CN113693083A

CN113693083A - Application of providencia bacterial strain in preparation of ferrous oxidant

Info

Publication number: CN113693083A
Application number: CN202111022310.XA
Authority: CN
Inventors: 李丁; 陈金媛; 胡远艺; 丁哲旭; 罗军; 易慧娟; 陈莎
Original assignee: Hunan University of Technology
Current assignee: Hunan University of Technology
Priority date: 2021-09-01
Filing date: 2021-09-01
Publication date: 2021-11-26
Anticipated expiration: 2041-09-01
Also published as: CN113693083B

Abstract

The invention discloses an application of providencia bacterial strains in preparing ferrous oxidants, an application of providencia bacterial strains in preparing rice root system surface iron film forming agents and an application of providencia bacterial strains in preparing rice root system heavy metal cadmium absorption blocking agents. According to the invention, by a traditional ferrous sulfide gradient tube method and by using an improved mineral medium, called MWMM for short, after the strain LLDRA6 is cultured, an obvious iron oxide layer appears, and then the strain LLDRA6 is found to have Fe (II) oxidation capability by a phenanthroline spectrophotometry. Meanwhile, the iron film formed by the strain LLDRA6 on the surface of the rice root system has obvious barrier effect on the cadmium absorption of the rice.

Description

Application of providencia bacterial strain in preparation of ferrous oxidant

Technical Field

The invention belongs to the field of microbial environment restoration, and particularly relates to application of providencia bacterial strains in preparation of a ferrous oxidant and application of the bacterial strains in preparation of a rice root system surface iron film forming agent, so that the effect of preventing rice root systems from absorbing heavy metal cadmium is achieved.

Background

Cadmium is one of the main elements of heavy metal pollution of rice, and has stronger activity and is easier to be absorbed by plants compared with other heavy metals. Cadmium in soil can be roughly divided into two forms of water-soluble cadmium and water-insoluble cadmium, and the water-soluble cadmium has strong migration and is easily absorbed by plants. Cadmium can obstruct the absorption of plant nutrition and moisture, inhibit enzyme activity, disturb plant metabolism and the like after entering a plant body, further influence the speed of plant photosynthesis and transpiration and generate toxic action on the plant. For rice, the excessive cadmium concentration in the rice field can directly cause the reduction of the rice yield and influence the rice quality. In addition, cadmium flowing from crops to human body through food chain is also seriously harmful, and can be combined with some essential amino acids or proteins in human body to form cadmium sulfur protein which reaches various parts of human body through blood circulation and can damage tissues and organs of human body to a certain extent. Most importantly, the heavy metal cadmium is not degradable and is difficult to remove after being accumulated in organisms, and various diseases can be caused after a certain amount of cadmium is accumulated, so that the life safety of people is threatened.

The traditional cadmium pollution treatment method comprises physical remediation and chemical remediation. However, these techniques generally have the problems of complicated operation, high energy consumption, high cost, and easy secondary pollution. Bioremediation can effectively avoid the problems, and the bioremediation refers to a method for remedying heavy metal pollution by using organisms as main bodies, including phytoremediation, animal remediation and microbial remediation, and is increasingly widely regarded in the aspect of soil cadmium pollution treatment due to easy operation, good effect and low cost.

In recent years, microorganisms are used for repairing heavy metals in the environment, which attracts people's attention. Microbial remediation refers to the absorption, precipitation, oxidation and reduction of heavy metals in soil by microorganisms. On one hand, the cell wall of the microorganism provides a plurality of functional groups (carboxylic acid, hydroxyl, amino and phosphate groups and the like) capable of binding heavy metal ions, and the microorganism can directly repair the heavy metal in the environment by utilizing the processes of adsorption, enrichment, dissolution, precipitation and the like; on the other hand, the organic fertilizer can reduce the absorption and accumulation of heavy metals in the environment by plants through the interaction with other microorganisms and plants. The method solves the problem of heavy metal pollution of soil by utilizing microbial remediation, and has the advantages of high remediation efficiency, low cost and no pollution to the environment. Therefore, the screening of the bacterial strain which has stronger resistance to the soil heavy metal and can effectively prevent the plants from absorbing the heavy metal is of great significance.

