Biomass charcoal-loaded bacillus subtilis carbon-loaded microbial inoculum as well as preparation method and application of biomass charcoal-loaded bacillus subtilis carbon-loaded microbial inoculum

Abstract

The invention provides a charcoal-loaded microbial agent of biomass charcoal-loaded bacillus subtilis as well as a preparation method and application of the charcoal-loaded microbial agent, and belongs to the technical field of microorganisms. The carbon-loaded microbial inoculant formed by loading the bacillus subtilis on the pig manure biomass charcoal can effectively repair a microbial community structure in a cadmium-polluted soil environment, and is beneficial to restoration of ecological balance of soil, promotion of nutrient circulation of the soil, enhancement of soil resistance and improvement of physicochemical properties of the soil.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a carbon-loaded microbial agent with Bacillus subtilis loaded on biomass carbon, and a preparation method and application thereof. Background Art

[0002] Cadmium (Cd) is a highly toxic environmental heavy metal that widely exists in industrial wastewater, mine tailings and agricultural fertilizers. Due to its high mobility and bioaccumulation, cadmium enters the human body through the soil-plant-food chain, which may cause serious health problems such as kidney damage, osteoporosis and even cancer. In recent years, with the intensification of industrialization and agricultural activities, the problem of cadmium pollution in farmland soil in China has become increasingly serious. Cadmium pollution will seriously affect the soil microbial biomass carbon and nitrogen, soil basal respiration and soil enzyme activity, thus destroying the normal composition of soil microorganisms, inhibiting the metabolic function of soil microorganisms and reducing the soil microbial activity. This inhibitory effect may be related to the binding of cadmium to the active site of enzyme molecules to form a stable complex, and then produce competitive inhibition with the substrate. Cadmium pollution will also change the community structure of soil microorganisms, reducing the number of some cadmium-sensitive microbial species, while some cadmium-tolerant or cadmium-degrading microbial species may become dominant species. This change in community structure will affect the stability and function of the soil ecosystem, and further affect the health status of the soil.

[0003] Traditional heavy metal remediation technologies mainly include: physical remediation, chemical remediation and biological remediation. Physical and chemical remediation have high costs and often lead to secondary pollution and soil quality deterioration. Biological remediation is a natural, environmentally friendly and sustainable remediation method. Compared with traditional chemical and physical methods, it has lower costs and longer-term effects. Therefore, providing a biological remediation method to repair the community structure of soil microorganisms is beneficial to restoring the soil ecological balance, promoting soil nutrient cycling, enhancing soil resistance and improving soil physical and chemical properties. Summary of the Invention

[0004] The purpose of the present invention is to provide a carbon-loaded microbial agent with Bacillus subtilis loaded on biomass carbon, which is used to repair the microbial community structure in cadmium-polluted soil, restore the soil ecological balance and improve the soil physical and chemical properties.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a carbon-loaded microbial agent with Bacillus subtilis loaded on biomass carbon. The carbon-loaded microbial agent is mainly composed of Bacillus subtilis bacterial liquid and biomass carbon, and the biomass carbon is pig manure biomass carbon.

[0007] Preferably, the pig manure biomass carbon is obtained by pyrolyzing pig manure in a carbonization furnace, screening after cooling, and then sterilizing and drying.

[0008] Preferably, the pig manure is air-dried pig manure without composting; the temperature of pyrolysis is 500-600 °C, and the time is 2-3 h; the aperture of the sieve for sieving is 0.1-0.2 mm; the temperature of sterilization is 115-125 °C, and the time is 25-35 min; the temperature of drying is 60-70 °C, and the drying time is 20-24 h.

[0009] The present invention also provides a preparation method of the carbon-supported microbial agent as described above, comprising the following steps:

[0010] 1) Take pig manure biochar in a liquid medium, add Bacillus subtilis bacterial liquid and then culture;

[0011] 2) Use a cell sieve to separate the biochar;

[0012] 3) Take the biochar after washing to obtain the carbon-supported microbial agent.

