A method for improving saline-alkali land

Abstract

The invention belongs to the field of modern agricultural technology and relates to a method for soil improvement, and specifically relates to a method for improving saline-alkali land, wherein a composite bacterial agent of Huajinpu alkaline halobacter BH‑8 and coal gangue is applied to saline-alkali land soil, wherein the concentration of Huajinpu alkaline halobacter BH‑8 in the composite bacterial agent is 1×10 9 ~3×10 9 CFU / mL, the concentration of coal gangue particles is 5-10g / L; the present invention uses Huajinpu alkaline halobacter and coal gangue to prepare a composite bacterial agent for saline-alkali land improvement for the first time. By comparing with the blank control group, coal gangue alone, and microorganisms alone, the combination of Huajinpu alkaline halobacter and coal gangue improves the soil fertility and various enzyme activities of saline-alkali land, and optimizes the structure and function of soil microbial communities in saline-alkali land. The method of the present invention is scientific and reliable, has good repeatability, has important scientific value, provides an important reference for saline-alkali land improvement, helps to play an important role in saline-alkali land improvement, and improves the efficiency of saline-alkali land improvement.

Description

Technical field:

[0001] The invention belongs to the field of modern agricultural technology and relates to a method for soil improvement, and specifically relates to a method for improving saline-alkali land. The saline-alkali land is improved by combining microorganisms with coal gangue, which can significantly increase the content of organic matter, total nitrogen, available phosphorus, quick-acting potassium and other trace elements in the saline-alkali land, thereby achieving the purpose of improving the saline-alkali land soil. Background technology:

[0002] Saline-alkali soil is usually caused by natural or human factors that lead to excessive accumulation of soluble salts in the soil surface, such as sodium ions (Na + ), potassium ion (K + ) and calcium ions (Ca 2+ ) and so on, causing soil salinization. Among the main crops in my country, wheat, rice, corn, etc. are all glycophytes, which are very sensitive to changes in salt concentration in the soil. When the salt content in the soil is too high, key cellular processes such as photosynthesis, protein synthesis, and lipid metabolism of these crops will be disturbed by salt stress. This stress not only inhibits the normal growth and development of plants, but in severe cases can even lead to plant death.

[0003] As the global climate continues to change, under the influence of natural and human factors such as unreasonable irrigation and fertilization, soil salinization has shown a trend of increasing intensity. As a country with widespread saline-alkali land distribution, my country's saline-alkali land area is equivalent to 40% of the country's total cultivated land. The Northeast Plain, the Northwest Arid Region, the Huanghuaihai Plain and the eastern coastal areas are all deeply troubled by land salinization, especially the saline-alkali wasteland area in Shandong Province, which is as high as more than 600,000 hectares. At present, the area of ​​saline-alkali land in the world exceeds 833 million hectares, accounting for 8.7% of the earth's area (FAO 2021), and the area of ​​soil salinization is increasing at a rate of more than 2 million hectares per year. The continued growth of this number has seriously affected human food and environmental security. Therefore, how to effectively control saline-alkali land, reduce the degree of soil salinization, and restore and improve soil productivity has become an important issue to be solved at present. Summary of the invention:

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for improving saline-alkali land. By combining microorganisms with coal gangue to improve saline-alkali land, the content of organic matter, total nitrogen, available phosphorus, available potassium and other trace elements in the saline-alkali land can be significantly increased, thereby achieving the purpose of improving the soil in the saline-alkali land.

[0005] To achieve the above object, the present invention provides a method for improving saline-alkali land, wherein a composite bacterial agent (CB) of Huajinpu alkaline halobacter BH-8 and coal gangue is applied to saline-alkali land soil, wherein the concentration of Huajinpu alkaline halobacter BH-8 in the composite bacterial agent is 1×10 9~3×10 9 CFU / mL, the concentration of coal gangue particles is 5-10g / L, the particle size of the coal gangue is 1mm; the solvent in the bacterial agent is distilled water; the application amount is 100mL of bacterial agent per 1kg of soil.

[0006] The Hwajinpu alkaline halobacter BH-8 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), and the deposit number is CGMCC No. 30829.

[0007] The preparation method of the composite bacterial agent (CB) of Huajinpu alkaline halobacter BH-8 and coal gangue is as follows: 1 mm coal gangue and BH-8 strain are co-cultured in a modified LB medium until the bacterial cell logarithmic phase, the coal gangue is filtered, the bacterial solution is centrifuged, the precipitate is taken, sterile water is added and resuspended to 10 9 CFU / mL, the resuspended bacterial suspension was mixed with the filtered coal gangue and used as a composite bacterial agent.

[0008] The formula of the improved LB medium is: 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, 2-12 g / L sodium chloride; pH is 7.2.