Disclosure of Invention

The invention aims to develop Fe (II) oxidant on the basis of finding that Providencia sp.LLDRA6 has ferrous iron Fe (II) oxidation capacity, further develop a rice root system surface iron film forming agent and a rice root system heavy metal cadmium absorption blocking agent, and provide a method for blocking heavy metal cadmium (Cd) from being absorbed by rice roots.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the Providencia bacterial strain with iron oxidation capability is preserved in the China center for type culture collection (CCTCC M2018876) in 2018, 12 months and 10 days, and is named as Providencia sp. The method for preventing heavy metal cadmium from being absorbed by the root system of the rice is that ferrous iron and bacterial suspension of Providencia sp.LLDRA6 are added into the paddy field polluted by the cadmium at the same time; OD of the bacterial suspension₆₀₀1.5-2.5, preferably 2, and the addition amount of the divalent iron is 200-300 mmol/m²Preferably 250mmol/m²The addition amount of the bacterial suspension is 400-600 mL/m²Preferably 500mmol/m²。

The present invention uses a ferrous sulfide gradient tube method, i.e. the improved MWMM medium is used for culturing the strain LLDRA 6. Two layers of culture medium are added into the test tube, the bottom layer is ferrous sulfide, and the upper layer is semisolid modified mineral medium (MWMM for short). In this medium, the ferrous ions in the bottom layer diffuse upward to form an iron concentration gradient, while the oxygen diffuses downward to form an oxygen concentration gradient due to the presence of a small amount of air. At a certain height, the oxygen and ferrous ion concentration reach the proper range, at which time the iron oxidizing bacteria will start to grow and form a circular iron oxidation band at a certain height. The experiment was divided into treatment group and control group, treatment group: inoculation of strain LLDRA 6; control group: blank medium without treatment. The experiment shows that a circle of reddish brown iron oxide bands are formed in the test tubes of the treatment group inoculated with the strain LLDRA6, and the strain LLDRA6 is initially found to be a strain of iron-oxidizing bacteria. Further testing by phenanthroline spectrophotometry; the phenanthroline is Fe (II) developer, and the product generated after the color development reaction of Fe (II) has an absorption peak at 512nm, and the absorbance can reflect the concentration of Fe (II). The experiment was divided into treatment group and control group, treatment group: the strain was inoculated in LB medium (50mL), shake-cultured at 35 ℃ and 180rpm for 4 hours, and then Fe (II) was added to give a final concentration of 8mmol/L in the system. Control group: blank LB medium (50mL), 35 ℃, 180rpm constant temperature shaking culture 4h, adding Fe (II), the system of final concentration of 8 mmol/L. Sampling at different time points, reacting with phenanthroline indicator, measuring the absorbance at 512nm by using an ultraviolet spectrophotometer, obtaining the final concentration of Fe (II) by contrasting with a standard curve, and calculating the Fe (II) oxidation rate of a treatment group and a control group, thus proving whether the strain has Fe (II) oxidation capacity. The experiment shows that the oxidation rate of Fe (II) in the treated group is higher than that in the control group, which indicates that Providencia sp.

The method for finding that the strain can promote the rice root system to form an iron film so as to achieve the effect of efficiently preventing the rice root system from absorbing the heavy metal cadmium is based on soil pot culture and field experiments of two rice varieties of Huazhan and Taibei 309. Designing 4 treatment groups for each of the pot culture and the field, repeatedly treating each group for 5 times, transplanting the germinated rice seedlings into the pot culture and the field after different treatments, after growing for 90-120 days, collecting rice root systems of the pot culture treatment groups for observation, and characterizing root samples by using SEM-EDS; collecting rice samples of the overground parts of the field treatment groups, and detecting the cadmium content of grains, stems and leaves. The pot experiment shows that the rice root system of the treatment group added with the bacterial suspension and the Fe (II) has more obvious color change and is rust red than other treatment groups; SEM-EDS analysis is carried out on the rice root systems of the treatment groups, layered precipitates formed on the surfaces of the rice root systems of the treatment groups added with the bacterial suspension and the Fe (II) are most obvious, the Fe and O content of the regions of the precipitates is high, and the precipitates can be determined to be iron oxide films. In a field experiment, the cadmium content of grains, stems and leaves of two rice varieties is obviously lower than that of other treatment groups in the treatment group added with the bacterial suspension and the Fe (II). The results show that the strain LLDRA6 has the capability of forming an iron film on the surface of the rice root system, thereby having the effect of efficiently preventing the rice root system from absorbing the heavy metal cadmium.

The method for preventing the heavy metal cadmium (Cd) from being absorbed by the root system of the rice by using the Providencia sp.LLDRA6 bacterial strain has the advantages of simple and convenient operation and low cost, overcomes the defects of complex operation, high cost, low efficiency and the like of the existing physicochemical restoration technology, has no secondary pollution risk to the environment, and has wide application prospect.

