Citrobacter freundii and application thereof

Abstract

The invention discloses citrobacter freundii and an application thereof. The citrobacter freundii disclosed by the invention has the functions of high cadmium resistance and improvement of selenium bioavailability, and the strain is classified and named as Citrobacter freundii DS and is preserved in CCTCC (China Center For Type Culture Collection) on May 26, 2022. The strain has high tolerance to heavy metal cadmium, can still grow under the condition that the concentration of Cd < 2 + > is 6 mM, and can reach the maximum biomass within 40 hours. When the strain is applied to cadmium-polluted soil, the proportion of exchangeable cadmium in the soil and the content of effective cadmium in the soil can be remarkably reduced, and meanwhile, the content of soluble selenium and effective selenium in the soil can be remarkably increased. A rice potting experiment is carried out, so that the cadmium biological enrichment coefficient of each part of the rice is further reduced, and the selenium biological enrichment coefficient and the selenium transfer coefficient are improved. The strain has a remarkable effect in the application of repairing cadmium pollution of soil, improving the biological utilization of selenium and promoting crop growth.

Description

Technical Field

[0001] The invention belongs to the technical field of soil heavy metal bioremediation, and specifically relates to Citrobacter freundii DS with high cadmium resistance and the function of improving selenium bioavailability and application thereof. Background Art

[0002] Cadmium (Cd) is one of the most common inorganic soil pollutants in my country, with a point-to-point excess rate of 7%, ranking first. Cadmium is listed as a Class I carcinogen by IARC (International Agency for Research on Cancer). It has strong biological toxicity to both animals and plants and is easily enriched in organisms. Most of China's naturally selenium-rich areas are also facing serious cadmium pollution, which seriously threatens food safety and hinders the utilization of these selenium-rich soils. Compared with other heavy metals, cadmium has higher bioavailability and stronger migration ability. It can react with specific proteins and enzymes in the body, seriously threatening ecosystem functions and human health. Rice is one of the main food crops in people's lives. While it provides essential nutrients to the human body, the heavy metal toxic elements accumulated in its edible part also enter the human body through the mouth, causing harm to health. Studies have shown that rice is one of the main ways for cadmium to enter the human body. After cadmium accumulates in large quantities in rice grains, it is transmitted through the food chain, which seriously threatens the life and health of humans and animals and has a great impact on food security. Therefore, controlling soil heavy metal pollution and reducing the cadmium content in rice are of great significance to reducing human cadmium absorption and protecting human health. At present, the remediation measures for cadmium-contaminated soil are mainly divided into three categories: physical remediation, chemical remediation and biological remediation. Compared with physical and chemical remediation, biological remediation is an environmentally friendly remediation method with the advantages of being green and environmentally friendly, with little soil disturbance, no secondary pollution, and in line with the laws of ecological development. As an important component of bioremediation, microorganisms are of various types and reproduce quickly. They can reduce the toxicity of cadmium through adsorption, transformation, and fixation. Microbial remediation has the advantages of fast effect, convenient management and operation, and no damage to soil structure. It has attracted widespread attention and has become a hot spot for studying soil cadmium pollution remediation. Studies have shown that some cadmium-resistant microorganisms can significantly reduce the bioavailability of cadmium in the soil, reduce crop absorption, and ensure the safety of agricultural products. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a cadmium-resistant Citrobacter freundii and its application in soil, so as to solve the problems of how to effectively control cadmium in the environment, bioremediate cadmium pollution and improve rice quality.

[0004] Technical solution: The Citrobacter freundii strain described in the present invention is deposited as Citrobacter freundii DS, and the deposit number is CCTCC NO: M2022724. The strain was deposited in the China Center for Type Culture Collection on May 26, 2022, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0005] The present invention screens out microorganisms with high cadmium resistance and improved effective selenium capacity from the intestines of cadmium-resistant earthworms (Hubei far-blind earthworms) to reduce the bioavailability of heavy metal cadmium in the soil, thereby achieving the purpose of reducing heavy metal accumulation in the soil and various parts of rice, and improving grain yield and quality. The present invention can be used to develop microbial remediation technology for soil heavy metal pollution.