[0009] The present invention uses the combined application of Huajinpu alkaline halobacter BH-8 and coal gangue to reduce soil microbial diversity, significantly increase the relative abundance of Bacteroidota in the soil, and significantly reduce the relative abundance of Actinomycetota, thereby further changing the composition and structural function of the soil microbial community. After the combined treatment of Huajinpu alkaline halobacter BH-8 and coal gangue, environmental factors such as soil organic matter, available potassium, and alkaline nitrogen all respond positively, and Huajinpu alkaline halobacter BH-8 plays a more significant role in these positive responses. After the combined treatment, the relative abundance of the CBM50 functional module (enzyme that cuts chitin or peptidoglycan) is significantly improved.

[0010] Compared with the prior art, the present invention uses Huajinpu alkaline halobacter and coal gangue to prepare a composite bacterial agent for improving saline-alkali land for the first time, and uses Huajinpu alkaline halobacter and coal gangue in combination to improve the soil fertility of saline-alkali land. By comparing with a blank control group, using coal gangue alone, and using microorganisms alone, the combination of Huajinpu alkaline halobacter and coal gangue improves the soil fertility and various enzyme activities of saline-alkali land, and optimizes the structure and function of soil microbial communities in saline-alkali land. The method of the present invention is scientific and reliable, has good repeatability, has important scientific value, provides an important reference for saline-alkali land improvement, helps to play an important role in saline-alkali land improvement, and improves the efficiency of saline-alkali land improvement. Description of the drawings:

[0011] Figure 1 This is a morphological characteristic diagram of the BH-8 strain involved in the present invention.

[0012] Figure 2 The constructed Hwajinpo Alkalihalobacillus developmental tree involved in the present invention is identified as Alkalihalobacillus hwajinpoensis through 16Sr RNA sequencing. The strain belongs to the class Bacilli, the order Bacillales, the family Bacillaceae, and is a Hwajinpo Alkalihalobacillus.

[0013] Figure 3 This is the growth curve of BH-8 strain under different NaCl concentrations.

[0014] Figure 4 It is a schematic diagram of the changes in basic physical and chemical properties of soil after applying coal gangue (C), Hwajinpu alkaline Halobacter BH-8 bacterial solution (B) and Hwajinpu alkaline Halobacter BH-8 and coal gangue composite bacterial agent (CB) involved in the present invention.

[0015] Figure 5 The present invention relates to a schematic diagram of the change results of soil available phosphorus (OP) content in different treatment groups, wherein CK is a control group; C is a group only applying coal gangue; B is a group only applying BH-8 bacterial solution; CB is a group applying a composite bacterial agent of BH-8 and coal gangue.

[0016] Figure 6 The figure is a schematic diagram of the change results of soil organic matter (OM) content in different treatment groups involved in the present invention, CK is a blank group; C is a group only treated with coal gangue; B is a group only treated with BH-8 bacterial solution; CB is a group treated with a composite bacterial agent of BH-8 and coal gangue.

[0017] Figure 7 The figure is a schematic diagram of the change results of soil pH in different treatment groups involved in the present invention, wherein CK is the control group; C is the group only applying coal gangue; B is the group only applying BH-8 bacterial solution; CB is the group applying BH-8 and coal gangue composite bacterial agent.

[0018] Figure 8 The present invention relates to a schematic diagram of the change results of the soil available potassium (AK) content in different treatment groups, wherein CK is the control group; C is the group only applying coal gangue; B is the group only applying BH-8 bacterial solution; CB is the group applying BH-8 and coal gangue composite bacterial agent.

[0019] Fig. 9 The figure is a schematic diagram of the change results of soil catalase activity in different treatment groups involved in the present invention, wherein CK is the control group; C is the group only treated with coal gangue; B is the group only treated with BH-8 bacterial solution; CB is the group treated with BH-8 and coal gangue composite bacterial agent.

[0020] Fig.10 The present invention relates to a schematic diagram of the change in soil nitrate reductase activity in different treatment groups, wherein CK is a control group; C is a group only treated with coal gangue; B is a group only treated with BH-8 bacterial solution; and CB is a group treated with a composite bacterial agent of BH-8 and coal gangue.

[0021] Fig.11 The present invention relates to a schematic diagram of the change results of soil alkaline phosphatase activity in different treatment groups, wherein CK is a control group; C is a group only treated with coal gangue; B is a group only treated with BH-8 bacterial solution; and CB is a group treated with a composite bacterial agent of BH-8 and coal gangue.

[0022] Fig.12 The present invention relates to a schematic diagram of the changes in soil acid phosphatase activity in different treatment groups, wherein CK is a control group; C is a group only treated with coal gangue; B is a group only treated with BH-8 bacterial solution; and CB is a group treated with a composite bacterial agent of BH-8 and coal gangue.

