CN113560334B

CN113560334B - Remediation method for cesium-polluted soil

Info

Publication number: CN113560334B
Application number: CN202110871791.5A
Authority: CN
Inventors: 陈晓明; 王丹; 勾家磊; 唐运来; 陈珂
Original assignee: Southwest University of Science and Technology
Current assignee: Southwest University of Science and Technology
Priority date: 2021-07-30
Filing date: 2021-07-30
Publication date: 2022-09-13
Anticipated expiration: 2041-07-30
Also published as: CN113560334A

Abstract

The invention discloses a method for repairing cesium-polluted soil, which belongs to the field of microbial remediation and is characterized in that a microbial compound inoculant is used for repairing the cesium-polluted soil; the microbial compound inoculant consists of bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 1:0.8-1.2:0.8-1.2:0.8-1.2, or bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 0.8-1.2:2:2:2, or bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 2:0.8-1.2:2:0, or bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 0:2:0.8-1.2: 0. According to the method, the microorganisms with high tolerance to Cs are screened firstly, then the screened microorganisms are sieved again through a liquid culture medium, the tolerance of the microorganisms and the removal capacity of the microorganisms to Cs are further investigated, the screened microorganisms are subjected to orthogonal combination to form different microorganism combinations, and the tolerance and the removal rate of the different microorganism combinations are measured.

Description

Method for repairing cesium-polluted soil

Technical Field

The application relates to the field of microbial remediation, in particular to a remediation method of cesium-contaminated soil.

Background

The heavy metal pollution of the soil not only causes great harm to the environment and destroys the ecological balance, but also poses great threat to the human health. The problem of treating soil heavy metal pollution is becoming more and more urgent.

In recent years, uranium and thorium ore mining, uranium ore concentration, nuclear waste treatment, nuclear weapon explosion, nuclear test, coal-fired power plants, phosphate ore mining and processing, and the like have been used. The emissions from atmospheric nuclear tests can cause radioactive contamination of the soil, in which case, ¹³⁷ the half-life period of the Cs is long, the Cs is easy to be adsorbed by soil, and the retention time is long. Cesium contaminates food and water, can be absorbed into the human body through the food chain very easily by digestion, or similar dust is inhaled by the human body through the respiratory tract.

The nuclide pollution conditions of soil at home and abroad are complex and various, the principle of ecological environmental protection of phytoremediation is widely concerned and supported, but the problem that single bioremediation is difficult to solve well still has a plurality of defects, including long period, inconvenient application and the like.

Aiming at the problem of repairing the cesium-containing polluted soil, a corresponding solution is urgently needed.

Disclosure of Invention

The invention aims to provide a method for repairing cesium-polluted soil. The method adopts a solid flat plate primary screen to screen the microorganisms with higher tolerance to Cs, and the screened microorganisms are rescreened through a liquid culture medium to further investigate the tolerance of the microorganisms and the removal capacity to the Cs. And then, forming different microorganism combinations by orthogonal combination of the screened microorganisms, and determining the tolerance and the removal rate of the different microorganism combinations. Further, the method carries out combined remediation on the screened microorganism combination and the screened plant, sets soil with different concentrations of Cs, measures the dry weight and the nuclide enrichment amount of the plant on the upper part and the lower part under different combination states, and selects the optimal plant-microorganism combination.

In order to achieve the purpose, the following technical scheme is adopted in the application:

a method for repairing soil polluted by cesium comprises the steps of applying a microbial compound inoculant to repair the soil polluted by the cesium;

the microbial compound inoculant consists of bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 1:0.8-1.2:0.8-1.2:0.8-1.2, or bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 0.8-1.2:2:2:2, or bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 2:0.8-1.2:2:0, or bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 0:2:0.8-1.2: 0.

The microbial compound inoculant consists of bacillus subtilis: deinococcus radiodurans: bacillus cereus: pseudomonas fluorescens is composed of 1:1:1:1, or Bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 1:2:2:2, or the bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 2:1:2:0, or the bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 0:2:1: 0.

The microbial compound inoculant consists of bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 1:2:2:2, or the bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 2:1:2: 0.

In the process of growing the pasture, the microbial compound inoculant is inoculated to the root of the pasture, and the remediation of the cesium-polluted soil is realized through the combined action of the microbial compound inoculant and the pasture.

The method comprises the following steps:

(1) sowing the grass seeds in the cesium-containing polluted soil to be repaired, and growing the grass seeds to a seedling stage;

(2) inoculating the microbial compound inoculum to a plant root layer; the remediation of the soil polluted by cesium is realized through the combined action of the microbial compound inoculant and the pasture.

Preferably, the pasture is one or more of Sudan grass, Timmosis and hybrid pennisetum;

the microbial compound inoculant consists of bacillus subtilis: deinococcus radiodurans: bacillus cereus: pseudomonas fluorescens is composed of 1:2:2:2, or Bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 2:1:2: 0.

The pasture is sudan grass; the microbial compound inoculant consists of bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 2:1:2: 0.

Drawings

The invention will now be described, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 is a primary screening graph of solid plates with microbial tolerance to Cs at different concentrations.

FIG. 2 is a graph showing the growth of Bacillus subtilis under Cs stress.

FIG. 3 is a graph showing the growth of Bacillus cereus under Cs stress.

FIG. 4 is a graph of growth of deinococcus radiodurans under Cs stress.

FIG. 5 is a graph showing the growth of P.fluorescens under Cs stress.

FIG. 6 is a diagram showing the removing effect of Bacillus subtilis under Cs stress.