[0006] The second aspect of the present invention discloses the use of the above-mentioned Citrobacter freundii in passivating cadmium in soil.

[0007] The third aspect of the present invention discloses the use of the above-mentioned Citrobacter freundii in reducing the cadmium content in rice.

[0008] The fourth aspect of the present invention discloses the use of the above-mentioned Citrobacter freundii in improving the biological effectiveness of selenium in soil.

[0009] The fifth aspect of the present invention discloses the use of the above-mentioned Citrobacter freundii in increasing the selenium content in rice.

[0010] Furthermore, in the above application, the application method of the Citrobacter freundii comprises the following steps:

[0011] (1) activating the Citrobacter freundii of claim 1, collecting the bacterial cells after transfer culture, and resuspending the bacterial cells to obtain a live bacterial liquid;

[0012] (2) Mixing the live bacterial solution with soil containing selenium and / or cadmium.

[0013] Preferably, in step (1), the Citrobacter freundii is transferred and cultured to the late logarithmic growth phase, and the cells are collected by centrifugation.

[0014] Preferably, in step (1), the transfer culture conditions are culturing at 28-37°C and 100-200 rpm, the activation of the Citrobacter freundii adopts LB liquid culture medium or LB solid culture medium, and the transfer culture adopts LB liquid culture medium.

[0015] Preferably, in step (1), the method of resuspending the bacterial cells is: resuspending the bacterial cell pellet in sterile deionized water to obtain a bacterial cell concentration of at least 2.0×10 9 CFU / mL of live bacterial solution.

[0016] In some embodiments, the cell concentration is preferably 2.0×10 9 -4.0×10 9 CFU / mL.

[0017] Preferably, in step (2), the method for mixing the active bacterial solution with the soil containing selenium and cadmium is: mixing the active bacterial solution with the soil at a ratio of 80 mL / kg.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0019] The present invention isolates and screens a cadmium-resistant bacterial strain that can effectively block and control cadmium in the environment. The bacterial strain has a fast growth rate and a high tolerance to heavy metal cadmium, with a tolerance concentration of cadmium reaching 6mM, and can passivate cadmium.

[0020] The application of Citrobacter freundii DS strain in soil contaminated by heavy metal cadmium can significantly reduce the effective cadmium content in the soil, while reducing the absorption of cadmium by various parts of rice, significantly improving rice biomass and quality, and alleviating the physiological stress of cadmium on rice. The cadmium-resistant strain that can efficiently remove cadmium in the environment separated and screened by the present invention reduces the cadmium content in rice through microbial remediation measures, thereby providing a scientific basis for ensuring the quality safety of food crops such as rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A phylogenetic tree diagram of the Citrobacter freundii of the present invention;

[0022] Figure 2 The present invention is a method for determining the effect of Citrobacter freundii on Cd in a culture medium under different cadmium concentration conditions. 2+ Removal rate graph;

[0023] Figure 3 The bar graph is a graph showing the changes in the effectiveness of soil Se and Cd caused by the Citrobacter freundii of the present invention in a rice pot experiment;

[0024] Figure 4 It is a bar graph showing the changes in Se and Cd contents in various parts of rice caused by the Citrobacter freundii of the present invention in a potted experiment;

[0025] Figure 5 The bar graph shows the changes in the biomass of rice and the bioavailability of Se and Cd caused by the Citrobacter freundii of the present invention in a pot experiment. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0027] Example 1: Isolation and Identification of Citrobacter freundii

[0028] (1) Sample collection: The Hubei earthworm was collected from farmland soil with a high background of selenium and cadmium in Dashan Village, Chizhou City, Anhui Province.