[0023] Fig.13 The figure is a schematic diagram of the change results of soil urease activity in different treatment groups involved in the present invention, wherein CK is the control group; C is the group only treated with coal gangue; B is the group only treated with BH-8 bacterial solution; CB is the group treated with BH-8 and coal gangue composite bacterial agent.

[0024] Fig.14 The figure is a chord diagram of relative abundance of species at the phylum level for different treatment groups involved in the present invention, wherein CK is the control group; C is the group only treated with coal gangue; B is the group only treated with BH-8 bacterial solution; and CB is the group treated with a composite bacterial agent of BH-8 and coal gangue. Specific implementation method:

[0025] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0026] Embodiment 1:

[0027] This embodiment relates to the screening and identification of Halobacterium alkaliensis, and the specific steps are as follows:

[0028] (I) Screening and isolation of Hwajinpo alkaline halobacter: Take 1 g of soil sample and add it into a 250 mL conical flask containing 100 mL of enrichment medium, shake and mix, and place it in a constant temperature shaker at 30°C for shaking culture for 2 to 3 days; use physiological saline to gradient dilute the cultured bacterial solution and spread it on a plate, place the plate in a constant temperature and humidity chamber at 30°C and culture until colonies grow on the plate; use an inoculation loop to pick up colonies with good growth and repeatedly streak them on a solid enrichment medium to obtain purified single colonies.

[0029] The formula of the enrichment medium is: 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate and 3 g / L NaCl, with a pH of 7.2.

[0030] The preparation method of the enrichment medium is as follows: 5g yeast powder; 10g peptone; 5g magnesium sulfate heptahydrate; 2.5g potassium chloride; 0.05g ferric sulfate heptahydrate; 3g NaCl. Adjust the pH to 7.2, add distilled water to 1L, sterilize with high temperature and wet heat for 30min, and place at room temperature for use.

[0031] The sodium chloride concentration in the acclimation medium was set to 3 g / L, 5 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 18 g / L, and 20 g / L. The purified single colony was streaked on the solid acclimation medium, and the sodium chloride concentration was gradually increased until a single colony that could tolerate a sodium chloride concentration of 20 g / L was obtained.

[0032] The formula of the acclimation medium is 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, and the NaCl content is adjusted according to experimental requirements; the pH is 7.2.

[0033] The culture medium composition is: 5g yeast powder; 10g peptone; 5g magnesium sulfate heptahydrate; 2.5g potassium chloride; 0.05g ferrous sulfate heptahydrate; the NaCl content is adjusted according to the experimental requirements. Adjust the pH to 7.2, add distilled water to 1L. After high temperature and wet heat sterilization for 30 minutes, place it at room temperature for use.

[0034] After enrichment culture, 8 different strains were isolated. After domestication, a moderately halophilic bacterium was finally screened out. This strain can grow on a medium with a NaCl concentration of 20g / L. It was named BH-8, and the colony morphology of the BH-8 strain was recorded, its physiological and biochemical characteristics and enzyme activity characteristics were analyzed, and the BH-8 strain was sequenced to determine its species.

[0035] (II) Physiological, biochemical and enzymatic characteristics of BH-8 strain: Figure 1 As shown, observing the colony morphology, it can be seen that the BH-8 colonies are yellow, have no flagella, cannot move, the surface of the colonies is moist and smooth, and the Gram staining result is positive.

[0036] The results of physiological and biochemical and enzyme activity characteristics are shown in Table 1. The VP test results of BH-8 strain were negative. It could not use glucose to produce pyruvate; it could not produce indole; it could produce H2S; it could not reduce nitrate; it could not use citrate; it had catalase and oxidase activities, but no urease activity. It could not use inositol and D(+)-galactose, but could use glucose, sucrose, lactose, D(-)-fructose, mannitol, and starch as the only carbon source.

[0037] Table 1 Biochemical and enzymatic characteristics of BH-8 strain

[0038] Experiment Name result VP - <![CDATA[ Indole Generate]]> - <![CDATA[H2S generation]]> + Nitrate reduction - Citrate Utilization - Starch hydrolysis + Catalase + <![CDATA[ Urease ]]> - Oxidase +

[0039] (III) 16srDNA sequencing results: The total DNA of the BH-8 strain was extracted using a bacterial genomic DNA kit, and the extracted DNA group products were subjected to agarose gel electrophoresis, and clear bands were visible, proving that the extracted bacterial DNA group was intact and not broken.

