A cadmium-resistant cellulomicrobium Cd-1 and its cultivation method and application

Abstract

The present invention relates to the field of microbial application technology, and in particular to a cadmium-resistant fiber microbacterium Cd-1 and a culture method and application thereof. The strain preservation number of the cadmium-resistant fiber microbacterium Cd-1 is CGMCC No. 25219. It can be used to promote the growth and development of ryegrass and the restoration of cadmium-contaminated soil. The cadmium-resistant fiber microbacterium Cd-1 provided by the present invention can increase the activity of soil urease and luciferin hydrolase, increase the microbial activity of the soil, increase the chlorophyll content in ryegrass leaves, and promote the antioxidant system of ryegrass to remove excessive active oxygen in ryegrass, alleviate the oxidative stress reaction of ryegrass under adverse stress, promote the growth of ryegrass in cadmium-contaminated soil, increase the biomass of ryegrass, and increase the absorption and enrichment of cadmium by ryegrass, which helps to promote the restoration of ryegrass to cadmium-contaminated soil.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to a cadmium-resistant cellulomicrobium Cd-1 and a culture method and application thereof. Background Art

[0002] With the development of economy and society, the rapid development of mining, metal smelting and chemical industry has led to the release of a large amount of heavy metals into the natural environment, polluting the soil and water system, causing the heavy metals in the environment to exceed the standard. The heavy metals released into the soil and water system will be absorbed by plants, which will pose a serious threat to the ecosystem.

[0003] Cadmium is toxic and a heavy metal pollutant. It is not easily biodegraded or chemically degraded in the soil, and it is bioaccumulative. Cadmium distributed in the soil can be absorbed by plants. When the plants that absorb cadmium are eaten by herbivores, herbivores are eaten by carnivores, and herbivores and carnivores are eaten by humans, cadmium is transferred to the human body through the food chain and accumulates in the human body, causing people to suffer from diseases such as cancer, which directly endangers human health.

[0004] Microbial soil remediation technology is a method of using microorganisms to fix, move or transform heavy metals in the soil to change the content of heavy metals in the soil. In the existing technology, the tolerance concentration of cadmium in cadmium-contaminated soil by microorganisms is 20-40 mg / kg. In a higher concentration of cadmium contaminated environment, the survival rate of existing cadmium-resistant microorganisms is low.

[0005] Ryegrass is a perennial plant with a stem height of 30-90 cm and soft roots at the base nodes. When planted in cadmium-contaminated soil, it can absorb cadmium through its roots. Therefore, ryegrass can be used to repair cadmium-contaminated soil. However, the growth of ryegrass will be restricted under high cadmium concentrations, resulting in a decrease in biomass, which is not conducive to the absorption and accumulation of cadmium. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a cadmium-resistant cellulosic microorganism Cd-1, which can survive in cadmium pollution at a concentration of 0 to 100 mg / L, can increase the activity of soil urease and luciferin hydrolase, increase the microbial activity of the soil, promote the growth of ryegrass, thereby promoting the enrichment of cadmium in ryegrass, and achieve the purpose of repairing cadmium-contaminated soil.

[0007] To solve the above technical problems, the embodiments of the present invention adopt the following technical solutions:

[0008] In the first aspect, the present invention provides a cadmium-resistant cellulosimicrobium Cd-1, whose strain preservation number is CGMCC No. 25219. The strain belongs to Cellulosimicrobium sp., and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 1, 2022. The storage address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0009] Compared with the prior art, the cadmium-resistant cellulosic microbe Cd-1 provided by the present invention can survive under a cadmium concentration of 0 to 100 mg / L, can increase the activities of soil urease and luciferin hydrolase, thereby increasing the biological activity of the soil, increasing the chlorophyll content in ryegrass leaves, and promoting the antioxidant system of ryegrass to remove excessive active oxygen in ryegrass, alleviating the oxidative stress response of ryegrass under adverse conditions, and promoting the growth of ryegrass in cadmium-contaminated soil; increasing the biomass of ryegrass, increasing the enrichment of cadmium by ryegrass, and helping to promote the restoration of ryegrass to cadmium-contaminated soil.

[0010] The present invention obtains differentially expressed genes by sequencing the Cd-1 transcription, and obtains a total of 148 screened differentially expressed genes, of which 142 are upregulated and 6 are downregulated. After performing GO enrichment analysis and KEGG pathway enrichment analysis on the screened differentially expressed genes, it is found that the enrichment pathway of the differentially expressed genes is most significant in the ribosome pathway.

[0011] In a second aspect, the present invention provides a method for culturing the cadmium-resistant cellulomicrobium Cd-1, wherein the cadmium-resistant cellulomicrobium Cd-1 is cultured in a culture medium with a pH of 6 to 7.

[0012] Preferably, the cadmium-resistant cellulobacterium Cd-1 is cultured at a temperature of 30-40°C.

[0013] The culturing method of the cadmium-resistant cellulobacterium Cd-1 provided by the present invention can increase the reproduction speed of the strain and allow Cd-1 to grow normally in a high-concentration cadmium environment.

[0014] In a third aspect, the present invention provides an application of the cadmium-resistant cellulosic microorganism Cd-1 in promoting the growth of ryegrass in cadmium-contaminated soil.

[0015] In a cadmium-contaminated environment, the active oxygen in ryegrass increases, causing ryegrass to produce oxidative stress, increasing the secretion of superoxide dismutase and glutathione to coordinate redox balance. Studies have found that inoculating cadmium-tolerant fiber microbes Cd-1 in cadmium-contaminated soil with ryegrass can increase the chlorophyll content in ryegrass leaves and promote the antioxidant system of ryegrass to remove excessive active oxygen in ryegrass, thereby reducing the superoxide dismutase activity and glutathione content in ryegrass leaves. This result shows that cadmium-tolerant fiber microbes Cd-1 can alleviate the oxidative stress response under adverse stress, allowing ryegrass plants to grow normally, thereby increasing the biomass of ryegrass and promoting the growth and development of ryegrass. The underground and aboveground parts of well-grown ryegrass have increased enrichment coefficients and transport coefficients for cadmium, thereby reducing the residual amount of cadmium in cadmium-contaminated soil and improving the removal rate of cadmium in contaminated soil.

