Citrobacter freundii and use thereof

Abstract

Provided are Citrobacter freundii and the use thereof. The Citrobacter freundii has a high cadmium tolerance and the function of enhancing selenium bioavailability. The strain is classified as and named Citrobacter freundii DS, which has been deposited in the CCTCC on 26 May 2022. The strain has a high tolerance to the heavy metal cadmium. The strain can still grow under the conditions of a Cd2+ concentration of 6 mM, reaching the maximum biomass at 40 h. By means of applying the strain to cadmium-contaminated soil, the proportion of exchangeable cadmium and the content of available cadmium in the soil can be significantly decreased, while significantly increasing the content of soluble selenium and available selenium in the soil. By means of performing a rice pot experiment, the cadmium bioaccumulation factor in various parts of rice is further reduced, while increasing the selenium bioaccumulation factor and translocation factor. The strain has significant effects in the applications of remediating cadmium-contaminated soil, enhancing the bioavailability of selenium and promoting crop growth.

Description

Citrobacter freundii and its application Technical Field

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

[0002] Cadmium (Cd) is one of the most common inorganic soil pollutants in my country, with a 7% exceedance rate, ranking first. Cadmium, classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC), is highly toxic to both animals and plants and readily accumulates in organisms. Most naturally selenium-rich areas in China also face severe cadmium pollution, posing a serious threat to food safety and hindering the utilization of these selenium-rich soils. Compared to other heavy metals, cadmium has a higher bioavailability and stronger mobility, reacting with specific proteins and enzymes in the body, posing a serious threat to ecosystem function and human health. Rice is a staple food crop. While it provides essential nutrients, toxic heavy metals accumulated in its edible parts can also enter the human body through the mouth, posing a health hazard. Research has shown that rice is a major route for cadmium to enter the human body. Cadmium accumulates in large quantities in rice grains and is then transferred through the food chain, posing a serious threat to the health of humans and animals and significantly impacting food security. Therefore, controlling heavy metal pollution in soil and reducing cadmium levels in rice are crucial for minimizing cadmium absorption and safeguarding human health. Currently, 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, causing little soil disturbance, not generating secondary pollution, and complying with the laws of ecological development. Microorganisms, as an important component of bioremediation, are diverse and reproduce rapidly. They can reduce the toxicity of cadmium through adsorption, transformation, and fixation. Microbial remediation has the advantages of rapid effectiveness, easy management and operation, and no damage to soil structure. It has attracted widespread attention and has become a hot topic in the research of soil cadmium contamination remediation. Studies have shown that some cadmium-tolerant microorganisms can significantly reduce the bioavailability of cadmium in 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, with a deposit number of 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 tolerance and the ability to increase effective selenium from the intestines of cadmium-tolerant earthworms (Hubei far-blind earthworms) to reduce the bioavailability of heavy metal cadmium in the soil, thereby reducing the accumulation of heavy metals in the soil and various parts of rice, and improving grain yield and quality. The method 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 bioavailability 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 described in claim 1, collecting the cells after transfer culture, and resuspending the cells to obtain a live bacterial solution;

[0012] (2) Mix 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 bacteria 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 for 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 bacterial 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 strain has a fast growth rate and a high tolerance to heavy metal cadmium, with a tolerance concentration of cadmium reaching 6 mM, and can passivate cadmium.

[0020] The application of the Citrobacter freundii DS strain in soil contaminated by heavy metal cadmium can significantly reduce the available cadmium content in the soil, while also reducing cadmium absorption by various parts of rice plants, significantly improving rice biomass and quality, and alleviating the physiological stress posed by cadmium on rice. The cadmium-tolerant strain isolated and screened by the present invention, which can efficiently remove cadmium from the environment, can reduce cadmium content in rice through microbial remediation measures, thus providing a scientific basis for ensuring the quality and safety of rice and other food crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1 is a phylogenetic tree diagram of Citrobacter freundii of the present invention;

[0022] FIG2 is a graph showing the effect of Citrobacter freundii on Cd in culture medium under different cadmium concentration conditions. 2+ Removal rate graph;

[0023] FIG3 is a bar graph showing changes in the effectiveness of Se and Cd in soil in a rice pot experiment using Citrobacter freundii of the present invention;

[0024] FIG4 is a bar graph showing changes in Se and Cd content in various parts of rice caused by the Citrobacter freundii of the present invention in a pot culture experiment;

[0025] FIG5 is a bar graph showing changes in rice biomass and Se and Cd bioavailability induced by the Citrobacter freundii of the present invention in a pot culture experiment.