Drawings

FIG. 1 is a comparison of test tubes of a control group not inoculated with strain LLDRA6 and a treatment group inoculated with strain LLDRA6 in the ferrous sulfide gradient tube method (A: control group; B: treatment group);

FIG. 2 is the iron oxidation rate at different time points with the addition of only Fe (II) and with the simultaneous addition of strains LLDRA6 and Fe (II);

FIG. 3 is a real-time image of the root system of the Huazhan variety in the potted plant experiment (A: a control group; B: only bacterial suspension is added; C: only Fe (II) is added; D: both Fe (II) and bacterial suspension are added);

FIG. 4 is a SEM-EDS representation of the root system of the Huazhan variety in the potting experiment (A: control group; B: only bacterial suspension is added; C: only Fe (II) is added; D: Fe (II) and bacterial suspension are added at the same time);

FIG. 5 shows the cadmium content of rice grains in a field experiment (a first treatment group is a control group, a second treatment group is a treatment group only added with bacterial suspension, a third treatment group is a treatment group only added with Fe (II), and a fourth treatment group is a treatment group simultaneously added with Fe (II) and bacterial suspension);

FIG. 6 shows the cadmium content of rice stem and leaf in the field experiment (first treatment group: control group; second treatment group: only bacterial suspension was added; third treatment group: only Fe (II) was added; fourth treatment group: both Fe (II) and bacterial suspension were added).

Detailed Description

The providencia bacterial strain provided by the invention is preserved in China center for type culture Collection, Wuhan, China with the preservation number of CCTCC M2018876.

Providencia sp

1. Ferrous sulfide gradient tube process

1) The main reagents are as follows:

firstly, ammonium chloride; magnesium sulfate heptahydrate; ③ calcium chloride dihydrate; potassium hydrogen phosphate; FeS; sixthly, trace element solution and vitamin solution.

2) Configuration of bidirectional gradient test tube culture medium

Modified mineral medium (MWMM liquid medium): 1g of ammonium chloride (NH) was weighed in turn₄Cl), 0.2g magnesium sulfate heptahydrate (MgSO)₄.7H₂O), 0.1g of calcium chloride dihydrate (NH)₄Cl.2H₂O), 0.5g dipotassium hydrogen phosphate (K)₂HPO₄) Dissolved in 100mL of ultrapure water and then made up to 1L.

② bidirectional gradient test tube bottom layer culture medium: consists of FeS and MWMM liquid medium in a ratio of 1:1, and 1% (w/v) agarose is added.

③ two-way gradient test tube upper layer culture medium: 0.15% (w/v) agarose was added to the MWMM liquid medium.

Fourthly, bidirectional gradient test tube culture medium: and (3) after the bottom layer culture medium is sterilized, adding 1mL of FeS at the bottom of each test tube while the FeS is hot as a source of ferrous ions, and standing until the FeS is cooled and solidified. Sterilizing the upper layer culture medium, cooling to about 40 deg.C, adding 1% vitamin solution and 1% microelement solution, shaking, and adding NaHCO solution with final concentration of 0.3%₃And (4) uniformly mixing. NaHCO 2₃Provides a carbon source for the growth of the iron oxidizing bacteria and acts as a buffer. Inserting a sterile pipette tip into a sterilized absorbent cotton ball, and connecting to a carbon dioxide (CO) bottle₂Content 30%), add CO to the upper medium₂Adjusting the pH of the medium to 6.0, CO₂And NaHCO₃Forming a buffer system. After the bottom medium in the tubes had completely solidified, 9mL of the top medium was added to each tube, slowly added along the tube wall with a disposable sterile pipette, and then the tubes were moved into a refrigerator for refrigeration, waiting for the top medium to solidify.

3) Experimental treatment

The experimental set was a control group and a treatment group. Control group: the bidirectional gradient test tube culture medium is not processed; treatment group: the bidirectional gradient tube medium was inoculated with strain LLDRA 6. Each treatment of 3 groups was performed in parallel, and the inoculated group was cultured in a 35 ℃ incubator.