[0013] 5. The preparation method according to claim 4, wherein in step 1), the temperature of the culture is 25-35 °C, the rotation speed during the culture is 160-200 r / min, and the culture time is 20-24 h.

[0014] Preferably, in step 1), the temperature of the culture is 25-35 °C, the rotation speed during the culture is 160-200 r / min, and the culture time is 20-24 h.

[0015] Preferably, the concentration of the Bacillus subtilis bacterial liquid in step 1) is 5×10 8 ~6×10 8 cfu / ml.

[0016] Preferably, the mass-volume ratio of the pig manure biochar to the Bacillus subtilis bacterial liquid in step 1) is 1-2 g:0.6-1.2 ml.

[0017] Preferably, the aperture of the cell sieve in step 2) is 30-40 μm.

[0018] The present invention also provides an application of the carbon-supported microbial agent as described above or the carbon-supported microbial agent prepared by the preparation method as described above in repairing the microbial community in cadmium-polluted soil.

[0019] The carbon-supported microbial agent provided by the present invention selects Bacillus subtilis and pig manure biochar as raw materials, significantly changes the community structure of soil microorganisms, significantly improves the abundance and diversity of fungi in the soil, improves the physical and chemical properties of the soil, significantly increases the content of soil nutrients such as total nitrogen, organic carbon, available phosphorus, and available potassium in the soil, effectively promotes the metabolic activity of soil microorganisms, and has a significant promoting effect on the restoration of cadmium-polluted soil to health. Description of the Drawings

[0020] Figure 1 Scanning electron micrographs of Bacillus subtilis suspension (BS), carbon-supported bacterial agent (BCS), wet biochar (BCW) and sterile dry biochar (BCD) in Example 2;

[0021] Figure 2 PCoA analysis chart of soil bacterial communities under different treatments in Example 4;

[0022] Figure 3 PCoA analysis chart of soil fungal communities under different treatments in Example 4;

[0023] Figure 4 Relative abundance chart of soil bacterial communities at the phylum level under different treatments in Example 4;

[0024] Figure 5 Relative abundance chart of soil fungal communities at the phylum level under different treatments in Example 4;

[0025] Figure 6 Relative abundance chart of soil bacterial communities at the family level under different treatments in Example 4;

[0026] Figure 7 Relative abundance chart of soil fungal communities at the family level under different treatments in Example 4;

[0027] Figure 8 Relative abundance chart of soil bacterial communities at the genus level under different treatments in Example 4;

[0028] Figure 9 Relative abundance chart of soil fungal communities at the genus level among different treatments in Example 4;

[0029] Figure 10 Influence chart of different treatments on total cadmium and available cadmium contents in soil in Example 5. Specific implementation mode

[0030] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0031] Example 1

[0032] 1.1 Preparation of biochar

[0033] The air-dried pig manure without composting was pyrolyzed at 550 °C for 2 h in a rotary kiln continuous carbonization furnace, cooled and passed through a 0.150 mm nylon sieve. The material under the sieve was sterilized with high-temperature steam at 121 °C for 30 min and dried in an oven at 65 °C for 24 h before use. The basic properties of the prepared biochar are shown in Table 1.

[0034] Table 1 Basic properties of biochar

[0035] Item pH C(％) N(％) O(％) H(％) Yield (%) BCD 9.66±0.01 72.20±0.16 2.90±0.06 15.47±0.09 2.36±0.01 24.84±0.11