[0016] In a fourth aspect, the present invention also provides a method for promoting the growth of ryegrass in cadmium-contaminated soil: inoculating the cadmium-resistant cellulobacterium Cd-1 into the cadmium-contaminated soil where ryegrass is planted.

[0017] Preferably, the concentration of cadmium in the cadmium-contaminated soil is ≤100 mg / L.

[0018] Preferably, the method further comprises, after inoculating cadmium-resistant cellulobacteria Cd-1, culturing at a light intensity of 10-14 h / d and a darkness intensity of 8-10 h / d.

[0019] Preferably, the time of inoculating the cadmium-resistant cellulobacterium Cd-1 is before sowing ryegrass seeds.

[0020] Preferably, the moisture content of the cadmium-contaminated soil inoculated with cadmium-resistant cellulobacteria Cd-1 is maintained at 40%-60%.

[0021] In a fifth aspect, the present invention also provides a method for repairing cadmium-contaminated soil, comprising planting ryegrass in cadmium-contaminated soil inoculated with cadmium-tolerant cellulobacterium Cd-1; collecting cadmium-enriched ryegrass plants to complete the repair of cadmium-contaminated soil.

[0022] The method of planting ryegrass can be to directly plant ryegrass plants or sow ryegrass seeds. The planting time should be after inoculating cadmium-tolerant fiber microorganisms Cd-1. Cadmium-tolerant fiber microorganisms Cd-1 can increase the activity of soil urease and luciferin hydrolase, thereby increasing the activity of soil microorganisms, promoting the growth of ryegrass seeds into ryegrass plants and the continued growth of ryegrass plants. Ryegrass plants absorb cadmium in the enriched soil through their roots and transfer the cadmium in the soil to ryegrass plants. Finally, by collecting ryegrass plants that absorb cadmium, the remediation of cadmium pollution in the soil is completed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1It is the growth curve and pH change diagram of the cadmium-resistant cellulobacterium Cd-1 in the embodiment of the present invention;

[0024] Figure 2 This is a graph showing the growth characteristics of the cadmium-resistant cellulobacterium Cd-1 under different cadmium concentrations in an embodiment of the present invention;

[0025] Figure 3 This is a graph showing the growth characteristics of the cadmium-resistant cellulobacterium Cd-1 at different pH values ​​in an embodiment of the present invention;

[0026] Figure 4 This is a graph showing the growth characteristics of the cadmium-resistant cellulobacterium Cd-1 at different temperatures in an embodiment of the present invention;

[0027] Figure 5 It is a volcano map of differentially expressed genes of cadmium-resistant cellulobacterium Cd-1 in the embodiment of the present invention;

[0028] Figure 6 GO functional classification diagram of differentially expressed genes of cadmium-resistant cellulobacterium Cd-1 in the embodiment of the present invention;

[0029] Figure 7 This is a GO function enrichment map of differentially expressed genes of cadmium-resistant cellulobacterium Cd-1 in an embodiment of the present invention;

[0030] Figure 8 is a KEGG pathway enrichment map of the cadmium-resistant cellulobacterium Cd-1 in an embodiment of the present invention;

[0031] Fig.9A This is the dynamic change of ryegrass biomass of cadmium-tolerant cellulobacterium Cd-1 under 4 mg / kg cadmium concentration in the embodiment of the present invention;

[0032] Fig. 9B This is the dynamic change of ryegrass biomass under 20 mg / kg cadmium concentration of cadmium-tolerant cellulobacterium Cd-1 in the embodiment of the present invention;

[0033] Fig. 10A It is the dynamic change of chlorophyll content of ryegrass in the cadmium-resistant cellulosic bacteria Cd-1 under cadmium concentration of 4 mg / kg in the embodiment of the present invention;

[0034] Fig. 10B It is the dynamic change of chlorophyll content of ryegrass under cadmium concentration of 20 mg / kg by cadmium-resistant cellulosic bacteria Cd-1 in the embodiment of the present invention;

[0035] Fig.11A The dynamic changes of SOD activity of ryegrass under 4 mg / kg cadmium pollution by cadmium-tolerant cellulosic bacteria Cd-1 in the embodiment of the present invention;

[0036] Fig. 11BThe dynamic changes of SOD activity of ryegrass under 20 mg / kg cadmium pollution of cadmium-resistant cellulosic bacteria Cd-1 in the embodiment of the present invention;

[0037] Fig. 12A The dynamic changes of the POD activity of ryegrass under 4 mg / kg cadmium pollution of the cadmium-tolerant cellulobacterium Cd-1 in the embodiment of the present invention;

[0038] Fig. 12B It is the dynamic change of ryegrass POD activity of cadmium-tolerant cellulobacterium Cd-1 under 20 mg / kg cadmium pollution in the embodiment of the present invention;

[0039] Fig.13A This is the dynamic change of GHS content in ryegrass under 4 mg / kg cadmium pollution by cadmium-tolerant cellulobacterium Cd-1 in the embodiment of the present invention;

[0040] Fig. 13B The dynamic changes of GHS content in ryegrass under 20 mg / kg cadmium pollution by cadmium-tolerant cellulosic bacteria Cd-1 in the embodiment of the present invention;

[0041] Fig.14A It is the dynamic change of soil urease activity of cadmium-resistant cellulosic bacteria Cd-1 under 4 mg / kg cadmium pollution in the embodiment of the present invention;

[0042] Fig. 14B It is the dynamic change of soil urease activity of cadmium-resistant cellulobacterium Cd-1 under 20 mg / kg cadmium pollution in the embodiment of the present invention;

[0043] Fig.15A It is the dynamic change of soil luciferase activity of cadmium-tolerant cellulobacterium Cd-1 under 4 mg / kg cadmium pollution in the embodiment of the present invention;

[0044] Fig. 15B It is the dynamic change of soil luciferase activity of the cadmium-tolerant cellulomicrobacterium Cd-1 under 20 mg / kg cadmium pollution in the embodiment of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] Example 1

[0047] 1. Study on the growth characteristics and resistance mechanism of cadmium-resistant bacteria

[0048] 1.1 Experimental Materials

[0049] The test strains were screened from the tailings soil of Fenghuang Mountain in Yi'an District, Tongling City, Anhui Province.