2. Phenanthroline spectrophotometry

1) The main reagents are as follows:

ammonium fluoride solution (2 mol/L); 14.816g of ammonium fluoride (NH) were weighed out₄F) Dissolving in ultrapure water, and then fixing the volume to 200 mL; hydrochloric acid solution (6 mol/L): mixing 100mL of concentrated hydrochloric acid (12mol/L) with ultrapure water according to the proportion of 1: 1; ③ ammonium acetate buffer solution: 250g of ammonium acetate (NH) are weighed out₄C₂H₃O₂) Dissolving in 150mL of ultrapure water, then diluting to 1L with glacial acetic acid, and adjusting the pH to 4.2; 0.20% phenanthroline solution: weighing 0.20g of phenanthroline to be dissolved in 100mL of ultrapure water, and adding 2 drops of dilute hydrochloric acid to completely dissolve the phenanthroline (for use in preparation); storage solution of Fe (II): 0.9941g FeCl was weighed₂·4H₂O (AR) dissolved in ultrapure water, and finally the volume is adjusted to 50mL, wherein the concentration of Fe (II) is 0.1 mol/L.

2) Drawing of Fe (II) Standard Curve

2mL of 6mol/L hydrochloric acid, 0, 0.25mL, 0.5mL, 1.25mL, 2mL, 2.5mL of Fe (II) stock solution, and then 2mL of NH₄F, 2mL of phenanthroline and 5mL of ammonium acetate, and finally adding ultrapure water to a constant volume of 25mL to prepare standard solutions with concentration gradients of 0, 1mmol/L, 2mmol/L, 5mmol/L, 8mmol/L and 10 mmol/L. After a reaction time of 15min the absorbance was measured at a wavelength of 512 nm.

3) Sample processing

Setting a control group: 50mL of LB medium was cultured at 180rpm and 35 ℃ for 4 hours, then Fe (II) (Fe (II)) was added to the medium to a final concentration of 8mmol/L, and the treatment group: the strain is inoculated into 50mL LB culture medium, cultured for 4h at 180rpm and 35 ℃, and then added with Fe (II) (Fe (II)) with the final concentration of 8 mmol/L. The control group and the treatment group were placed on a shaker at 180rpm and 35 ℃, 2mL samples were aspirated at different time points, and after centrifugation at 10000rpm and 10min, the supernatant was taken for use. 2mL of hydrochloric acid solution, 1mL of supernatant, 2mL of ammonium fluoride solution, 2mL of 0.2% phenanthroline solution and 5mL of ammonium acetate solution are sequentially added into a 25mL volumetric flask, and finally ultrapure water is added to achieve a constant volume of 25 mL. After being inverted and mixed evenly, the mixture reacts for 15min, the absorbance A is measured at the wavelength of 512nm of a spectrophotometer, and the Fe (II) oxidation rate at different time points is calculated according to the following formula.

C＝3.003A+0.09566

R％＝(C₀-C_t)/C₀×100％

Wherein C is₀Is the initial concentration of Fe (II), C_tThe Fe (II) concentration was determined after various incubation times.

Secondly, the rice root system forms an iron film to prevent the rice plants from absorbing heavy metal cadmium

1) Rice seed pretreatment

Selecting two rice varieties of Taibei 309 and Huazhan, disinfecting the surface of the seeds by using a 3% sodium hypochlorite solution, washing the seeds by using ultrapure water after 15min, and culturing the seeds in a greenhouse with the temperature of 28 ℃ and the relative humidity of 70% until the seeds germinate. And selecting three-week-old seedlings with good growth vigor for transplanting.

2) Preparation of the bacterial suspension

A single colony of the strain LLDRA6 is picked on an LB solid plate and inoculated into an LB liquid medium (11L), and the culture is carried out for 12 to 16 hours at 180rpm and 35 ℃.

3) Experiment of potting

Firstly, collecting cadmium-polluted rice soil (the cadmium content is 0.92mg/Kg), air-drying, sieving by a 200-mesh sieve, filling 5Kg of dry soil into each pot (before planting, 150mg of ammonium nitrogen, 100mg of phosphorus (used as single super phosphate) and 120mg of potassium (used as potassium fertilizer) are applied to each pot), and standing for two weeks.

② four treatments are set

Processing group one: control (no addition);

and a second treatment group: addition of bacterial suspension (OD) only₆₀₀2.0, 50 mL/pot);

and (3) treatment group III: the amount of addition of only Fe (II) (Fe (II)) was 5 mmol/kg;

treatment group four: simultaneously adding Fe (II) and bacterial suspension (Fe (II)) in an amount of 5mmol/kg, and adding bacterial suspension OD₆₀₀2.0, 50 mL/pot).