[0040] The 16S rRNA sequencing results were compared with the known species sequences in Genbank, and the 16S rRNA analysis phylogenetic tree was constructed using Mega 11 software ( Figure 2 ), it was found that the BH-8 strain belonged to the same branch as Alkalihalobacillus hwajinpoensis JSM076093 in the phylogenetic tree, had high similarity with Salibacterium nitratireducens SMB4, Alkalihalobacillus algicola AB423f, and Pseudalkalibacillus caeni HB172195, and had the highest sequence homology with Alkalihalobacillus hwajinpoensis SW72 (Pseudalkalibacillus hwajinpoensisSW-72), which was 99.30%. It was known that the BH-8 strain was an Alkalihalobacillus hwajinpoensis.

[0041] The Huajinpu alkaline halobacter BH-8 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​May 31, 2024, with the deposit number CGMCC No. 30829.

[0042] Embodiment 2:

[0043] This embodiment relates to a preparation method of a composite bacterial agent of Huajinpu alkaline halobacter BH-8 and coal gangue, combining the Huajinpu alkaline halobacter BH-8 strain with coal gangue taken from Henan Province, and optimizing the preparation conditions, which ensures the growth activity of the strain and can give full play to the characteristics of coal gangue, opening up a new way for the efficient utilization of coal gangue and the wide application of moderate halophilic bacteria, and showing the great potential of the composite bacterial agent in saline-alkali soil treatment. The specific steps are:

[0044] (I) Determination of the optimal salt concentration of BH-8 strain: 100 mL of liquid culture medium was placed in each 250 mL conical flask, and the NaCl concentration was adjusted to 2 g / L, 5 g / L, 8 g / L, 10 g / L, 12 g / L, and 15 g / L, respectively. Three replicates were prepared in each group. After high-temperature sterilization, the culture was cooled to room temperature and inoculated with 1 mL of thawed preserved BH-8 bacterial solution. The culture was placed in a 30°C constant temperature shaker for shaking culture. The absorbance of the bacterial solution at 600 nm was measured every 2 hours until the absorbance value no longer increased. The growth curve was fitted using Origin 2021 software, and the growth curve of the BH-8 strain under different NaCl conditions was plotted to determine the NaCl concentration tolerance range of the BH-8 strain.

[0045] The formula of the liquid culture medium is 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, and the NaCl content is adjusted according to experimental requirements; the pH is 7.2.

[0046] The growth curve of BH-8 strain under different NaCl concentration environments was drawn ( Figure 3 ), it can be clearly observed that the BH-8 strain has the ability to grow and reproduce in the range of NaCl concentration between 2g / L and 12g / L. However, as the NaCl concentration gradually increases, the growth rate of the absorbance value of the bacterial solution gradually slows down, and its maximum absorbance value also decreases, indicating that the growth of the strain is significantly inhibited. Despite this, the BH-8 strain still shows a high salt tolerance and can withstand a maximum NaCl concentration of 12g / L. When the NaCl concentration reaches 15g / L, the absorbance value of the bacterial solution remains almost unchanged, indicating that the strain cannot grow effectively in this environment. It is worth mentioning that when the NaCl concentration is in the range of 2g / L to 5g / L, the growth condition of the BH-8 strain is most ideal, so the following selection of a NaCl culture medium with a concentration of 3g / L is used to conduct an in-depth exploration of the co-culture conditions with the BH-8 strain and coal gangue.

[0047] (ii) Co-cultivation method of BH-8 strain and coal gangue: Coal gangue with different particle sizes (0.15 mm, 0.3 mm, 1 mm) and different concentrations (5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L) were co-cultivated with BH-8 strain in 100 mL liquid culture medium. Three parallels were set up in each group. The inoculation amount of BH-8 strain was 1 mL. The culture was carried out in a constant temperature shaker at 30°C. The absorbance value of the bacterial solution at 600 nm was measured, and the growth curve was fitted using Orgin 2021 software.

[0048] Under the condition that the concentration of NaCl in the liquid culture medium was 3g / L, the effect of different concentrations (0, 5g / L, 10g / L, 15g / L, 20g / L, 25g / L) of coal gangue on the growth of BH-8 strain was investigated. The results showed that with the increase of coal gangue concentration, the growth curve of BH-8 strain gradually flattened, the maximum absorbance gradually decreased, and the growth of the strain was gradually inhibited. When the concentration of coal gangue was higher than 10g / L, it was difficult for BH-8 strain to grow normally; when 5g / L concentration of coal gangue was used to co-culture with BH-8 strain, although the growth rate of the strain was slower than that when no coal gangue (CK) was added, it could still maintain normal growth. Therefore, 5g / L concentration of coal gangue was finally selected to investigate the effect of different particle sizes of coal gangue on the growth of BH-8 strain.