[0050] 1) Cultivation of resistant bacteria:

[0051] Use CdCl2 to prepare a solution containing 5 mg / L Cd 2+ 50 mL of liquid LB medium was placed in a 250 mL conical flask and sterilized by high pressure steam at 121°C for 20 min. In the clean bench, a small amount of soil samples (5 g each of uncovered, shallowly covered, and deeply covered soil samples) were taken and placed in 50 mL of a flask containing Cd. 2+ The cells were cultured in a conical flask containing liquid LB medium, and the conical flask was placed in a shaking incubator (SHZ-82 air bath constant temperature oscillator) for 3 days. The shaking incubator was set at 30°C and 150 rpm.

[0052] 2) Purification of strains:

[0053] Dilution of culture medium: In the clean bench, take 0.5mL of bacterial solution from the conical flask culture medium and put it into a test tube containing 4.5mL of pure water, mix well, and the culture medium is diluted to 10 -1 After mixing, transfer the diluted solution to another tube containing 4.5 mL of pure water and dilute to 10 -2 Concentration, and so on, dilute step by step.

[0054] Coating plate: In the clean bench, dip the applicator into a small amount of alcohol and put it on the alcohol lamp to dry, then place it on the rack to cool. Take 100μL of bacterial solution and drop it on the sterile plate, and evenly coat it on the surface of the culture medium with the cooled applicator. After coating, burn it to sterilize, and cool it for the next use.

[0055] Plate streaking: In the clean bench, burn the alcohol-soaked inoculation loop with alcohol. After cooling, pick an independent colony from the plate and quickly streak back and forth on a quarter of a blank plate, then burn the inoculation loop. After cooling, streak the inoculation loop from the last part of the first streak to the second quarter of a circle and streak back and forth to form a second area, and burn the inoculation loop again. Repeat the above process to complete the streaking of the third interval, and the last streak does not touch the starting line. After completion, burn the inoculation loop.

[0056] 3) Domestication of strains:

[0057] Prepare the same concentration of Cd as the initial liquid LB medium. 2+ Solid LB medium. After 3 days of culture, the bacterial solution was diluted to 10 -3 , 10 -4 , 10 -5 Concentration, coat 3 identical plates for each concentration, place them in a constant temperature incubator at 30℃ for 3 days, and observe the experimental results.

[0058] Double Cd 2+ The concentration is 10 mg / L, and liquid LB medium is prepared. 1 mL of the culture solution from the previous batch is drawn into the newly prepared medium, and the shaking culture is continued. The above screening process is repeated to obtain colony plates at different dilution concentrations. The cultured plates are observed in the clean bench, and all colonies with different morphological structures, colors, etc. are picked from the LB plates containing cadmium, and streaked into the solid LB plates. The plates are placed in an incubator and cultured at 30°C for 3 days, the morphology is observed, and the plates are temporarily stored in a refrigerator at 4°C.

[0059] Pick a single colony from the streaked plate, add liquid LB medium containing double the cadmium ion concentration, and culture it in a shaker under the same conditions. After one week, use the spread plate method to test whether the strain can survive in the presence of cadmium ions at this concentration. -1 Concentration dilution, take 100 μL to coat the LB solid culture medium plate containing the cadmium concentration, place the coated plate in a 30°C incubator, and observe whether colonies can grow. If there are colonies, pick the colonies from the plate with a sterilized inoculation loop in the clean bench, add them to the culture medium with a higher concentration and continue to culture on the shaking table. If there are no colonies, abandon the culture of the strain. This screening process continues until the two strains with the strongest cadmium tolerance are screened out. Set Cd 2+ The concentration gradients from low to high are:

[0060] Cd 2+ :20mg / L, 40mg / L, 80mg / L, 150mg / L, 200mg / L, 300mg / L.

[0061] In the process of increasing the concentration step by step, different strains were inoculated into liquid LB medium at each concentration. After shaking culture, they were diluted and coated on plates and placed in an incubator for culture and detection. It was gradually found that some strains died. After a period of screening, the strain Cd-1 with the strongest tolerance was finally obtained. It was identified that strain Cd-1 belongs to Cellulosimicrobium sp. (as shown in SEQ ID NO.1), and its 16SrDNA sequence is:

[0062] GGACGGGGGCCGGGCGTGGCTTACACATGCAGTCGAACGATGATGCCCAGCTTGCTGGGCGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTTCGGGATAACTCCGGGAAACCGGGGCTAATACCGGATATGAGCCGTCCTCGCATGGGGGTGGTTGGAAAGTTTTTCGGTCAGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGAAGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGGTGTGAAAACTCGAGGCTCAACCTCGAGCTTGCATCGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGG.

[0063] 1.2 Experimental design

[0064] In order to understand the growth characteristics of Cd-1, the OD values ​​at 24h and 48h were measured under different temperatures, initial cadmium concentrations and initial pH conditions. 600 value and pH.

[0065] In order to explore the resistance mechanism of cadmium-resistant bacteria, cadmium-resistant bacteria were cultured in normal LB medium and medium containing 50 mg / L cadmium. 2+LB medium was added to the logarithmic growth phase, and the bacterial solution was centrifuged at 4°C and 5000r / min for 10min. The precipitate was washed with phosphate buffer and repeated 3 times. The cells were quick-frozen in liquid nitrogen and the cells were collected for transcriptome sequencing and numbered B1 and B1C respectively.