After the soil treatment, the rice seedlings (3 weeks) were transplanted into pots and cultured.

Third post-processing

After the rice is mature, collecting rice root systems of different treatment groups, and carrying out object photographing and SEM-EDS characterization analysis.

4) Field experiment

Selecting a paddy field (the cadmium content is 0.92mg/Kg) polluted by cadmium, and separating a plurality of paddy fields with the areas of 1m by using ditches²And 5 modules are processed differently.

② four treatments are set

Processing group one: control (no addition);

and a second treatment group: addition of bacterial suspension (OD) only₆₀₀The addition amount is 500mL/m²)；

And (3) treatment group III: the amount of Fe (II) (Fe (II)) added alone was 250mmol/m²)；

Treatment group four: the addition amount of the Fe (II) and the bacterial suspension (Fe (II)) is 250mmol/m²(ii) a Bacterial suspension OD₆₀₀The addition amount is 500mL/m²)。

Third post-processing

After the rice is mature, samples of stem leaves and seed parts are collected, dried for 72 hours at 65 ℃, ground into powder, and the cadmium content in the samples is detected.

Results and analysis

The results of Providencia sp. LLDRA6 iron oxidation capacity are shown in fig. 1 and fig. 2, in the ferrous sulfide gradient tube method, a circle of obvious circular iron oxidation bands are formed in test tubes of a treatment group (inoculated strain LLDRA6), and no obvious change is caused in a control group (not inoculated strain LLDRA 6); in the phenanthroline spectrophotometry, the oxidation rate of the treatment group (simultaneously adding the bacterial suspension and Fe (II)) is higher than that of the control group (only adding Fe (II)), and the result shows that Providencia sp.LLDRA6 has the capability of oxidizing Fe (II)).

In the pot experiment, the real shot image of the root system of the Huazhan variety rice is shown in figure 3 (A: a first treatment group, B: a second treatment group, C: a third treatment group, and D: a fourth treatment group), and the root system of the rice in the fourth treatment group (simultaneously added with bacterial suspension and Fe (II)) has obvious color change and is red iron rust compared with the root systems of the other three treatment groups; the SEM-EDS characterization results are shown in FIG. 4 (A: treatment group I; B: treatment group II; C: treatment group III; and D: treatment group IV), wherein layered precipitates formed on the surfaces of rice roots in the treatment group IV (simultaneously adding bacterial suspension and Fe (II)) are most obvious, and the precipitates are considered to be iron oxide films because the Fe and O contents in the regions of the precipitates are high.

In a field experiment, cadmium content in rice grain and stem leaf samples of each treatment group is detected, and results are shown in fig. 5 and fig. 6, cadmium content of grain and stem leaf parts of two rice varieties of a treatment group four (simultaneously adding bacterial suspension and Fe (II)) is obviously lower than that of other three treatment groups, and cadmium content of grain of Huazhan and Taibei 309 grains in the treatment group four is 0.115mg/kg and 0.042mg/kg respectively, and meets the requirement that grain cadmium residue is less than 0.2mg/kg specified in national standard GB 2762-2012.

Claims

1. The application of providencia bacterial strain in preparing ferrous oxidant is characterized in that the providencia bacterial strain isProvidenciaLldra6, strain accession number CCTCCM 2018876.

2. The application of providencia bacterial strain in preparing rice root system surface iron film forming agent is characterized in that the providencia bacterial strain isProvidenciaLldra6, strain accession number CCTCCM 2018876.

3. The application of providencia bacterial strain in preparing rice root system heavy metal cadmium absorption blocking agent is characterized in that the providencia bacterial strain isProvidenciaLldra6, strain accession number CCTCCM 2018876.

4. The use of claim 3, wherein the providencia bacterial strain is used for preventing the rice root system from absorbing the heavy metal cadmium by oxidizing ferrous iron to form an iron film on the surface of the rice root system.

5. A method for preventing heavy metal cadmium from being absorbed by rice root systems is characterized in that ferrous iron and providencia bacterial strains are simultaneously added into cadmium-polluted rice fieldsProvidenciaA bacterial suspension of lldra 6; OD of the bacterial suspension₆₀₀1.5-2.5, and the addition amount of the ferrous iron is 200-300 mmol/m²The addition amount of the bacterial suspension is 400-600 mL/m²。

6. The method of claim 5, wherein the OD of the bacterial suspension₆₀₀2.0, and the addition amount of the ferrous iron is 250mmol/m²The addition amount of the bacterial suspension is 500mL/m²。