[0049] When the NaCl concentration was 3g / L and the coal gangue concentration was 5g / L, the growth of BH-8 strain was inhibited to a certain extent when co-cultured with coal gangue of different particle sizes (0.15mm, 0.3mm, 1mm) compared with the control group (CK), which was manifested as the smaller the coal gangue particle size, the higher the degree of inhibition on the growth of BH-8 strain. Compared with the 0.3mm and 0.15mm coal gangue groups, when 1mm coal gangue was used to co-culture with BH-8 strain, the growth of the strain was inhibited only in that it reached the logarithmic phase later than the CK group and grew slightly slower, but it could still reach an absorbance value of about 0.8 in the end. The addition of 0.3mm and 0.15mm particle size coal gangue not only slowed down the growth of BH-8 strain but also affected the maximum absorbance value of the bacterial solution in the platform period. Therefore, coal gangue with a particle size of 1mm was selected to prepare BH-8 and coal gangue composite bacterial agent.

[0050] Embodiment 3:

[0051] This example involves an experiment on the effect of Huajinpu alkaline halobacterium BH-8 and coal gangue composite bacterial agent on saline-alkali soil remediation, and the specific steps are as follows:

[0052] 1. Experimental design

[0053] This experiment was conducted indoors. 12 portions of 200g air-dried coastal saline-alkali soil were placed in black plastic flower pots. Filter paper was placed at the bottom to prevent soil loss during irrigation. The basic physical and chemical properties of the soil are as follows: soil conductivity 91.7ms / m, density 2.64g / cm3, total salt content 2.6g / kg, total nitrogen 0.89g / kg. An equal amount of sterile water was added every 3 days to maintain soil moisture at 60%. After incubation for 3 days, the soil was divided into 4 groups for treatment, namely the control group (CK), which was treated with 20mL of sterile water; the group (C) that only applied coal gangue, which was treated with 20mL of sterilized coal gangue particles with a concentration of 5g / L; the group (B) that only applied Huajinpu alkaline halobacterium BH-8, which was treated with 20mL of 10 9 CFU / ml of BH-8 bacterial solution; Huajinpu alkaline halobacter BH-8 and coal gangue composite bacterial agent group (CB), 20mL Huajinpu alkaline halobacter BH-8 and coal gangue composite bacterial agent was applied, and the composite bacterial agent contained coal gangue particles with a concentration of 5g / L and a concentration of 1×10 9 CFU / mL of BH-8 strain. After adding different treatments, the topsoil and the applied bacterial agent need to be stirred evenly. Three replicates were added to each group. Four samples were taken on the 1st (treatment day), 7th, 14th, and 21st day after treatment. Each time, 25g of the sample was sampled using the five-point sampling method. The 15g soil sample was air-dried under natural conditions, ground and sieved, and the changes in soil physical and chemical properties and enzyme activity were measured. The results are as follows Figure 4-Figure 13 The remaining 10 g of fresh soil sample was stored in a -80 °C refrigerator for subsequent soil microbial community analysis.

[0054] The preparation method of the bacterial solution used in the Hwajinpu alkaline Halobacter group (B) is as follows: after culturing the BH-8 strain in a modified LB medium to the logarithmic phase, the bacterial solution is centrifuged, and the precipitate is added with sterile water and resuspended to 10 9 CFU / mL of bacterial suspension.

[0055] The preparation method of the composite bacterial agent (CB) of Huajinpu alkaline halobacter BH-8 and coal gangue is as follows: 1 mm coal gangue and BH-8 strain are co-cultured in a modified LB medium until the bacterial cell logarithmic phase, the coal gangue is filtered, the bacterial solution is centrifuged, the precipitate is taken, sterile water is added and resuspended to 10 9 CFU / mL, and the resuspended bacterial suspension and filtered coal gangue were added to the treatment group.

[0056] The formula of the improved LB medium is: 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, 3 g / L NaCl; pH is 7.2.

[0057] (II) Effects of Huajinpu alkaline halobacterium BH-8 and coal gangue composite bacterial agent on the physical and chemical properties of saline-alkali soil:

[0058] By applying coal gangue (C), Hwajinpu alkaline Halobacterium BH-8 bacterial solution (B), and Hwajinpu alkaline Halobacterium BH-8 and coal gangue composite bacterial agent (CB), the basic physical and chemical properties of the soil changed to varying degrees, such as Figure 4-Figure 8 shown.