[0026] DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] Example 1: Isolation and Identification of Citrobacter freundii

[0029] (1) Sample collection: The Hubei far-blind earthworm was collected from farmland soil with a high background of selenium and cadmium in Dashan Village, Chizhou City, Anhui Province.

[0030] (2) Isolation and screening of cadmium-resistant strains: Take earthworms that have been cleansed for 24 hours, clean them and fix them in a wax tray, add 2-3 drops of 75% ethanol solution to their heads, dissect the earthworms, take out their intestinal contents, and grind the intestines thoroughly with a sterilized grinding rod. Add sterile water at a weight (g): volume (mL) ratio of 1:9, grind them thoroughly 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 0.1 mM was added to LB liquid medium and cultured at 28 °C with shaking at 180 r / min 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 LB solid screening medium was 1 mM and cultured in a bacterial incubator at 28 ° C for 48 h. Observe the colony morphology, color, wetness, glossiness, etc., pick a single colony on the plate, and 2+ Streak cultures on plates at concentrations of 2, 4, 6, 8, and 10 mM to obtain pure cultures of bacteria with the highest cadmium tolerance. Prepare the following LB liquid medium (1 L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, adjust the pH to 7.0. Add distilled water to 1 L. Sterilize under autoclave at 121°C for 20 min.

[0031] (3) Identification of cadmium-resistant strains: Identification was performed by amplifying the 16S rDNA nucleotide sequence of the bacteria. The 16S rDNA primers used were forward primer 27F (AGTTTGATCMTGGCTCAG) and reverse primer 1492R (GGTTACCTTGTTACGACTT). The PCR products were sequenced, and the results were as follows:

[0032] Citrobacter freundiiDS 16S ribosomal DNA gene

[0033]

[0034] The sequencing results were compared with sequences of closely homologous strains using BLAST in the EzBioCloud database (www.ezbiocloud.net). The Citrobacter isolated in the present invention showed the highest homology with Citrobacter freundii. A phylogenetic tree (shown in Figure 1) was constructed using MEGA 6.0 software using the Neighbor-Joining method, identifying the strain as Citrobacter freundiiDS.

[0035] (4) Morphological 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, with raised surfaces, flat edges, and smooth and moist surfaces. They grow faster on LB agar medium.

[0036] (5) Preservation of Citrobacter freundii: Citrobacter freundii can be cultured on LB liquid or solid medium at 37°C. After culture, it can be transferred to slant medium at 4°C for short-term preservation. For long-term preservation, the bacterial liquid can be mixed with equal volumes 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.

[0037] Example 2: Cadmium removal efficiency by Citrobacter freundii

[0038] After LB liquid culture medium and 1 mol / L CdCl2 solution were sterilized by high temperature and high pressure, 2+ The concentration range was 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 into the Cd-containing 2+ The cells were cultured 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 used to determine the Cd 2+ As shown in Figure 2, with the increase of culture time, the Citrobacter freundii 2+ The removal rate of Cd increased significantly, and the change of removal rate was slow after 24 h. 2+ The higher the concentration, the lower the final removal rate. 2+ When the concentration was 2 mmol / L, the removal rate was 63.61%.2+ When the concentration was 6 mmol / L, the removal rate reached 51.88%, and the removal rate remained above 50%.