[0036] 1.2 Preparation of Bacillus subtilis bacterial suspension

[0037] Take an appropriate amount of Bacillus subtilis powder and place it in a conical flask containing sterile water and an internal small glass bead. Then place it in a constant temperature shaker at 30 °C and shake it at 180 r·min -1 at a rotation speed for 20 min to disperse it. After dilution, streak plate coating is carried out. After separation and purification, a single colony is obtained, and then strain identification is carried out to determine it as Bacillus subtilis. Pick a single colony of Bacillus subtilis into a liquid medium, and then place it in a constant temperature shaker at 30 °C and cultivate it at 180 rmin -1 at a rotation speed for 20 h. Transfer it to a sterile centrifuge tube, and after ultra-high speed refrigerated centrifugation (5000×g, 5 min), suspend the cells with the liquid medium to obtain the bacterial suspension (BS). Place it in a refrigerator at 4 °C for storage. The concentration of Bacillus subtilis measured by the dilution plate counting method is 5.8×10 8 cfu·mL -1 .

[0038] 1.3 Preparation of biomass carbon loaded with Bacillus subtilis

[0039] Take 3 g of biomass carbon after sterilization and drying and place it in 60 mL of beef extract peptone liquid medium. Add 1.8 mL of Bacillus subtilis bacterial suspension to each bottle, for a total of three bottles. Place it in a constant temperature shaker at 30 °C and shake it at 180 r·min -1 at a rotation speed for 24 h. After cultivation, separate the medium and biomass carbon with a 40 μm cell sieve, rinse it with sterile water, and then centrifuge it in an ultra-high speed freezer (5000×g, 5 min), discard the upper liquid, and obtain the carbon-loaded bacterium agent (BCS) of biomass carbon loaded with Bacillus subtilis for standby.

[0040] Example 2

[0041] Observe the Bacillus subtilis suspension (BS), carbon-loaded bacterium agent (BCS), wet biomass carbon (BCW, the preparation process is the same as BCS except without adding Bacillus subtilis loading), and sterile dry biomass carbon (BCD) prepared in Example 1 under a tungsten filament lamp scanning electron microscope (JSM-6360LV), and the results are as Figure 1 shown (Figures a and b are for BS, Figures c and d are for BCS, Figures e and f are for BCW, Figures g and h are for BCD):

[0042] From Figure 1It can be seen that in Figures a and b, the cell morphology of Bacillus subtilis is relatively plump, rod-shaped, and round at both ends. From Figures c and d, it can be seen that Bacillus subtilis attaches to the surface of biochar or penetrates into the pores of biochar, and there is little difference in cell morphology and size compared with those in a and b. From Figures e, f, g, and h, it can be found that biochar has abundant pores and a rough surface. There are more surface debris on BCD than on BCW. The possible reason is that BCW was soaked in the culture medium and washed with water, washing away the fine particles on the surface.

[0043] Example 3

[0044] Take 1.000 g of the carbon-supported bactericide prepared in Example 1 above and soak it in 100 mL of beef extract peptone liquid medium for 5 min. Extract the Bacillus subtilis fixed on the biochar by ultrasonic bath (10 min) and vortex mixing (3 min, 2 times). Finally, use the dilution plate coating counting method to calculate the loading amount of Bacillus subtilis on the biochar (after culturing at 30 °C for 48 h, count the single colonies on the plate). The number of Bacillus subtilis fixed per gram of biochar is 6.7×10 6 cfu.

[0045] Example 4

[0046] Based on pot experiments, using high-throughput sequencing technology, study the effects of different material additions (CK, BS, BCW, BCS, BCD) on the bacterial and fungal community structures and species richness in Cd-contaminated soil (total Cd: 6.63 mg kg -1 ) and reveal the effects of different materials on the soil microbial community structure. The experiment was conducted in an artificial climate chamber at Northwest A&F University. During the experiment, the indoor temperature was controlled at 15 - 25 °C. Each treatment had 4 replicates, and each pot was filled with 1.75 kg of soil. Among them, the addition amounts of the three biochars BCW, BCS, and BCD were 1% (dry basis mass ratio). The viable bacteria counts in the BS and BCS treatments were equal, and the addition amount per pot was 1.2×10 8 cfu. BCW, BCS, and BCD materials were dried and ground in an oven at 35 °C, thoroughly mixed with the soil, and then filled into the pots. BS was the treatment with bactericide, added in the form of a bacterial suspension, which was diluted with distilled water and evenly applied to the soil with a syringe. The soil water content was maintained at 60% - 65% of the field water holding capacity by the weighing method.