[0059] from Figure 4 It can be seen that compared with the control group (CK), after applying the Huajinpu alkaline halobacter BH-8 bacterial solution (B) and the Huajinpu alkaline halobacter BH-8 and coal gangue composite bacterial agent (CB), the alkaline nitrogen (AN) content of the soil increased significantly on the day of use, 7 days, 14 days, and 21 days, and was the highest on the day of application. The soil alkaline nitrogen content of the CB group increased by 277.43% on the first day, and that of the B group increased by 181.47%. With the passage of time, the soil alkaline nitrogen content gradually decreased and reached a stable level after the 14th day; the application of coal gangue alone had no significant effect on the content of soil alkaline nitrogen.

[0060] from Figure 5 From the changes in soil available phosphorus (OP) content, it can be seen that compared with the control group, the soil available phosphorus content increased significantly on the 1st, 7th and 14th days after the application of BH-8 bacterial solution (B) and BH-8 coal gangue composite bacterial agent (CB), and on the 1st day, the soil available phosphorus content of the BH-8 and coal gangue composite bacterial agent group (CB) increased by 207.42%, with the best effect; the application of Huajinpu alkaline halobacter BH-8 (B) alone had a more stable effect on the increase of soil available phosphorus content, and only decreased on the 21st day; the application of coal gangue alone (C) had no obvious effect on the soil available phosphorus content.

[0061] from Figure 6 From the changes in soil organic matter (OM) content, it can be seen that the effects of different treatment groups on soil organic matter content showed a positive trend. Compared with the control group, groups C, B and CB all significantly increased the organic matter content in the soil. This improvement effect reached its best state on the 14th day after treatment, indicating that these treatment methods have a significant effect on increasing soil organic matter. The increase in organic matter content not only plays an important role in improving soil fertility, but also has a positive effect on improving soil water retention and aeration. At the same time, this also helps to promote the activity of soil microorganisms and further enhance the ecological function of the soil.

[0062] from Figure 7 From the changes in soil pH, it can be seen that the soil pH decreased significantly on the 1st and 7th days after the application of BH-8 bacterial solution (B) and Hwajinpu alkaline halobacter BH-8 and coal gangue composite bacterial agent (CB), which is of positive significance for improving the alkaline environment of coastal saline soil; however, the improvement effect gradually weakened after the 14th day, which may be caused by the loss of soil salt due to irrigation.

[0063] from Figure 8 From the changes in the content of available potassium (AK) in the soil, it can be seen that the content of available potassium in the soil increased significantly in the first 14 days after the application of BH-8 bacterial solution (B) and the combined bacterial agent of Huajinpu Alkaline Halobacterium BH-8 and coal gangue (CB). Available potassium is the main form of potassium absorbed and utilized by plants, and the increase in its content helps to enhance the stress resistance and yield of plants. However, the treatment of applying coal gangue alone had no obvious effect on the content of available potassium in the soil, which may be closely related to the potassium release characteristics of coal gangue itself.

[0064] In summary, compared with the control group (CK), the addition of BH-8 bacterial agent (B) and coal gangue composite bacterial agent (CB) significantly increased the content of organic matter and alkaline nitrogen in the soil (p < 0.01), and significantly reduced the pH of coastal saline soil (p < 0.05). For the content of available potassium and available phosphorus in the soil, the application of group B and group CB significantly increased their content from 0 to 14 days (p < 0.05), but these effects decreased at 21 days, which may be due to soil nutrient loss caused by irrigation. The improvement effect of applying coal gangue (C) was not significant compared with the control group (CK).

[0065] (III) Effects of the combination of Hwajinpu alkaline Halobacterium BH-8 and coal gangue on enzyme activity in saline-alkali soil:

[0066] The nitrate reductase, alkaline phosphatase, acid phosphatase, urease and catalase activities of the soil after each treatment were detected using existing technology. The results are as follows: Figure 9-13 shown.

[0067] from Fig. 9 It can be seen from the changes in the catalase activity (S-CAT) of the soil that the catalase activity was basically not affected by the treatments, and was only slightly higher than the control group (CK) on the first day after the application of the Huajinpu alkaline Halobacter BH-8 bacterial solution (B) and the Huajinpu alkaline Halobacter BH-8 coal gangue composite bacterial agent (CB).

[0068] from Fig.10 From the changes in soil nitrate reductase activity (S-NR), it can be seen that the application of Huajinpu alkaline halobacter BH-8 and coal gangue composite bacterial agent group (CB) significantly increased nitrate reductase activity compared with the control group (CK) in the first 14 days; there were no significant differences between the other treatment groups (Group C and Group B) and the control group (CK).