[0066] 2 Results Analysis

[0067] 2.1 Growth curve of cadmium-resistant cellulomicrobium Cd-1

[0068] Figure 1 It is the growth curve and pH change diagram of the cadmium-resistant cellulobacterium Cd-1 in the embodiment of the present invention;

[0069] like Figure 1 As shown in the figure, the OD of cadmium-resistant cellulomicrobacterium Cd-1 in the early stage of culture was 600 The value did not change significantly; at 4h, the cadmium-resistant cellulosic bacteria Cd-1 began to grow rapidly until it stopped growing after 30h. At this time, the OD of Cd-1 600 The growth value reached a peak of about 2.9. The initial pH value was 6.8. When the cadmium-resistant cellulomicrobium Cd-1 began to accelerate its growth, the pH of the liquid culture medium changed dynamically. Within 4h-30h, the pH of the liquid culture medium increased from neutral to alkaline. At 30h, the pH was about 8.7, and it began to gradually decrease to about 8.3 from 30h to 36h.

[0070] 2.2 Growth characteristics of cadmium-resistant cellulomicrobium Cd-1 under different initial conditions

[0071] During the cultivation of cadmium-tolerant cellulobacteria Cd-1 under different cadmium concentrations, as the initial cadmium concentration increased, the OD of Cd-1 600 The value shows a downward trend, such as Figure 2 shown.

[0072] Figure 2 1 is a graph showing the growth characteristics of the cadmium-resistant cellulosic bacteria Cd-1 under different cadmium concentrations in the embodiment of the present invention; Figure 2 It can be seen that at 48h, the OD of Cd-1 treated with 10-100mg / L cadmium 600 The OD value of 10 mg / L cadmium treatment was significantly lower than that of 0 mg / L cadmium treatment. 600 The OD of 20mg / L cadmium treatment was reduced by about 9.1% compared with 0mg / L cadmium treatment. 600 The OD value of 50mg / L cadmium treatment was lower than that of 0mg / L cadmium treatment by about 15.8%. 600 The OD value of 100 mg / L cadmium treatment was lower than that of 0 mg / L cadmium treatment by about 43.7%. 600 The value decreased by about 79.7%.

[0073] Growth characteristics of cadmium-resistant cellulobacterium Cd-1 in different pH ranges, such as Figure 3 shown. Figure 3 : is a graph showing the growth characteristics of cadmium-resistant cellulosic bacteria Cd-1 at different pH values ​​in an embodiment of the present invention; Figure 3 It can be seen that within the five pH ranges set, Cd-1 could not grow except at pH 4, but could grow in other pH environments. At 48h, as the pH increased, the OD of Cd-1 600 The OD of Cd-1 treated with pH 7 and 8 decreased gradually. 600 The OD of Cd-1 treated with pH 6 was 600 The OD of Cd-1 treated with pH 10 was significantly reduced by 7.6% and 21.2%, respectively. 600 The OD of Cd-1 treated with pH 6 was 600 The value was significantly reduced by about 80.3%.

[0074] Growth characteristics of cadmium-resistant cellulobacterium Cd-1 at different temperatures, such as Figure 4 shown.

[0075] Figure 4 1 is a graph showing the growth characteristics of cadmium-resistant cellulosic bacteria Cd-1 at different temperatures in an embodiment of the present invention; Figure 4 It can be seen that within the set temperature range, at pH 4-6, temperature has a significant effect on the growth of Cd-1. As the temperature increases, the OD 600 At 48h, the OD of Cd-1 treated at 40℃ 600 The values ​​were significantly higher than the OD of Cd-1 treated at other temperatures. 600 The values ​​were about 23.0%, 20.4%, 4% and 13.7%. At the same time, Cd-1 grown under three different initial conditions could increase the pH of the liquid culture medium from neutral to alkaline, which was consistent with the law obtained in the growth curve of Cd-1.

[0076] 2.3 Differential expression of Cd-1 genes in cadmium-tolerant cellulobacteria

[0077] 2.3.1 Sequencing data quality assessment and genome alignment

[0078] The average mass concentrations of samples B1 and B1C were 399 ng / μL and 269.3 ng / μL, respectively; the integrity values ​​were 9.4 and 8.9, respectively, which met the requirements for library construction and sequencing. The transcriptome sequencing data are shown in Table 1.

[0079] Table 1 Data output quality statistics

[0080] Sample name Raw data Filtered data Error rate Comparison ratio GC content B1 16128177 15943262 0.03％ 87.6％ 74.16 B1C 16361579 16162315 0.03％ 87.81％ 71.74

[0081] As shown in Table 1, B1 and B1C obtained an average of 16128177 and 16361579 raw sequences respectively. After filtering the raw data, the CleanReads obtained were 15943262 and 16162315 respectively; their error rates were both 0.03%; the alignment results of genome positioning analysis of B1 and B1C were 87.6% and 87.81% respectively; and the GC contents were 74.16 and 71.74 respectively. This shows that the data has high accuracy and can be directly used for subsequent analysis.

[0082] 2.3.2 Differential gene expression analysis

[0083] The differentially expressed genes of each sample were screened based on the criteria of |Log2(FoldChange)|>1 and P<0.005. Figure 5 shown.

[0084] Figure 5 It is a volcano map of differentially expressed genes of cadmium-resistant cellulobacterium Cd-1 in the embodiment of the present invention; Figure 5 In the figure, dots represent specific genes, the dots in region A represent significantly up-regulated genes, the dots in region B represent significantly down-regulated genes, and the dots in region C represent genes with non-significant differential expression.

[0085] from Figure 5 It can be seen that a total of 148 differentially expressed genes were screened from the B1 and B1C comparison groups, of which 142 were upregulated and 6 were downregulated.

[0086] 2.3.3 GO enrichment analysis and KEGG pathway analysis of differentially expressed genes

[0087] Figure 6 GO functional classification diagram of differentially expressed genes of cadmium-resistant cellulobacterium Cd-1 in the embodiment of the present invention; Figure 6As shown in the figure, after GO functional annotation of the screened differentially expressed genes, it was found that 37 significantly enriched Goterms were annotated in the B1C / B1 group, mainly distributed in 7 secondary Goterms. Among them, there were 2 terms with a total of 494 differential genes in biological processes, and these differential genes were mainly involved in functions such as cell processes and metabolic processes; there were 2 terms with a total of 278 differential genes in cell composition, and these genes were mainly involved in functions such as cell anatomical entities, cell waste unit components, and protein-containing complexes; there were 2 terms with a total of 50 differential genes in molecular biological functions, and these genes were mainly involved in functions such as structural molecular activity and binding. Further analysis of these differentially expressed genes revealed that the most significantly enriched genes in biological processes were translation, peptide metabolism, peptide biosynthesis, amide biosynthesis, cellular amide metabolism, protein metabolism, organic nitrogen compound biosynthesis, gene expression and organic nitrogen metabolism; the most significantly enriched genes in cellular compositions were ribosomes, ribosomal protein complexes, intracellular organelles without membrane boundaries, cytoplasm, intracellular organelles, membraneless organelles, organelles, cytoplasm, protein-containing complexes and intracellular parts.