[0039] Example 3: Rice pot test

[0040] The soil for the potted experiment 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 2 mm sieve and 5 kg of sterilized soil samples were packed into each pot (specification: 14 cm high and 19.7 cm in diameter). The rice pot experiment was used to explore the classified management of selenium-rich soil with different cadmium pollution levels. The experimental design was a completely randomized block experiment. The experimental treatments included a control group and three treatment groups: a Citrobacter freundii inoculated group (CF group), a rapeseed straw biochar added group (BC group) and a lime group (LM group). All treatments were repeated 4 times. The potted experiment started in May 2021. First, 5 kg of air-dried soil was weighed and put into a plastic pot (26.5 cm in diameter and 28 cm high). In the CF group, the prepared Citrobacter freundii bacterial solution (bacterial solution concentration 2.0×10 9 CFU / mL) was mixed thoroughly with the soil at a ratio of 80 mL / kg to ensure that the microbial agent was evenly distributed and the content reached at least 1×10 8 CFU / g; for the BC group, 50 g of rapeseed straw biochar was added to each pot at a ratio of 1 wt% of the total soil mass and mixed thoroughly; for the LM group, 5 g of lime was added at a ratio of 0.1 wt% and mixed thoroughly. Before transplanting rice, soil moisture was adjusted to a stable equilibrium at field capacity for one week. Rice seedlings with relatively consistent growth conditions were then selected and planted at a rate of 4 per pot. To reduce ineffective tillering and ensure normal grain maturation after grain filling, drainage was implemented during the late tillering and grain filling stages, and a 2-3 cm water layer was maintained throughout the rest of the growth period. At harvest, rice plants were removed from the soil as intact as possible, along with their roots, rinsed with deionized water, oven-dried at 105°C for 30 minutes, and then dried at 70°C to constant weight. The rice plants were weighed, recorded, and ground into brown rice, including brown rice, husk, leaves, stems, and roots, for total selenium and cadmium determination and analysis of their bioavailability in brown rice. Rice rhizosphere soil samples were collected and 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.

[0041] Citrobacter freundii DS strain (CF), lime (BC), and biochar (LM) were used as passivation agents to compare the regulatory effects and mechanisms of different passivation materials on cadmium accumulation in rice. The main findings were based on soil physicochemical properties, soil selenium and cadmium occurrence forms, rice selenium and cadmium accumulation and distribution, and bioavailability. The effects of different passivation treatments on the migration and transformation of selenium and cadmium in the soil-rice system were compared.

[0042] Example 4: Changes in Selenium and Cadmium Availability in Rhizosphere Soil in Potted Plant Experiments

[0043] The available cadmium in the rice rhizosphere soil in Example 3 was extracted using the diffusive gradients in thin-films technique (DGT). The DGT method specifically involves the following steps: harvest rice after six months of cultivation, collect soil samples, weigh 80 g of soil sample passed through a 2 mm sieve, place in a 150 mL beaker, add deionized water to 60% of the soil's maximum field water holding capacity, stir and homogenize, and let stand for two days. Deionized water is then added to 100% of the soil's maximum field water holding capacity and allowed to stand for 24 hours. A small amount of soil is then carefully applied to the sampling window (on the filter membrane) of the DGT device. The DGT device is then placed with the sampling window facing downward and slowly rotated left and right into the soil to ensure full contact between the device and the soil. The device is then placed in a ziplock bag containing a small amount of deionized water for 24 hours. The DGT device is then removed, the adsorption membrane is removed, and placed in a 1.5 mL centrifuge tube. Elution is performed by adding 1 mL of ultrapure nitric acid and shaking at 150 rpm for 2 hours. The eluate is then stored and the cadmium content in the eluate is determined using a flame atomic absorption spectrophotometer.

[0044] Determination of available selenium in rhizosphere soil: Weigh 5 g of soil and add 0.1 mol / L KH2PO4 at a soil-to-liquid ratio of 1:5 (mass-to-volume ratio). Shake horizontally at 180 rpm for 2 h at 25°C and centrifuge. Then, remove 10 mL of the supernatant and add 0.5 mL of 4.5 mol / L HCl and 1.0 mL of 5% K2S2O8. Shake well and heat in a slightly boiling water bath for 1 h. Add 1.0 mL of 3% H2C2O4 and continue heating for 30 min. Add 7.5 mL of concentrated HCl and heat for an additional 15 min. After cooling, dilute to 25 mL and filter into a collection tube for later use. Determine the total soil selenium in the collected solution using a graphite furnace atomic absorption spectrophotometer.