[0029] (2) Isolation and screening of cadmium-resistant strains: Take earthworms that have been cleaned for 24 hours, wash them and fix them in a wax tray, add 2 to 3 drops of 75% ethanol solution to their heads, dissect the earthworms, take out their intestinal contents, and fully grind the intestines with a sterilized grinding rod. Add sterile water in a ratio of weight (g): volume (mL) = 1:9, grind them fully in a mortar under ice water bath conditions, mix them on a vortex oscillator, and prepare a 10% tissue homogenate. Take 2 mL of the intestinal content tissue homogenate and add Cd-containing 2+ The concentration of 0.1mM was in LB liquid medium, and the culture was shaken at 180r / min at 28℃ until the exponential growth phase. The inoculum was inoculated into the Cd-containing 2+ The culture was carried out in LB liquid medium with concentrations of 0.3, 0.5, and 1.0 mM. 0.1 mL of bacterial solution was added to 0.9 mL of sterile water and diluted to 10 -6 , draw the diluted bacterial suspension and apply it on the Cd 2+ The concentration of 1mM was LB solid screening medium, and cultured in a bacterial incubator at 28℃ for 48h. The colony morphology, color, wetness, glossiness, etc. were observed, and a single colony on the plate was picked and plated with Cd 2+ Streak culture on plates with concentrations of 2, 4, 6, 8, and 10 mM to obtain pure cultures of bacteria with the strongest tolerance to cadmium. Prepare commonly used LB liquid culture medium with the following formula (1 L): 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and adjust the pH to 7.0. Add distilled water to 1 L. Sterilize at 121°C under high pressure steam for 20 min.

[0030] (3) Identification of cadmium-resistant strains: Identification was performed by amplifying the 16S rDNA nucleotide sequence of the bacteria. The 16S rDNA primers selected were forward primer 27F (AGTTTGATCMTGGCTCAG) and reverse primer 1492R (GGTTACCTTGTTACGACTT). The PCR product was sequenced, and the results were as follows:

[0031] Citrobacter freundii DS16S ribosomal DNA gene

[0032]

[0033] The sequencing results were compared with the sequences of strains with close homology using BLAST in the EzBioCloud database (www.ezbiocloud.net). The Citrobacter isolated in the present invention has the highest homology with Citrobacter freundii. The phylogenetic tree (e.g. Figure 1 The strain was identified as Citrobacter freundii DS.

[0034] (4) Morphology and physiological and biochemical characteristics of Citrobacter freundii: Citrobacter freundii is a Gram-negative bacterium. Its colonies are milky white, with the same color on both sides. They are round in shape, with protrusions on the surface, flat edges, and smooth and moist surfaces. They grow faster on LB agar medium.

[0035] (5) Preservation of Citrobacter freundii: Citrobacter freundii can be cultured on LB liquid or solid culture medium at 37°C, and after culture, it can be transferred to slant culture medium at 4°C for short-term storage. For long-term storage, the bacterial liquid can be mixed with an equal volume of diluted glycerol, placed in a centrifuge tube and frozen at -20°C. The strain was submitted to the China Center for Type Culture Collection (CCTCC) for preservation on May 26, 2022, with the preservation number CCTCC M 2022724; the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0036] Example 2: Removal rate of cadmium by Citrobacter freundii

[0037] After LB liquid culture medium and 1 mol / L CdCl2 solution were sterilized by high temperature and high pressure, 2+ The concentration range is 0, 2, 4 to 6 mmol L -1 CdCl2 solution was added to LB liquid medium. The concentration of the DS strain seed solution was 2.0×10 9 CFU / mL, inoculated at 2% of the inoculum into the Cd 2+ The culture was carried out in LB medium at 180 rpm and 35 °C for 24 h. The bacterial solution was centrifuged at 1000 r / min for 10 min, and the supernatant was taken to determine Cd by graphite furnace atomic absorption spectrophotometry. 2+ The concentration of Figure 2 As shown in Figure 2, with the increase of culture time, the activity of Citrobacter freundii to Cd 2+ The removal rate of Cd increased significantly, and the removal rate changed slowly after 24 hours. 2+ The higher the concentration, the lower the final removal rate. 2+When the concentration was 2mmol / L, the removal rate reached 63.61%, and the initial Cd 2+ When the concentration was 6mmol / L, the removal rate reached 51.88%, and the removal rate remained above 50%.