[0088] Figure 7 This is a GO function enrichment map of differentially expressed genes of cadmium-resistant cellulobacterium Cd-1 in an embodiment of the present invention; Figure 7 In the figure, the horizontal axis represents different GO functions; the vertical axis represents significance, and the larger the value, the more significant it is. Different colors represent different functional branches. Figure 7 As shown, the functions significantly enriched in molecular biological functions are mainly ribosome structure composition, structural molecule activity, iron-sulfur cluster binding, metal cluster binding, endopeptidase activity, electron transfer activity, DNA binding transcription factor activity, transcriptional regulation activity, metal ion binding and cation transmembrane transport activity.

[0089] In order to determine the most important biochemical metabolic pathways and signal transduction pathways involved in the differentially expressed genes, KEGG pathway enrichment analysis was performed on the differentially expressed genes, such as Figure 8 shown.

[0090] Figure 8 is a KEGG pathway enrichment map of the cadmium-resistant cellulobacterium Cd-1 in an embodiment of the present invention;

[0091] Figure 8 The horizontal axis represents the enrichment ratio. The larger the enrichment ratio, the greater the degree of enrichment. The vertical axis represents the pathway. The size of the dot represents the number of genes in this pathway. Figure 8It can be seen that the 20 most significantly enriched pathways were plotted as differential gene KEGG pathway enrichment scatter plots. Under the culture conditions of 50 mg / L cadmium treatment and 0 mg / L cadmium treatment, the top five pathways with the most enriched differentially expressed genes in cadmium-resistant cellulobacteria Cd-1 were: ribosome pathway, secondary metabolite biosynthesis pathway, oxidative phosphorylation pathway, arginine biosynthesis pathway, and amino acid biosynthesis pathway. The number of enriched differentially expressed genes was 26, 10, 9, 6, and 6, respectively. Among them, the ribosome pathway was the most significantly enriched.

[0092] 3. Summary

[0093] The cadmium-resistant cellulomicrobium Cd-1 has good tolerance to cadmium and can grow normally in a cadmium environment of 100 mg / L. Under the set environmental conditions, the optimal pH is 6-7 and the optimal temperature is 35℃-40℃.

[0094] Under different cadmium concentrations, the functions of genes upregulated in cadmium-tolerant cellulobacteria Cd-1 are mainly concentrated in compound synthesis and metabolism, cell membrane and its related components, transmembrane transport and heavy metal binding; specifically, the top five pathways enriched in differentially expressed genes are: ribosome pathway, secondary metabolite biosynthesis pathway, oxidative phosphorylation pathway, arginine biosynthesis pathway and amino acid biosynthesis pathway. Among them, the most significant pathway enriched in Cd-1 differential genes is the ribosome pathway.

[0095] Example 2

[0096] 1. Dynamic effects of inoculation with cadmium-tolerant cellulobacterium Cd-1 on the physiology and biochemistry of ryegrass in cadmium-contaminated soil

[0097] 1.1 Experimental Materials

[0098] The test soil was collected from clean soil in the 0-20cm soil layer of a farmland. After the fresh soil was collected, stones, plant roots, large and medium-sized soil animals were removed, and the soil was air-dried and sieved through a 2mm sieve for later use. The soil pH was 5.51, the total nitrogen content was 0.81g / kg, the total phosphorus content was 0.48g / kg, the total potassium content was 1.63g / kg, and the cadmium content was not detected.

[0099] The test plant was ryegrass (Lolium perenne L.), and ryegrass seeds were purchased from Suqian Horticulture Co., Ltd., Jiangsu Province.

[0100] 1.2 Experimental design

[0101] This experiment was designed with 4 treatments, each with 18 replicates, for a total of 72 pots. The experimental design is shown in Table 2.

[0102] Table 2 Experimental design

[0103]

[0104] The test soil was contaminated (cadmium was added in the form of CdCl2·2.5H2O solution), and 0.5 g / LCd was accurately prepared. 2+ As the mother solution, it was evenly irrigated into the potting soil, and the Cd pollution concentration was adjusted to 0.4 mg / kg (agricultural soil control value) and 20 mg / kg (a type of construction land screening value) according to the soil environmental quality standard (GB15618-2018). 2+ Each pot (8cm×8cm×10cm) was filled with 500g of soil (dry weight), and 50 perennial ryegrass seeds were evenly planted in each pot. According to the conventional application rate of N 20kg / mu and P 10kg / mu, the water-soluble fertilizer was diluted and applied as basal fertilizer. Each pot was inoculated with 9mL of cadmium-resistant fiber microbe Cd-1 (10 9 CFU / mL), with 14h / d of light and 10h / d of darkness, for a 2-month indoor culture test. During this period, distilled water was added daily according to the soil moisture status of each pot, so that the soil moisture content of the potted plants was maintained at 60% of the saturated moisture content. Destructive collection sampling was carried out at 20d, 40d, and 60d. Six replicates were randomly selected for each treatment each time, of which three replicates were used to analyze indicators such as plant physiology and biochemistry and soil microbial activity, and the other three replicates were used to determine related indicators such as biomass and heavy metal content.