[0047] 1.1 Effects of different treatments on the diversity of soil bacteria and fungi

[0048] Table 2 Alpha diversity of soil bacteria under different treatments

[0049]

[0050] Data are presented as mean ± standard deviation (n = 4). Different letters indicate significant differences among treatments at the significance level of P < 0.05

[0051] Table 3 Alpha diversity of soil fungi under different treatments

[0052]

[0053] Data are presented as mean ± standard deviation (n = 4). Different letters indicate significant differences among treatments at the significance level of P < 0.05

[0054] As shown in Table 2 and Table 3, there were no significant differences in the Alpha diversity indices of soil bacteria among treatments, and there were no significant differences in the Chao 1 index and Pd faith index of soil fungi. Compared with BCD, the Shannon index and Simpson index of soil fungi under the BCS treatment were significantly increased, and the Simpson index under the BS treatment was significantly increased. There were no significant differences among other treatments

[0055] 1.2 PCoA analysis was performed on the soil bacterial OUT composition under five treatments. The contribution rates of the first principal coordinate and the second principal coordinate were 11% and 10% respectively. The CK group was located in the first and third quadrants, the BS group was located in the first and second quadrants, the BCW group was located in the second, third, and fourth quadrants, the BCS group was located in the first, third, and fourth quadrants, and the BCD was located in the first, second, and third quadrants. There was no obvious distribution pattern among the five treatments, and there were significant differences in the bacterial community composition among treatments. The results are as Figure 2 shown

[0056] PCoA analysis was performed on the soil fungal OUT composition under five treatments. The contribution rates of the first principal coordinate and the second principal coordinate were 17% and 12% respectively. The samples of the CK group and the BCD group were both located on the right side of the first principal coordinate, indicating that the fungal community compositions of the CK and BCD groups were similar. The CK group and the BS group were close, indicating that the fungal community compositions of the CK and BS groups were similar. The samples of the CK group and the BCS group were located on the right and left sides of the first principal coordinate respectively, indicating that the fungal community compositions of the CK and BCS groups were quite different. The CK group and the BCW group were far apart, indicating that the fungal community compositions of the CK and BCW groups were quite different. The BCS group was far from the BS and BCD groups, indicating that the fungal community compositions of the BCS group were quite different from those of the BS and BCD groups. The BCS group was located on the left side of the first principal coordinate axis, and most of the BCW group was located on the left side of the first principal coordinate, indicating that the fungal community compositions of the BCS group and the BCW group were similar and also different. The BCD group and the BCW group were far apart, indicating that the fungal community compositions of the BCD group and the BCW group were quite different. The BCW group was located in the first, second, and third quadrants, and the BS group was located in the second, third, and fourth quadrants, indicating that there were both similarities and differences in the soil fungal community compositions between the BCW and BS treatments. The results are Figure 3 shown

[0057] 2.1 Effects of different treatments on the phylum-level composition of soil bacterial and fungal communities