[0038] Example 3: Rice pot test

[0039] The soil for the potted test was taken from the paddy field with high selenium and cadmium background values ​​in Dashan Village, Shitai County. After natural air drying, it was ground through a 2mm sieve and 5kg of sterilized soil samples were filled in each pot (specification: 14cm high and 19.7cm in diameter). The classified management of selenium-rich soil with different cadmium pollution levels was explored through rice pot experiments. The experimental design was a completely randomized block experiment. The experimental treatments included a control group and three treatment groups inoculated with Citrobacter freundii (CF group), a group with rapeseed straw biochar added (BC group) and a lime group (LM group). All treatments were repeated 4 times. The potted test began in May 2021. First, 5kg of air-dried soil was weighed and put into a plastic pot (26.5cm in diameter and 28cm in height). In the CF group, the prepared Citrobacter freundii bacterial solution (bacterial solution concentration 2.0×10 9 CFU / mL) was mixed with the soil at a ratio of 80 mL / kg to ensure that the fungus was evenly distributed and the content reached at least 1×10 8 CFU / g; for the BC group, 50g of rape straw biochar was added to each pot at a ratio of 1wt% of the total soil mass and mixed thoroughly, and 5g of lime was added to the LM group at a ratio of 0.1wt% and mixed thoroughly. Before transplanting rice, the soil moisture content was adjusted to a stable balance of field water holding capacity for one week. Then, rice seedlings with relatively consistent growth conditions were selected and planted at 4 plants / pot. In order to reduce ineffective tillering and ensure normal maturity of grains after filling, drainage management was carried out in the late tillering and late filling stages, and a 2-3cm water layer was maintained in the remaining growth stages. When harvesting, the rice was removed from the soil as intact as possible along with the root system, cleaned with deionized water, and dried at 70℃ to constant weight after being sterilized at 105℃ in an oven for 30min. The rice plants were weighed, recorded and ground according to the brown rice, rice husk, leaves, stems and roots, and reserved for the determination of the total amount of selenium and cadmium and the analysis of the bioavailability of selenium and cadmium in brown rice. Rice rhizosphere soil samples were collected, placed in a ventilated and cool place indoors to dry naturally, the residual roots were removed, and the samples were ground and passed through 10-mesh and 100-mesh nylon sieves respectively, and placed in ziplock bags for later use.

[0040] Citrobacter freundii DS strain (CF), lime (BC) and biochar (LM) were selected as passivators to compare the regulatory effects and mechanisms of different passivation materials on rice cadmium accumulation. The main research results were based on soil physicochemical properties, soil selenium and cadmium occurrence forms, rice selenium and cadmium accumulation and distribution, and bioavailability analysis, and compared the effects of different passivation treatments on the migration and transformation of selenium and cadmium in the soil-rice system.

[0041] Example 4: Changes in the availability of selenium and cadmium in rhizosphere soil in potted plant experiments

[0042] The effective cadmium in the rhizosphere soil of rice in Example 3 was extracted by gradient film diffusion technique (DGT). The specific steps of the DGT method are as follows: harvest rice after six months of rice cultivation, collect soil samples, weigh 80g of soil samples through a 2mm sieve in a 150mL beaker, add deionized water to 60% of the maximum field water holding capacity of the soil, stir and homogenize, leave for two days, add deionized water to 100% of the maximum field water holding capacity and leave for 24h, take out a small amount of soil, carefully apply it on the sampling window of the DGT device (on the filter membrane), then turn the sampling window of the DGT device downward, and slowly press it into the soil in a left-right rotation to make the device fully contact with the soil, place it in a ziplock bag filled with a little deionized water for 24h, take out the DGT device, take out the adsorption membrane, put it in a 1.5mL centrifuge tube, add 1mL of ultrapure nitric acid for elution, shake at 150rpm for 2h, save the eluate, and use a flame atomic absorption spectrophotometer to determine the cadmium content in the eluate.