[0105] 2 Results Analysis

[0106] 2.1 Effects of Cd-1 on ryegrass biomass at different cadmium concentrations

[0107] Fig.9A This is the dynamic change of ryegrass biomass of cadmium-tolerant cellulobacterium Cd-1 under 4 mg / kg cadmium concentration in the embodiment of the present invention; Fig. 9B The dynamic changes of ryegrass biomass under 20 mg / kg cadmium concentration of cadmium-tolerant cellulosic bacteria Cd-1 in the embodiment of the present invention; Figures 9A-9B It can be seen that the biomass of ryegrass treated with different Cd concentrations showed an increasing trend over time, and there were certain differences in the biomass of ryegrass treated with different Cd concentrations. Fig.9A It can be seen that under the treatment of 4 mg / kg cadmium, at 40 days, the Cd-1 inoculation treatment significantly increased the ryegrass biomass by about 56.3% (P = 0.001) compared with the non-inoculation treatment; at 60 days, the Cd-1 inoculation treatment significantly increased the biomass by 58.6% (P = 0.001) compared with the non-inoculation treatment. Fig. 9BIt can be seen that under the 20 mg / kg Cd treatment, at 40 days, the Cd-1 inoculation treatment significantly increased the ryegrass biomass by about 186.5% (P=0.001) compared with the non-Cd-1 treatment; at 60 days, the Cd-1 inoculation treatment significantly increased the ryegrass biomass by about 20.0% (P=0.017) compared with the non-Cd-1 treatment.

[0108] 2.2 Effects of Cd-1 on chlorophyll content of ryegrass at different cadmium concentrations

[0109] Fig. 10A It is the dynamic change of chlorophyll content of ryegrass under cadmium concentration of 4 mg / kg by cadmium-resistant cellulosic bacteria Cd-1 in the embodiment of the present invention; Fig. 10B The dynamic changes of chlorophyll content of ryegrass in the cadmium-resistant cellulosic bacteria Cd-1 under cadmium concentration of 20 mg / kg in the embodiment of the present invention; Fig. 10A -B shows that under different Cd concentration treatments, the chlorophyll content of ryegrass generally showed a downward trend with the increase of cultivation time, and with the increase of Cd concentration, the chlorophyll content of ryegrass generally showed a downward trend. Fig. 10A It can be seen that under the treatment of 4 mg / kg Cd, at 40 days, the chlorophyll content of ryegrass inoculated with Cd-1 was significantly increased by about 24.8% (P=0.004) compared with the treatment without Cd-1 inoculation; at 60 days, the chlorophyll content of ryegrass inoculated with Cd-1 was significantly increased by 94.8% (P=0.001) compared with the treatment without Cd-1 inoculation. Fig. 10B It can be seen that under the 20 mg / kg Cd treatment, at 20 days, the chlorophyll content of ryegrass inoculated with Cd-1 was significantly increased by about 22.0% (P=0.001) compared with the non-inoculated with Cd-1 treatment; at 40 days, the chlorophyll content of the inoculated with Cd-1 treatment was significantly increased by 41.0% (P=0.041) compared with the non-inoculated with Cd-1 treatment.

[0110] 2.3 Effects of Cd-1 on SOD activity of ryegrass at different cadmium concentrations

[0111] Fig.11A The dynamic changes of SOD activity of ryegrass under 4 mg / kg cadmium pollution by cadmium-tolerant cellulosic bacteria Cd-1 in the embodiment of the present invention; Fig. 11B The dynamic changes of SOD activity of ryegrass in the cadmium-tolerant cellulosic bacteria Cd-1 under 20 mg / kg cadmium pollution in the embodiment of the present invention; Fig.11A -B shows that under different Cd concentration treatments, the SOD activity of ryegrass generally increased with the increase of treatment time. Fig.11AIt can be seen that under the treatment of 4 mg / kg Cd, at 60 days, the SOD activity of ryegrass in the treatment of Cd-1 inoculation was significantly reduced by about 38.9% (P = 0.012) compared with the treatment without Cd-1 inoculation. Fig. 11B It can be seen that under the treatment of 20 mg / kg Cd, at 40 days, the superoxide dismutase (SOD) activity of ryegrass in the Cd-1 inoculation treatment was significantly reduced by about 34.2% (P=0.012) compared with the non-inoculation treatment; at 60 days, the superoxide dismutase activity of ryegrass in the Cd-1 inoculation treatment was extremely significantly reduced by about 20.8% (P=0.007) compared with the non-inoculation treatment.

[0112] 2.4 Effects of Cd-1 on POD activity of ryegrass at different cadmium concentrations

[0113] Fig. 12A The dynamic changes of the POD activity of ryegrass under 4 mg / kg cadmium pollution of the cadmium-tolerant cellulobacterium Cd-1 in the embodiment of the present invention; Fig. 12B The dynamic changes of the POD activity of ryegrass under 20 mg / kg cadmium pollution of cadmium-resistant cellulomicrobacterium Cd-1 in the embodiment of the present invention; Fig. 12A -B shows that under different Cd concentration treatments, the POD activity of ryegrass generally showed a downward trend with the increase of treatment time. Fig. 12A It can be seen that under the treatment of 4 mg / kg Cd, at 20 days, the Cd-1 inoculation treatment significantly increased the ryegrass peroxidase (POD) activity by about 38.5% (P=0.007) compared with the non-Cd-1 treatment; at 40 days, the Cd-1 inoculation treatment significantly reduced the ryegrass peroxidase activity by 56.7% (P=0.005) compared with the non-Cd-1 treatment; at 60 days, the Cd-1 inoculation treatment significantly increased the ryegrass peroxidase activity by 74.8% (P=0.003) compared with the non-Cd-1 treatment. Fig. 12B It can be seen that under the treatment of 20 mg / kg Cd, at 20 days, the POD activity of ryegrass inoculated with Cd-1 was significantly increased by about 57.3% (P=0.002) compared with the treatment without Cd-1 inoculation.