[0058] Figure 4 Figure 1 shows the relative abundances of bacteria in different soil samples at the phylum level. A total of 39 phyla, 283 families, and 532 genera were identified in the soil bacteria of the five treatment groups. Proteobacteria was the dominant phylum in each treatment group, with an average relative abundance ranging from 28.34% to 35.43% in each treatment group. Compared with CK, the relative abundance of Proteobacteria in the BCS treatment group increased by 2.99%, while the relative abundances in the BS, BCW, and BCD treatment groups decreased by 3.61%, 2.49%, and 4.10% respectively; Actinobacteriota was the second dominant phylum, with an average relative abundance ranging from 18.37% to 21.51% in each treatment group. Compared with CK, the relative abundance of Actinobacteriota in the BCW treatment group increased by 0.15%, while those in the BS, BCS, and BCD treatment groups decreased by 2.99%, 1.39%, and 1.65% respectively; Acidobacteriota was the third dominant phylum, with an average relative abundance ranging from 15.40% to 22.90% in each treatment group. Compared with CK, the relative abundances of Acidobacteriota in the BS, BCW, and BCD treatment groups increased by 5.08%, 3.90%, and 6.56% respectively, while that in the BCS treatment group decreased by 0.94%. Generally speaking, the differences in the relative abundances of the top 10 species at the bacterial phylum level among the treatments were not significant. In addition, it was detected that outside the top 10 and within the top 30, there was only one bacterial taxon with a significant difference in phylum-level abundance: Bdellovibrionota, with an average relative abundance ranging from 0.21% to 0.34% in each treatment group. The BCS treatment was significantly higher than the CK group, and there were no significant differences among the other treatment groups.

[0059] Figure 5The relative abundances of fungi in different soil samples at the phylum level. A total of 7 phyla, 192 families, and 458 genera were identified in the soil fungi of the five treatment groups. Ascomycota was the dominant phylum in each treatment group, with an average relative abundance ranging from 57.76% to 71.57% in each treatment group. Compared with the CK treatment, there was no significant difference in the relative abundance of Ascomycota among the treatment groups. The difference in the relative abundance of Ascomycota between the BCS and BCD treatment groups reached an extremely significant level, and the average relative abundance of Ascomycota in the BCD treatment group increased by 13.8% compared with the BCS treatment group; Mucoromycota was the second dominant phylum, with an average relative abundance ranging from 13.14% to 26.66% in each treatment group. Compared with CK, the average relative abundances of Mucoromycota in the BS, BCW, BCS, and BCD treatment groups increased by 4.49%, 5.91%, 13.52%, and 0.83%, respectively. There was no significant difference in the relative abundance of Mucoromycota between the CK group and the BS, BCW, and BCD groups. The difference in Mucoromycota between the CK and BCS treatment groups reached an extremely significant level; Basidiomycota was the third dominant phylum, with an average relative abundance ranging from 11.89% to 15.80% in each treatment group. Compared with CK, the average relative abundances of Basidiomycota in the BS, BCW, BCS, and BCD treatment groups decreased by 3.65%, 2.40%, 3.06%, and 3.91%, respectively. There was no significant difference among the treatment groups. The difference values of the relative abundances of species in the remaining four phyla were not significant among the treatments.

[0060] 2.2 Effects of different treatments on the family-level composition of soil bacteria and fungal communities

[0061] From Figure 6It can be seen that the top 10 dominant bacterial groups in the soil bacteria at the family classification level under each treatment mainly include: Sphingomonadaceae, Vicinamibacteraceae, Pyrinomonadaceae, Micrococcaceae, Gemmatimonadaceae, Xanthobacteraceae, Roseiflexaceae, Chitinophagaceae, Geodermatophilaceae, and Comamonadaceae. The only significant difference among the treatment groups at the above family level is Comamonadaceae, and its average relative abundance in each treatment group ranges from 1.67% to 2.72%. The average relative abundance of Comamonadaceae in the treatment groups BS and BCW decreased significantly by 0.97% and 1.06% compared with the CK group, and the treatment groups BCS and BCD also decreased to varying degrees, by 0.74% and 0.64% respectively compared with CK.