[0069] Soil alkaline phosphatase (S-AKP, Fig.11 ), acid phosphatase (S-ACP, Fig.12 ) and urease activity (S-UE, Fig.13) were significantly increased within 21 days after the application of BH-8 bacterial solution (B) and BH-8 and coal gangue composite bacterial agent (CB). The application of BH-8 coal gangue composite bacterial agent (CB) had the most significant effect on improving soil urease activity. The application of BH-8 bacterial solution (B) alone increased the activities of soil acid phosphatase and alkaline phosphatase to a greater extent within 14 days. The application of BH-8 coal gangue composite bacterial agent (CB) had the best effect on improving the activities of soil acid phosphatase and alkaline phosphatase at 21 days. The application of coal gangue alone (C) had a significant effect on improving soil urease activity, and also increased soil nitrate reductase activity on the 1st and 14th days.

[0070] In terms of changes in soil enzyme activity, each treatment had no significant effect on soil catalase activity; the CB group could significantly increase soil nitrate reductase activity in the first 14 days; soil acid phosphatase, alkaline phosphatase, and urease activities were significantly increased within 21 days after the application of BH-8 bacterial solution (B) and BH-8 coal gangue composite bacterial agent (CB).

[0071] The above experiments prove that the application of BH-8 bacterial agent can significantly increase the content of alkaline nitrogen, organic matter and available phosphorus in coastal saline soil, and enhance the activity of soil nitrate reductase, acid phosphatase, alkaline phosphatase and urease, while the application effect of BH-8 strain and coal gangue composite bacterial agent is better than BH-8 bacterial agent, indicating that coal gangue and BH-8 strain have a synergistic effect, and BH-8 and coal gangue composite bacterial agent can effectively improve the nutrient content of coastal saline-alkali soil.

[0072] Embodiment 4:

[0073] This example involves an experiment on the effect of Huajinpu alkaline halobacter BH-8 and coal gangue composite bacterial agent on the microbial community structure of saline-alkali soil, and the specific steps are as follows:

[0074] 1. Experimental design

[0075] This experiment was conducted in an indoor environment. 12 portions of 200g of air-dried coastal saline-alkali soil were collected and placed in black plastic pots. Filter paper was laid at the bottom of the pots to prevent soil loss during irrigation. An equal amount of sterile water was added every three days to maintain soil moisture at 60%. After three days of incubation, the soil was divided into four groups for different treatments: the control group (CK) was treated with 20mL of sterile water; the gangue treatment group (C) was treated with 20mL of a 5g / L sterilized gangue particle solution; the Huajinpu alkaline halobacterium BH-8 treatment group (B) was treated with 10 9 CFU / mL of BH-8 bacterial solution 20mL; Huajinpu alkaline halobacter BH-8 and coal gangue composite bacterial agent group (CB), added Huajinpu alkaline halobacter BH-8 and coal gangue composite bacterial agent, the concentration of coal gangue in the composite bacterial agent was 5g / L, the concentration of BH-8 bacteria was 109 CFU / ml. Three replicate samples were set up for each group, and soil sampling was performed on the 1st day (treatment day), 7th day, 14th day and 21st day after treatment. Each sampling followed the five-point sampling method, and 10 g of fresh soil samples were collected and immediately stored in an ultra-low temperature refrigerator at -80°C for subsequent sequencing.

[0076] (II) Effects of Huajinpu Alkaline Halobacterium BH-8 and coal gangue composite bacterial agent on soil microbial diversity:

[0077] Table 2 Differences in Alpha diversity index among groups

[0078]

[0079] Note: abcd are significant differences.

[0080] Table 2 shows the Alpha diversity index of different sample groups. The indices used here are the Shannon diversity index, Simpson index, Chao1 species richness estimator, and ACE richness estimator. In the Shannon diversity index, the value of the control group (CK) was 6.758±0.183, indicating that the diversity of the control group was relatively high; the diversity of the treatment with single application of coal gangue (C) was slightly lower, at 6.469±0.132; while the value of the group with single application of Huajinpu alkaline Halobacter BH-8 (B) was 5.983±0.201, which was lower than the control; the Shannon index of the group with salt Bacillus and coal gangue composite bacteria (CB) was 5.426±0.148, which indicated that the combined treatment of salt Bacillus and coal gangue could not change the effect of the single application of Huajinpu alkaline Halobacter BH-8 (B) on diversity. The value of the control group (CK) was very similar to the Simpson index of the single application of coal gangue (C), indicating that the species dominance or uniformity did not change much; the value of the single application of Huajinpu alkaline Halobacter BH-8 group (B) was lower, at 0.950±0.007, indicating that the dominance of one or several species increased slightly; the value of the composite bacterial agent group of salt Bacillus and coal gangue (CB) was 0.933±0.005, indicating that the combined treatment of Huajinpu alkaline Halobacter and coal gangue resulted in a more uneven distribution of species, which may further aggravate the dominance effect. The value of Chao1 species richness control group (CK) was 3264.128±19.229, showing a higher species richness; the value of single application of coal gangue (C) was 3344.226±15.617, which may indicate that single application of coal gangue can slightly increase species richness; the value of single application of Huajinpu alkaline Halobacter BH-8 group (B) was lower, at 3497.598±6.757, which means that the estimated species richness decreased; and the value of Huajinpu alkaline Halobacter and coal gangue composite bacteria (CB) was 3451.400±43.283, which indicates that compared with single application of Huajinpu alkaline Halobacter BH-8 (B), the combined treatment did not significantly increase species richness. In the ace richness estimation, the recorded value of the control group (CK) was 3246.862±16.566, which was similar to the estimated value of Chao1, indicating a high species richness; the value of the single application of coal gangue (C) was 3323.715±12.102, which was again slightly higher than the control group; the value of the single application of Huajinpu Alkaline Halobacter BH-8 group (B) was 3477.981±5.112, indicating a decrease in richness; the value of the combined treatment of Huajinpu Alkaline Halobacter and coal gangue (CB) was 3429.857±40.909, and the species richness did not recover significantly.