[0043] Determination of available selenium in rhizosphere soil: Weigh 5g of soil, add 0.1mol / LKH2PO4 at a soil-liquid ratio of 1:5 (mass-to-volume ratio), oscillate horizontally at 180rpm for 2h at 25℃, and centrifuge. Then take 10mL of supernatant, add 0.5mL4.5mol / LHCl and 1.0mL 5% K2S2O8, shake well, heat in a slightly boiling water bath for 1h, add 1.0mL 3% H2C2O4, and continue heating for 30min. Add 7.5mL concentrated HCl, heat for another 15min, cool and dilute to 25mL, filter into a collection tube for use, and use a graphite furnace atomic absorption spectrophotometer to determine the total selenium in the soil in the collected liquid.

[0044] The available cadmium content in the rice rhizosphere soil is Figure 3As shown. The effective cadmium content in the rhizosphere soil of the control group was 0.45 mg / kg. Compared with the control group, the effective cadmium content measured by the DGT method in the soil of the treatment group with the addition of Citrobacter freundii was significantly reduced, and the CF group had the most obvious reduction effect among all treatment groups. The addition of Citrobacter freundii DS strain significantly reduced the exchangeable cadmium content and the effective cadmium content in the soil (DGT method), and the addition of Citrobacter freundii DS strain significantly increased the soluble selenium content and the effective selenium content in the soil. The results showed that the addition of Citrobacter freundii DS strain can reduce the effective cadmium concentration in the soil, thereby achieving the effect of passivating cadmium. The addition of different materials significantly increased the selenium content in various parts of rice (p<0.05). The increase in selenium content in brown rice ranged from 0.02 mg / kg to 0.17 mg / kg, among which the inoculation of Citrobacter freundii (CF) had the strongest enhancement effect on the selenium accumulation of brown rice, with an increase of 154.55%. The selenium content of brown rice in the LM and BC groups also increased by nearly 20% and 40%, respectively. Similarly, selenium is more likely to accumulate in the roots of rice.

[0045] Example 5: Changes in selenium and cadmium content in various parts of rice in potted experiments

[0046] After the rice in Example 3 grew for 120 days, the rice was harvested by cutting the aboveground part above the soil surface, and the roots were separated from the soil. These samples were divided into roots, leaves, rice husks and brown rice. The leaves and roots were thoroughly washed with tap water, then rinsed with deionized water 3-4 times, and dried to constant weight at 70°C. The dried samples were ground with a stainless steel mill before analysis. Determination of total selenium in rice roots, leaves, rice husks and brown rice: Accurately weigh 0.3g (accurate to 0.0001g) of dry plant samples passed through a 100-mesh sieve, placed in a dry 50mL polytetrafluoroethylene digestion tank, added 8mL of mixed acid (HNO3:HClO4=4:1), covered with a small funnel and cold digested overnight in a fume hood, placed on a graphite digestion instrument for digestion the next day, and the total selenium in the same soil was determined on the machine. Determination of total cadmium in rice roots, leaves, rice husks, and brown rice: Accurately weigh 0.3 g (accurate to 0.0001 g) of dry plant sample that has passed through a 100-mesh sieve, place it in a 50 mL polytetrafluoroethylene digestion tank, add 8 mL of mixed acid (HNO3:HClO4=4:1), cover with a small funnel and cold digest in a fume hood overnight. The next day, place it on a graphite digester for digestion, and determine the total cadmium in the same way as in the soil.

[0047] The results are as follows Figure 4 As shown, Figure a shows the cadmium content in various tissues of rice under different repair treatments, and Figure b shows the selenium content in various tissues of rice under different repair treatments. Figure 4It can be seen that, compared with the control group (CK), the cadmium contents in the leaves and roots of rice in each treatment group were significantly reduced, and the reduction effect was most obvious in the treatment group with the addition of Citrobacter freundii. The cadmium contents in different tissues showed the following pattern: CF = BC < LM < CK, and the cadmium contents in brown rice of each treatment group met the edible safety standards; the addition of the DS strain of Citrobacter freundii significantly increased the selenium contents in various tissues of rice, and the selenium contents in different tissues showed the pattern of CF = BC > LM > CK. The results indicate that the addition of Citrobacter freundii has a positive effect on reducing the cadmium content in rice.