[0114] 2.5 Effects of cadmium-tolerant cellulomicrobium Cd-1 on GSH content in ryegrass at different cadmium concentrations

[0115] Fig.13A This is the dynamic change of GHS content in ryegrass under 4 mg / kg cadmium pollution by cadmium-tolerant cellulobacterium Cd-1 in the embodiment of the present invention; Fig. 13B The dynamic changes of GHS content in ryegrass of cadmium-tolerant cellulosic bacteria Cd-1 under 20 mg / kg cadmium pollution in the embodiment of the present invention; Fig.13A-B shows that under different Cd concentration treatments, the GSH content of ryegrass inoculated with cadmium-tolerant cellulobacteria Cd-1 is generally lower than that in the non-inoculated Cd-1 treatment. Fig.13A It can be seen that under the treatment of 4 mg / kg Cd, at 40 days, the glutathione (GSH) content of ryegrass in the treatment of Cd-1 inoculation was significantly reduced by about 54.2% (P=0.001) compared with the treatment without Cd-1 inoculation; at 60 days, the glutathione content of ryegrass in the treatment of Cd-1 inoculation was significantly reduced by 39.7% (P=0.023) compared with the treatment without Cd-1 inoculation. Fig. 13B It can be seen that under the treatment of 20 mg / kg Cd, at 20 days, the glutathione content of ryegrass inoculated with Cd-1 was extremely significantly reduced by about 20.8% (P=0.001) compared with the treatment without Cd-1; at 60 days, the glutathione content of ryegrass inoculated with Cd-1 was extremely significantly reduced by about 29.6% (P=0.001) compared with the treatment without Cd-1.

[0116] 2.6 Effects of Cd-1 on the accumulation of cadmium in ryegrass

[0117] The accumulation characteristics of cadmium in ryegrass are shown in Table 2.

[0118] Table 2 Characteristics of cadmium accumulation and transport in ryegrass

[0119]

[0120]

[0121] Note: On the 20th day, the underground biomass of ryegrass was insufficient to determine the Cd content in the underground part; Different letters in the same column indicate significant differences (P<0.05).

[0122] As shown in Table 2, at different sampling periods, with the increase of Cd treatment concentration, the Cd content in the underground and aboveground parts of ryegrass also increased, and there were significant differences between different Cd concentration treatments, showing that the Cd concentrations in the underground and aboveground parts of the 20 mg / kg treatment were higher than those in the 4 mg / kg treatment. At the same sampling time and the same Cd concentration treatment, the Cd concentrations in the aboveground and underground parts of the inoculated Cd-1 treatment were higher than those in the non-inoculated Cd-1 treatment to a certain extent. At 20 days, the Cd concentration in the aboveground part of the C1B1 treatment was significantly higher than that in the C1B0 treatment by about 19.6% (P=0.039); at 40 days, the Cd concentration in the aboveground part of the C1B1 treatment was significantly higher than that in the C1B0 treatment by about 31.9% (P=0.026); at 60 days, the Cd concentration in the underground part of the C1B1 treatment was significantly lower than that in the C1B0 treatment by about 20.6% (P=0.004). The transport coefficient and enrichment coefficient also showed some differences under different sampling time and different cadmium concentration treatments. At 40 days, the enrichment coefficient of C2B1 treatment under 20 mg / kg Cd treatment was significantly higher than that of C2B0 treatment, about 81.6%. At 60 days, the inoculation of Cd-1 treatment under 4 mg / kg and 20 mg / kg Cd treatments significantly increased the enrichment coefficient of ryegrass to cadmium, by 32.9% and 27.5%, respectively. Except for the inoculation of cadmium-tolerant cellulobacteria Cd-1 treatment under 20 mg / kg Cd treatment at 60 days, the other treatments could improve the transport coefficient and enrichment coefficient of ryegrass to Cd to a certain extent, but not significantly. The accumulation of cadmium in ryegrass and the removal rate of cadmium in soil are shown in Table 3.

[0123] Table 3 The accumulation of cadmium in ryegrass and its removal rate from soil

[0124]

[0125]

[0126] * indicates that the difference between inoculated and non-inoculated treatments at the same Cd concentration is significant; ** indicates that the difference between inoculated and non-inoculated treatments at the same Cd concentration is extremely significant.

[0127] As shown in Table 3, at different sampling periods and different Cd concentrations, inoculation with Cd-1 can increase the accumulation of cadmium in ryegrass, and there are certain differences between different Cd concentrations. At 20 days, the cadmium accumulation in the treatment of inoculation with Cd-1 at 4 mg / kg Cd concentration was significantly increased by 85.7% (P=0.024) compared with the treatment without inoculation with Cd-1; at 40 days, the cadmium accumulation in the treatment of inoculation with Cd-1 at 4 mg / kg and 20 mg / kg Cd concentrations was significantly increased by 159.2% (P=0.007) and 383.9% (P=0.001) compared with the treatment without inoculation with Cd-1; at 60 days, the cadmium accumulation in the treatment of inoculation with Cd-1 at 4 mg / kg Cd concentration was significantly increased by 136.3% (P=0.032) compared with the treatment without inoculation with Cd-1.

[0128] 3 Summary

[0129] The experimental results show that inoculating cadmium-tolerant fiber microorganisms Cd-1 in cadmium-contaminated soil with ryegrass can increase the chlorophyll content and peroxidase activity in ryegrass leaves; and reduce the superoxide dismutase activity and glutathione content in ryegrass leaves. The reason for this phenomenon may be that in a cadmium-contaminated environment, the active oxygen in ryegrass continues to increase, causing ryegrass to produce oxidative stress, increasing the secretion of superoxide dismutase and glutathione to coordinate redox balance. Studies have found that inoculating cadmium-tolerant fiber microorganisms Cd-1 in cadmium-contaminated soil with ryegrass can increase the chlorophyll content in ryegrass leaves and promote the antioxidant system of ryegrass to remove excessive active oxygen in ryegrass, thereby reducing the superoxide dismutase activity and glutathione content in ryegrass leaves. This result shows that cadmium-tolerant fiber microorganisms Cd-1 can alleviate the oxidative stress response under adverse stress, allowing ryegrass plants to grow normally, and then can increase the biomass of ryegrass and promote the growth and development of ryegrass. The underground and aboveground parts of well-grown ryegrass increase the enrichment coefficient and transport coefficient of cadmium, thereby reducing the residual cadmium in cadmium-contaminated soil and improving the removal rate of cadmium in contaminated soil.