[0062] There are also bacterial groups with significant differences in family-level abundance within the top 30: namely, Xanthomonadaceae, Steroidobacteraceae, Rhizobiaceae, and Beijerinckiaceae. The average relative abundance of Xanthomonadaceae in each treatment group ranges from 0.69% to 2.49%. The relative abundance of Xanthomonadaceae in the BCS treatment group is significantly higher than that in the blank group and other treatment groups, and its relative abundance increased by 1.66% compared with the blank. Secondly, the relative abundance of the BCW group increased significantly by 0.50% compared with the BS group; the average relative abundance of Steroidobacteraceae in each treatment group ranges from 0.90% to 1.37%. The relative abundance of the BCS treatment group increased significantly by 0.47% compared with the BS group, and there were no significant differences among other treatments; the average relative abundance of Rhizobiaceae in each treatment group ranges from 0.78% to 1.63%. The average relative abundance of the BCS group is significantly higher than that of other treatment groups, increasing by 0.59% compared with CK. The average relative abundance of Beijerinckiaceae in each treatment group ranges from 1.41% to 2.39%. Compared with CK, the average relative abundances of the BS, BCW, and BCD treatment groups decreased by 0.77%, 0.28%, and 0.47% respectively, while the BCS group increased by 0.22%. The average relative abundance of the BCS treatment group is significantly higher than that of the BS group.

[0063] Generally speaking, the top 10 species composition abundances of each treatment are similar at the bacterial family level, and there are only significant differences in the relative abundances of the Comamonadaceae flora; within the top 10 to the top 30, there are 4 bacterial groups with significant differences in abundances among different treatment groups at the family level. The results show that different treatments have little effect on the relative abundances of the dominant families of soil bacteria, but will have a relatively greater impact on the abundances of a few species. The BCS treatment significantly increased the relative abundances at the family levels of Xanthomonadaceae and Rhizobiaceae.

[0064] It can be seen from Figure 7 that the top 10 dominant families of each treatment group of fungi are Nectriaceae, Mortierellaceae, Chaetomiaceae, Piskurozymaceae, Lasiosphaeriaceae, Plectosphaerellaceae, Olpidiaceae, Pleosporaceae, Bolbitiaceae, and Herpotrichiellaceae. Among them, the first four families have significant differences in relative abundances among groups. The average relative abundance of Nectriaceae in each treatment group is between 22.71% and 35.07%. The average relative abundance of the BCD group is significantly increased by 12.35% compared with the BCS group, and there are no significant differences among other treatments; the average relative abundance of Mortierellaceae in each treatment group is between 13.12% and 26.57%. Compared with CK, the average relative abundances of the BS, BCW, and BCS groups are increased by 4.44%, 5.91%, and 13.45% respectively, and the BCS treatment group is significantly higher than CK; the average relative abundance of Chaetomiaceae in each treatment group is between 11.37% and 17.09%. The average relative abundance of the BS group is significantly increased by 5.72% and 5.06% compared with the BCS and BCD groups, and there are no significant differences among other treatments; the average relative abundance of Piskurozymaceae in each treatment group is between 6.79% and 9.70%. The average relative abundance of the BS group is significantly increased by 2.92% compared with the BCS group, and there are no significant differences among other treatments. In addition, there are no fungal groups with significant differences at the family level within the top 30. In summary, at the family level, there are significant differences in the abundances of some dominant flora species among different treatment groups of fungi, and the changes in other flora except the dominant flora are not significant.

[0065] 2.2 Effects of different treatments on the genus-level composition of soil bacteria and fungi