[0048] Example 6: Changes in rice biomass and bioaccessibility of selenium and cadmium

[0049] The bioaccessibility of selenium and cadmium in brown rice was evaluated using a simplified PBET model. To simulate the process of residents using rice, the brown rice samples of each treatment group were placed in plastic petri dishes and steamed with a household rice cooker, and the steamed rice samples were freeze-dried to a constant weight in a -40 °C vacuum dryer, and then the samples were ground into particles with a mortar to prepare PBET test samples. The determination of the bioaccessible contents of selenium and cadmium was the same as that of total selenium and total cadmium in soil. The bioaccessibility of selenium and cadmium in brown rice in the simulated gastrointestinal tract was calculated according to the following formula:

[0050] Bioaccessibility of selenium and cadmium (%) = Bioaccessible content in the gastrointestinal tract (mg / kg) / Content of selenium and cadmium in brown rice (mg / kg) × 100%

[0051] The measurement results are as Figure 5 shown, where Figure a is the statistical result of the changes in the bioaccessibility of Se and Cd, and Figure b is the statistical result of the changes in the biomass of each tissue of rice. It can be Figure 5 seen that the bioaccessibility of cadmium in the intestinal phase was significantly lower than that in the gastric phase, the addition of the CF treatment could significantly reduce the bioaccessibility of cadmium in brown rice in the gastric phase, and the addition of the CF or BC treatment could significantly reduce the bioaccessibility of cadmium in brown rice in the intestinal phase; the bioaccessibility of selenium in the intestinal phase was significantly higher than that in the gastric phase, and the addition of the CF treatment could significantly increase the bioaccessibility of selenium in brown rice in the gastric and intestinal phases. The bioaccessible contents of selenium and cadmium were significantly positively correlated with the total amounts of selenium and cadmium in brown rice.

[0052] The above experimental results show that the addition of the Citrobacter freundii in the present invention can promote the growth of rice, relieve the oxidative stress suffered by rice, and is significantly better than the other three passivators.

Claims

1. A Citrobacter freundii, characterized in that The deposited name of the strain is Citrobacter freundiiDS, and the deposited number is CCTCC NO: M2022724. The strain was deposited in the China Center for Type Culture Collection on May 26, 2022.

2. A use of the Citrobacter freundii described in claim 1 in passivating cadmium in soil.

3. Use of the Citrobacter freundii described in claim 1 in reducing the cadmium content in rice.

4. Use of the Citrobacter freundii according to claim 1 in improving the biological effectiveness of selenium in soil.

5. Use of the Citrobacter freundii according to claim 1 in increasing the selenium content in rice.

6. The use according to any one of claims 2 to 5, characterized in that: The steps include: (1) activating the Citrobacter freundii of claim 1, collecting the bacterial cells after transfer culture, and resuspending the bacterial cells to obtain a live bacterial liquid; (2) Mixing the live bacterial solution with soil containing selenium and / or cadmium.

7. The use according to claim 6, characterized in that: In step (1), the Citrobacter freundii is transferred and cultured to the late logarithmic growth phase, and the cells are collected by centrifugation.

8. The use according to claim 6, characterized in that: In step (1), the transfer culture conditions are culturing at 28°C-37°C and 100-200rpm, the activation of the Citrobacter freundii adopts LB liquid culture medium or LB solid culture medium, and the transfer culture adopts LB liquid culture medium.

9. The use according to claim 6, characterized in that: In step (1), the method of resuspending the bacterial cells is: resuspending the bacterial cell pellet in sterile deionized water to obtain a bacterial cell concentration of at least 2.0×10 9 CFU / mL of live bacterial solution.

10. The use according to claim 6, characterized in that: In step (2), the method for mixing the active bacterial solution with the soil containing selenium and cadmium is: mixing the active bacterial solution with the soil at a ratio of 80 mL / kg.

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