[0081] In summary, the analysis in Table 2 shows that treatments may have different effects on biodiversity. The application of H. chinensis BH-8(B) alone reduced the diversity of all indices; the combined treatment of H. chinensis and coal gangue (CB) did not change the effect of H. chinensis BH-8(B) alone on the diversity of indices.

[0082] (III) Effects of the combination of Hwajinpu alkaline halobacterium BH-8 and coal gangue on the composition of saline-alkali soil microbial communities:

[0083] Fig.14 The changes in the relative abundance of species at the phylum level between the treatment group and the control group are shown. Pseudomonas was the dominant phylum at the phylum level. The relative abundance of Bacteroidetes changed significantly between the different treatment groups. The relative abundance of Bacteroidetes increased by 6.86% in the group (B) treated with Hwajinpu alkaline Halobacter and the control group (CK), and increased by 15.35% in the group (CB) treated with Hwajinpu alkaline Halobacter and coal gangue composite bacteria compared with the control group (CK). The relative abundance of species at the phylum level did not change significantly in the group (C) treated with only coal gangue compared with the control group (CK).

[0084] After applying the composite bacterial agent of Huajinpu alkaline halobacter BH-8 and coal gangue, the microbial community of the soil showed obvious changes. The overall gene number and diversity of the soil decreased, but the relative abundance of communities such as Bacteroidetes increased significantly. The application of the composite bacterial agent also had a significant impact on the community structure of soil microorganisms, and was affected by important environmental factors such as organic matter, available potassium, and alkaline nitrogen. In terms of soil microbial function, after applying the composite bacterial agent, the relative abundance of some glycoside hydrolases and carbohydrate binding modules in the soil increased, especially the CBM50 module had a very obvious abundance change. The CBM50 module is an enzyme that cuts chitin or peptidoglycan.

[0085] The present invention improves the acid-base balance and nutrient environment of saline-alkali soil by applying the composite bacterial agent of Hwajinpu alkaline Halobacter BH-8 and coal gangue, thereby having a profound impact on the composition and function of soil microbial communities, effectively improving the fertility of saline-alkali soil, and demonstrating the application value and potential of Hwajinpu alkaline Halobacter and coal gangue in bioremediation of saline-alkali soil.

Claims

1. A method for improving saline-alkali land, characterized in that: A composite bacterial agent of Huajinpu alkaline halobacter BH-8 and coal gangue is applied to saline-alkali soil. The Huajinpu alkaline halobacter BH-8 is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee with a deposit number of CGMCC No. 30829. The particle size of the coal gangue is 1 mm.

2. The method for improving saline-alkali land according to claim 1, characterized in that: The concentration of the compound bacterial agent is 1×10 9 ~3×10 9 CFU / mL, and the concentration of coal gangue particles was 5-10g / L.

3. The method for improving saline-alkali land according to claim 1, characterized in that: The preparation method of the composite bacterial agent of Huajinpu alkaline halobacter BH-8 and coal gangue is as follows: use coal gangue with a particle size of 1 mm and the BH-8 strain to co-culture in a modified LB medium until the bacterial body is in the logarithmic phase, filter the coal gangue, centrifuge the bacterial solution, take the precipitate, add sterile water and resuspend it to 1×10 9 ~3×10 9 CFU / mL, the resuspended bacterial suspension was mixed with the filtered coal gangue and used as a composite bacterial agent.

4. The method for improving saline-alkali land according to claim 1, characterized in that: The formula of the improved LB medium is: 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, 2-12 g / L sodium chloride; pH is 7.

2.

5. The method for improving saline-alkali land according to claim 1, characterized in that: The application amount of the compound bacterial agent is 100 mL per 1 kg of soil.

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