[0045] Figure 3 shows the available cadmium content in the rhizosphere soil of rice. The available cadmium content in the rhizosphere soil of the control group was 0.45 mg / kg. Compared with the control group, the available cadmium content in the soil treated with Citrobacter freundii (CF) was significantly reduced, as measured by the DGT method. The CF treatment had the most significant reduction among all treatments. Addition of the DS strain of Citrobacter freundii significantly reduced the exchangeable cadmium content and available cadmium content in the soil (DGT method), while significantly increased the soluble selenium content and available selenium content in the soil. These results indicate that the addition of the DS strain of Citrobacter freundii can reduce the available cadmium concentration in the soil, thereby achieving the effect of cadmium inactivation. The addition of different materials significantly increased the selenium content in various rice parts (p < 0.05). The selenium content in brown rice increased from 0.02 mg / kg to 0.17 mg / kg. Inoculation with Citrobacter freundii (CF) had the strongest effect on enhancing selenium accumulation in brown rice, reaching a 154.55% increase. Selenium content in the LM and BC treatments also increased by nearly 20% and 40%, respectively. Similarly, selenium is more likely to accumulate in rice roots.

[0046] Example 5: Changes in Selenium and Cadmium Contents in Various Parts of Rice in a Potted Plant Experiment

[0047] After 120 days of growth, the rice plants in Example 3 were harvested by cutting the aboveground portion above the soil surface, and the roots were separated from the soil. These samples were separated into roots, leaves, husks, and brown rice. The leaves and roots were thoroughly washed with tap water, then rinsed 3-4 times with deionized water and dried at 70°C to a constant weight. The dried samples were ground using a stainless steel mill before analysis. Determination of total selenium in rice roots, leaves, husks, and brown rice: 0.3 g (accurate to 0.0001 g) of dried plant sample, passed through a 100-mesh sieve, was accurately weighed and placed in a dry 50 mL polytetrafluoroethylene digestion vessel. 8 mL of mixed acid (HNO₃:HClO₄ = 4:1) was added, and the sample was covered with a small funnel and digested overnight in a fume hood. The next day, the sample was digested in a graphite digester and assayed for total selenium in the same manner as the soil. 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 passed through a 100-mesh sieve and 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 overnight in a fume hood. The next day, place the sample in a graphite digester for digestion, and determine the total cadmium in the same manner as in soil.

[0048] The results are shown in Figure 4, where Figure a shows the cadmium content in various tissues of rice under different remediation treatments, and Figure b shows the selenium content in various tissues of rice under different remediation treatments. As can be seen from Figure 4, compared with the control group (CK), the cadmium content in the leaves and roots of rice in each treatment group was significantly reduced, and the treatment group with the addition of Citrobacter freundii had the most obvious reduction effect. The cadmium content in different tissues showed the following pattern: CF = BC < LM < CK, and the cadmium content in brown rice of each treatment group met the edible safety standard; the addition of the DS strain of Citrobacter freundii significantly increased the selenium content in various tissues of rice, and the selenium content in different tissues showed the pattern of CF = BC > LM > CK. The results show that the addition of Citrobacter freundii has a positive effect on reducing the cadmium content in rice.

[0049] Example 6: Changes in rice biomass and selenium and cadmium bioaccessibility

[0050] The simplified PBET model was used to evaluate the bioaccessibility of selenium and cadmium in brown rice. 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 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 content 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:

[0051] Bioaccessibility of selenium and cadmium (%) = Bioaccessible content in the gastrointestinal tract (mg / kg) / Content of selenium and cadmium in brown rice (mg / kg) × 100%

[0052] The measurement results are shown in Figure 5, where Figure a shows the statistical results of the changes in Se and Cd bioaccessibility, and Figure b shows the statistical results of the changes in the biomass of various tissues of rice. As can be seen from Figure 5, the bioaccessibility of cadmium in the intestinal phase was significantly lower than that in the gastric phase, and 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 content of selenium and cadmium was significantly positively correlated with the total amount of selenium and cadmium in brown rice.

[0053] The above experimental results show that the addition of 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. Use of the Citrobacter freundii according to claim 1 in passivating cadmium in soil.

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

4. Use of the Citrobacter freundii according to claim 1 in improving the bioavailability 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 described in claim 1, collecting the cells after transfer culture, and resuspending the cells to obtain a live bacterial solution; (2) Mix 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 bacteria are collected by centrifugation.

8. The use according to claim 6, characterized in that In step (1), the transfer culture conditions are 28°C-37°C, 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.

9. The use according to claim 6, characterized in that In step (1), the method for resuspending the bacterial cells is as follows: 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 and the soil at a ratio of 80 mL / kg.

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