mu L of basic salt culture medium containing 100-200-mM carbon equivalent is added into each well, and after 2-4 mu L of bacterial liquid is inoculated, the culture is carried out in a constant temperature shaking table at 25-30 ℃ and 150-200 rpm.
8. The method for restoring gangue soil based on CUE synthetic microorganism consortium as claimed in claim 3, wherein in the step (2), absorbance OD 600 at 600 nm is recorded every 1-3 hours by using an ultraviolet spectrophotometer for 40-50 hours before screening after inoculation and culture, and the growth rate of the screened high CUE candidate strain is 0.25-0.47 h - and the doubling time is 1.1-3.0 hours.
9. The method for restoring gangue soil based on CUE synthetic microorganism consortium as claimed in claim 3, wherein in the step (3), the step of inoculating and culturing candidate strain is to inoculate each strain in 4-6 mL TSB culture medium, culturing in shaking table of 35-38 ℃ and 150-200 rpm for 40-50 hours to stationary phase, centrifuging at 7000-9000 rpm for 8-12 minutes, filtering supernatant with 0.22 μm sterile filter membrane to obtain cell-free supernatant; Co-cultivation is to adjust each candidate strain after inoculation and cultivation to the same OD 600 value, co-cultivate each candidate strain with the supernatant of the other candidate strain respectively, wherein the ratio is that 1-2 mu L of the supernatant of the other candidate strain is added to 100-200 mu L of bacterial liquid, and in co-cultivation, the interaction intensity among quantified strains is 12 h, 36 h and 48 h, and then the OD 600 value of each strain in the supernatant of the other candidate strain is measured.
10. The method for restoring gangue soil based on CUE synthetic microbial communities, as claimed in claim 3, wherein in the step (3), the high CUE strains with strong forward interaction are selected to be assembled into different bacterial communities, the different bacterial communities are respectively inoculated into TSB culture medium and cultured overnight at the temperature of 25-30 ℃ at the rotating speed of 150-200 rpm, wherein the ratio of the OD 600 value of the strains in the supernatant of other candidate strains to the OD 600 value of the strains cultured in the supernatant of the strains is larger than 1.2, and the strains have strong interaction. After culturing, centrifuging for 3-10 min at 10000-15000 rpm, re-suspending with sterile water to OD 600 value of 0.1, repeating three times of re-suspending operations to remove residual TSB culture medium, and mixing the bacterial suspensions of the same bacterial strain with equal volume and equal volume to obtain different synthetic bacterial groups.

Description

Method for restoring gangue soil based on CUE (compute unified element) synthetic microbial community Technical Field The invention relates to the technical field of microbiology, in particular to a method for repairing gangue soil based on CUE synthetic microbial communities. Background Gangue is the main solid waste generated in the coal mining process, and the problems of heavy metal leaching, soil acidification, structural degradation and the like caused by the gangue can cause serious threat to a soil ecosystem. These hazards are widely present in areas of gangue pile-up, severely damaging soil functions, such as inhibiting nutrient circulation and carbon sequestration processes. In the existing restoration means, although the composite modifier combines the advantages of coal gangue and biomass, the problems of complex material pretreatment, high cost, weak pertinence to composite pollution and the like exist, the problems of high species matching requirement, easy inactivation of microorganisms in a field environment and the like exist in the plant-microorganism cooperative restoration, and the methods are limited by continuous physicochemical stress of tailings, especially carbon deficiency. Soil microbiome is the core for maintaining the health of ecological system, and can promote plant growth through nitrogen fixation, phosphorus dissolution, carbon conversion and other nutrient circulation processes. However, the extreme ecological environment of the gangue tailings can inhibit the diversity and activity of microorganisms, and prevent the artificial regulation of soil formation and sustainable restoration process. The synthetic microbial community provides a potential biotechnology means for sustainable development and environmental remediation of agriculture, and nutrient bioavailability can be improved, pathogenic bacteria can be inhibited and host stress resistance can be enhanced by directionally reconstructing the microbiome. The design of synthetic communities typically employs a "bottom-up" strategy, i.e., screening strains with specific functions (e.g., contaminant degradation, beneficial flora enrichment), or a "top-down" strategy, by compacting the communities to enrich for key clusters. Although this technology has significant success in agricultural settings, its application in tailings extreme environments is still quite limited. Carbon limiting habitat of mining contaminated soil is a typical technical bottleneck, for example, the combination of thiobacillus ferrooxidans and elemental sulfur can promote iron oxidation and soil formation in alkaline tailings, and organic modifiers such as compost, biochar and the like can also stimulate functional microorganism colonisation. These studies confirm the value of microbial inoculants in combination with physicochemical modification measures, however, policy optimization for efficient utilization of limited carbon sources by microorganisms still has significant gaps, and carbon source supplementation is a routine practice in tailings remediation. Carbon utilization efficiency refers to the proportion of assimilated carbon that microorganisms distribute to biomass synthesis, rather than respiratory consumption. In the tailing repairing scheme, external carbon sources such as biochar and glucose are often added to relieve physical and chemical stress and support microbial activity, but the carbon sources often reduce microbial colonization and ecological restoration effects due to insufficient utilization efficiency. Disclosure of Invention In order to solve or partially solve the problems in the related art, the invention provides a method for repairing coal gangue soil based on CUE synthetic microorganism community, which efficiently utilizes an exogenous carbon modifier, the method has the advantages that the method can solve the core problems of lack of carbon in gangue tailing soil, low microbial activity, blocked nutrient circulation and the like, and realize accurate and efficient restoration of polluted soil. In order to achieve the aim, the technical scheme of the invention is that the method for repairing the coal gangue soil based on the CUE synthetic microbial community is characterized in that biochar or glucose is added into the coal gangue contaminated soil and a synthetic flora is inoculated for repairing, wherein the synthetic flora comprises one or more of Arthrobacter (Arthrobacter), nocardia (Nocardioides), arthrobacter (Pseudarthrobacter), rhizobium (Pararhizobium), new grass snail (Noviherbaspirillum), bacillus (Cytobacillus), paenibacillus (Paenibacillus) and Bacillus (Bacillus). Preferably, the addition amount of the biochar or glucose in the gangue polluted soil is that 0.5-1.5% (m/m) of the biochar or 15-25 ppm glucose is added into the polluted soil containing 85-95% (m/m) of the gangue, the inoculation amount of the synthetic flora is that 40-60 mL bacterial liquid is applied to each kilogram of the polluted soi