[0130] Example 3

[0131] 1. Effects of cadmium-tolerant cellulobacterium Cd-1 on microbial activity in cadmium-contaminated soil

[0132] 1.1 Materials and methods

[0133] The experimental materials are the same as those in Example 2.

[0134] 1.2 Experimental design

[0135] The experimental design is the same as that of Example 2.

[0136] 2 Results and analysis

[0137] 2.1 Dynamic effects of cadmium-tolerant cellulomicrobium Cd-1 on soil urease activity under cadmium pollution

[0138] Fig.14A It is the dynamic change of soil urease activity of cadmium-resistant cellulosic bacteria Cd-1 under 4 mg / kg cadmium pollution in the embodiment of the present invention; Fig. 14B The dynamic changes of soil urease activity of cadmium-tolerant cellulobacteria Cd-1 under 20 mg / kg cadmium pollution in the embodiment of the present invention; Fig.14A -B shows that soil urease activity generally shows an upward trend with the increase of cultivation time, and inoculation of cadmium-resistant cellulosic bacteria Cd-1 can generally enhance soil urease activity. Fig.14A It can be seen that under the treatment of 4 mg / kg Cd concentration, at 40 days, the soil urease activity in the Cd-1 inoculated treatment was significantly increased by about 60.5% (P=0.002) compared with the non-Cd-1 inoculated treatment.

[0139] from Fig. 14B It can be seen that under the treatment of 20 mg / kg Cd concentration, at 20 days, the soil urease activity in the Cd-1 inoculated treatment was significantly increased by about 49.7% (P=0.042) compared with the non-Cd-1 inoculated treatment.

[0140] 2.2 Effects of Cd-tolerant Microbacterium cytochrome Cd-1 on luciferase activity in soils contaminated with cadmium

[0141] Fig.15A It is the dynamic change of soil luciferase activity of cadmium-tolerant cellulobacterium Cd-1 under 4 mg / kg cadmium pollution in the embodiment of the present invention; Fig. 15B The dynamic changes of soil luciferase activity of cadmium-tolerant cellulobacterium Cd-1 under 20 mg / kg cadmium pollution in the embodiment of the present invention; Fig.15A -B shows that the activity of soil luciferase hydrolase (FDA) generally shows a downward trend with the increase of culture time, and the inoculation of cadmium-resistant cellulosic bacteria Cd-1 also has a certain effect on the activity of soil luciferase hydrolase. Fig.15A It can be seen that under the treatment of 4 mg / kg Cd concentration, at 40 days, the soil luciferase activity of the Cd-1 inoculated treatment was significantly reduced by about 57.3% compared with the non-Cd-1 treatment (P = 0.018); at 60 days, the soil luciferase activity of the Cd-1 inoculated treatment was significantly increased by about 112.6% compared with the non-Cd-1 treatment (P = 0.001). Fig. 15BIt can be seen that under the treatment of 20 mg / kg Cd concentration, at 20 days, the soil luciferase hydrolase activity in the Cd-1 inoculation treatment was significantly reduced by about 38.8% (P=0.002) compared with the non-Cd-1 treatment; at 60 days, the soil luciferase hydrolase activity in the Cd-1 inoculation treatment was significantly reduced by about 72.0% (P=0.002) compared with the non-Cd-1 treatment.

[0142] 3 Summary

[0143] The experimental results show that under cadmium pollution conditions, inoculating cadmium-resistant fiber microbes Cd-1 in cadmium-contaminated soil with ryegrass can increase the activity of soil urease, and can increase the activity of luciferase hydrolase in the later stage of cultivation, that is, inoculating cadmium-resistant bacteria can increase the activity of soil microorganisms. From the above results, it can be seen that the cadmium-resistant fiber microbes Cd-1 provided by the present invention can increase the activity of soil urease and luciferase, increase the microbial activity of soil, increase the chlorophyll content in ryegrass leaves, and promote the antioxidant system of ryegrass to remove excessive active oxygen in ryegrass, thereby alleviating the oxidative stress reaction of ryegrass under adverse stress, and promoting the growth of ryegrass in cadmium-contaminated soil; increasing the biomass of ryegrass, increasing the enrichment of cadmium by ryegrass, and helping to promote the repair of ryegrass to cadmium-contaminated soil.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A cadmium-resistant cellulobacterium Cd-1, characterized in that: Its strain collection number is CGMCC No.25219.

2. The method for cultivating the cadmium-resistant cellulobacterium Cd-1 according to claim 1, characterized in that: The cadmium-resistant cellulobacterium Cd-1 is cultured in a culture medium with a pH of 6-7.

3. The culture method according to claim 2, characterized in that The cadmium-resistant cellulobacterium Cd-1 was cultured at a temperature of 30-40°C.

4. Use of the cadmium-resistant cellulobacterium Cd-1 described in claim 1 in promoting the growth of ryegrass in cadmium-contaminated soil.

5. A method for promoting the growth of ryegrass in cadmium-contaminated soil, characterized in that: The cadmium-resistant cellulomicrobium Cd-1 described in claim 1 is inoculated into the cadmium-contaminated soil where ryegrass is planted.

6. The method according to claim 5, characterized in that: The concentration of cadmium in the cadmium-contaminated soil is ≤100 mg / L.

7. The method according to claim 5, characterized in that: The method further comprises, after inoculating the cadmium-resistant cellulosic bacteria Cd-1 described in claim 1, culturing under light conditions of 10-14 h / d and darkness of 8-10 h / d.

8. The method according to claim 7, characterized in that: The water content of the cadmium-contaminated soil inoculated with the cadmium-resistant cellulosic microbe Cd-1 according to claim 1 is maintained at 40%-60%.

9. The method according to claim 5, characterized in that: The time for inoculating the cadmium-resistant cellulosic bacteria Cd-1 described in claim 1 is before sowing ryegrass seeds.

10. A method for repairing cadmium-contaminated soil, characterized in that: Planting ryegrass in cadmium-contaminated soil inoculated with the cadmium-resistant cellulobacterium Cd-1 described in claim 1; collecting ryegrass plants enriched in cadmium to complete the restoration of cadmium-contaminated soil.

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