[0066] It can be seen from Figure 8It can be seen that the top 10 dominant genera in each treatment group of bacteria are Sphingomonas, RB41, Pseudarthrobacter, Blastococcus, Microvirga, Rahnella1, Bryobacter, Skermanella, Massilia, and Bradyrhizobium. Except for Microvirga, there were no significant differences in the relative abundances among the treatment groups of the above dominant genera. The average relative abundance of Microvirga in each treatment group was between 1.09% and 1.84%. The average relative abundance in the BCS group increased significantly by 0.75% compared with the BS group. There were no significant differences in the relative abundances among the other groups. In addition, the bacterial taxa with significant differences at the genus level within the top 30 were Variovorax, Lysobacter, and Gemmatimonas. The average relative abundance of Variovorax in each treatment group was between 0.35% and 0.70%. Compared with CK, the average abundances in the BS, BCW, BCS, and BCD treatment groups decreased by 0.32%, 0.36%, 0.23%, and 0.33% respectively. There were significant differences between CK and the BS, BCW, and BCD treatments. The average relative abundance of Lysobacter in each treatment group was between 0.47% and 1.23%. Compared with CK, the average relative abundances in the BCW and BCS groups increased significantly by 0.36% and 0.68% respectively. The average relative abundance of Gemmatimonas in each treatment group was between 0.41% and 0.67%. The relative abundance in the BCD treatment group increased significantly by 0.26% compared with the BCS treatment group. There were no significant differences among the other treatment groups.

[0067] It can be seen from Figure 9It can be seen that the top 10 dominant genera in each treatment group of fungi are Fusarium, Mortierella, Solicoccozyma, Humicola, Chaetomium, Olpidium, Alternaria, Gibellulopsis, Podospora, and Conocybe. Among them, Fusarium, Mortierella, and Solicoccozyma showed significant differences among groups at the genus level. The average relative abundance of Fusarium in each treatment group was between 21.95% and 34.48%. Compared with CK, the average relative abundances in the BCW and BCD groups increased by 4.51% and 9.41% respectively, while those in the BS and BCS groups decreased by 0.36% and 3.11%. There was a significant difference in the average relative abundance between the BCS group and the BCD group; the average relative abundance of Mortierella in each treatment group was between 13.12% and 26.57%. Compared with CK, the average relative abundances in the BS, BCW, and BCS groups increased by 4.44%, 5.91%, and 13.45% respectively, and the average relative abundance in the BCS group was significantly higher than that in the CK group; the average relative abundance of Solicoccozyma in each treatment group was between 6.79% and 9.70%. Compared with the BS treatment, the BCS treatment significantly decreased by 2.92%. In addition, the only fungal group with significant differences at the genus level within the top 30 was Dokmaia, and the average relative abundance in each treatment group was between 0.49% and 1.03%. The BCS group significantly decreased by 0.54% compared with CK, and there were no significant differences in relative abundance among other treatments.

[0068] Example 5

[0069] 1.1 Effects of Different Treatments on Soil Cadmium Content

[0070] The effects of different treatments on the total cadmium and available cadmium contents in the soil are shown in Figure 10 . Compared with the blank control, there was no significant difference in the total cadmium content in the soil after applying different materials. However, the available cadmium content in the soil was significantly reduced. The available Cd contents in the BS, BCW, BCS, and BCD groups decreased by 6.1%, 7.0%, 6.7%, and 8.0% respectively compared with CK.

[0071] 1.2 Effects of Different Treatments on Soil Physical and Chemical Properties

[0072] Table 4 Soil Physical and Chemical Properties under Different Treatments

[0073]

[0074]

[0075] EC: Electrical conductivity; SOC: Soil organic carbon; TN: Total nitrogen; AP: Available phosphorus; AK: Available potassium. CK: Blank control; BS: Bacillus subtilis; BCW: Wet biochar; BCS: Bacillus subtilis loaded on biochar; BCD: Sterile biochar. The values in the table represent the mean ± standard deviation (n = 4), and different lowercase letters in the same row indicate significant differences (P < 0.05).

[0076] As can be seen from Table 4, compared with CK, the addition of the three biochar-containing materials (BCW, BCS, BCD) significantly increased the soil pH value, and BCD had the most obvious effect. Compared with CK, the soil pH values under the treatments of BCW, BCS, and BCD increased by 0.12, 0.13, and 0.23 units respectively. There were no significant differences among other treatments. The addition of BCW and BCS could significantly increase the soil electrical conductivity. Compared with CK, the increase ranges were 29.7% and 25.9% respectively. There were no significant differences between the BS and BCD treatments and the CK treatment. Compared with CK and BS, the addition of BCW, BCS, and BCD significantly increased the content of soil organic carbon. Compared with CK, the soil organic carbon increased by 26.9%, 57.6%, and 30.9% under the treatments of BCW, BCS, and BCD respectively. There was no significant difference between the BS treatment and the CK treatment. The addition of BCW, BCS, and BCD significantly increased the total nitrogen content of the soil. Compared with the CK treatment, the increase ranges were 17.1%, 19.2%, and 16.8% respectively. There was no significant difference in the total nitrogen content between the BS treatment and the CK treatment. However, it significantly reduced the soil ammonium nitrogen content. Compared with CK, the BCW, BCS, and BCD treatments significantly reduced it by 34.3%, 29.0%, and 35.3% respectively. There were no significant differences between the BS treatment and other treatments. Compared with CK, the treatments of BCW and BS significantly increased the soil nitrate nitrogen content by 46.9% and 20.9% respectively. There were no significant differences among other treatments. Compared with CK, the addition of BCW, BCS, and BCD significantly increased the content of available phosphorus in the soil. The increase ranges were 46.0%, 44.1%, and 58.6% respectively. Among them, the increase rate of available phosphorus in the soil under the BCD treatment was significantly higher than that of other treatments. There was no significant difference between the BS treatment and the CK treatment. Compared with CK, the treatments of BS, BCW, BCS, and BCD all significantly increased the content of available potassium in the soil. The increase ranges were 4.9%, 5.9%, 4.3%, and 11.5% respectively. Among them, the increase rate of available potassium in the soil under the BCD treatment was significantly higher than that of other treatments.

[0077] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A biochar-loaded bacterial agent containing Bacillus subtilis, characterized in that: The charcoal-supported bacterial agent is mainly composed of Bacillus subtilis bacterial liquid and biomass charcoal, and the biomass charcoal is pig manure biomass charcoal.

2. The charcoal-supported bacterial agent according to claim 1, characterized in that: The pig manure biomass charcoal is obtained by pyrolyzing pig manure in a carbonization furnace, sieving after cooling, and then sterilizing and drying.

3. The charcoal-supported bacterial agent according to claim 2, characterized in that: The pig manure is air-dried pig manure that has not been composted; the pyrolysis temperature is 500-600°C, and the time is 2-3 hours; the sieve mesh aperture is 0.1-0.2 mm; the sterilization temperature is 115-125°C, and the time is 25-35 minutes; the drying temperature is 60-70°C, and the drying time is 20-24 hours.

4. A method for preparing the charcoal-supported bacterial agent according to any one of claims 1 to 3, characterized in that: The steps include: 1) Take pig manure biochar in a liquid culture medium, add Bacillus subtilis liquid and culture; 2) Separation of biochar using cell sieves; 3) The biomass charcoal is washed to obtain a charcoal-supported bacterial agent.

5. The preparation method according to claim 4, characterized in that: The culture temperature in step 1) is 25-35° C., the rotation speed during culture is 160-200 r / min, and the culture time is 20-24 h.

6. The preparation method according to claim 5, characterized in that: The concentration of the Bacillus subtilis solution in step 1) is 5×10 8 ~6×10 8 cfu / ml.

7. The preparation method according to claim 6, characterized in that: In step 1), the mass volume ratio of the pig manure biomass charcoal to the Bacillus subtilis liquid is 1-2 g: 0.6-1.2 ml.

8. The preparation method according to claim 7, characterized in that: The pore size of the cell screen in step 2) is 30-40 μm.

9. Use of the charcoal-supported bacterial agent according to any one of claims 1 to 3 or the charcoal-supported bacterial agent prepared by the preparation method according to any one of claims 4 to 8 in repairing microbial communities in cadmium-contaminated soil.

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