FIG. 7 is a graph showing the effect of Bacillus cereus on removal under Cs stress.

FIG. 8 is a graph of the elimination effect of deinococcus radiodurans under Cs stress.

FIG. 9 is a diagram showing the effect of Pseudomonas fluorescens on removal under Cs stress.

FIG. 10 is a graph of growth of combinations of microorganisms under Cs stress.

FIG. 11 is a graph of the effect of microbial combinations on removal under Cs stress.

FIG. 12 is a standard curve of IAA secretion by microorganisms.

FIG. 13 is a standard curve for ACC deaminase secretion by microorganisms.

FIG. 14 shows a graph of the determination of the siderophore capacity of microorganisms under Cs stress.

FIG. 15 is a schematic diagram of a five-point inoculation method.

FIG. 16 is a graph showing the effect of microbial combinations on the dry aerial weight of Sudan grass under Cs stress.

FIG. 17 is a graph showing the effect of microbial combinations on dry weight of the aerial parts of Simplex under Cs stress.

FIG. 18 is a graph showing the effect of microbial combinations on the dry weight of top parts of hybrid pennisetum under Cs stress.

Fig. 19 is a pasture grass growth diagram.

FIG. 20 is a graph showing the effect of microbial combinations on the dry weight of the underground portion of Sudan grass under Cs stress.

FIG. 21 is a graph of the effect of microbial combinations on dry weight of the subsurface of Timochi under Cs stress.

FIG. 22 is a graph showing the effect of microbial combinations under Cs stress on the lower dry weight of hybrid pennisetum.

FIG. 23 is a graph showing the effect of microbial combinations on the enrichment of cesium in the aerial parts of Sudan grass under Cs stress.

FIG. 24 is a graph of the effect of microbial combinations on Cesium enrichment in Ammoxy under Cs stress.

FIG. 25 is a graph showing the effect of combinations of microorganisms on the enrichment of cesium in the top of hybrid pennisetum under Cs stress.

FIG. 26 is a graph showing the effect of microbial combinations on the cesium enrichment capacity of the underground portion of Sudan grass under Cs stress.

FIG. 27 is a graph of the effect of microbial combinations on Cesium enrichment in Thimoxidensin under Cs stress.

FIG. 28 is a graph showing the effect of combinations of microorganisms on the lower cesium enrichment capacity of hybrid pennisetum under Cs stress.

FIG. 29 is a graph of the effect of microbial combinations on total amount of Cesium sudanense enrichment under Cs stress.

FIG. 30 is a graph of the effect of microbial combinations on total enrichment of Timocisic cesium under Cs stress.

FIG. 31 is a graph of the effect of microbial combinations on the total cesium enrichment of hybrid pennisetum under Cs stress.

Detailed Description

All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.

Example 1

1. Material method

1.1 test strains

Based on previous studies, the inventors preliminarily selected the following five types of microorganisms: bacillus subtilis (Bacillus subtilis), Bacillus cereus (Bacillus cereus), Deinococcus radiodurans (Deinococcus radiodurans), Citrobacter (Citrobacter) and Pseudomonas fluorescens (Pseudomonas fluorescens) were used as the research strains.

1.2 solid plate prescreening of cesium tolerant microorganisms

Respectively setting the Cs concentration to be 0, 200 and 500mg L ^-1 Three-gradient TGY solid culture medium, culturing the microorganism obtained by primary screening at 30 deg.C under shaking, and measuring OD with ultraviolet spectrophotometer (analytikjena 200plus) ₆₀₀ At 0.8, 40. mu.L of the solution was applied to a solid plate. And (3) culturing the control group and the experimental group together under the same condition for 24h, observing the growth condition of the microorganisms, and comparing the growth condition with the control group to select the microorganisms with nuclide tolerance.

1.3 liquid rescreening of cesium tolerant microorganisms

And (3) culturing the microorganisms obtained by screening the solid plate in liquid culture media containing different heavy metal concentrations, and determining the OD value of the bacteria growth and the content of heavy metal in the culture media at the same time interval so as to evaluate the tolerance of the microorganisms to the heavy metal and the capability of removing the heavy metal in the liquid.

1.3.1 growth curves for cesium tolerant microbial fluid rescreening

Setting the concentration gradient of Cs as 0, 20, 50, 100, 200 and 500mg L ^-1 The three groups of the liquid culture medium are arranged in parallel for each sample, 134mL of corresponding blank culture medium is added into a 250mL triangular flask, 15mL of bacterial liquid with OD value of 0.8 measured under the condition of 600nm is added, and the mixture is uniformly mixed. 120r min at 30 DEG C ^-1 2mL of culture solution is taken every 24h, and the OD value is measured at the position of 600nm until the growth of the microorganisms stops and the OD value of the bacterial solution is kept unchanged or reduced. Taking 6mL of bacterial liquid at 3000r min ^-1 Centrifuging for 10min, taking supernatant for digestion, and measuring the concentration of nuclide in the supernatant by an atomic absorption spectrometer.

1.3.2 removal Rate of Cesium tolerant microbial liquid rescreening

And (3) centrifuging 6mL of bacterial liquid while measuring the OD value of the bacteria, adding 5mL of supernatant into a digestion tank, adding 2.5mL of perchloric acid and 5mL of nitric acid, digesting at 180 ℃, filtering, and fixing the volume in a 25mL volumetric flask. The Cs concentration of the digestion solution is measured by atomic absorption spectroscopy (Perkin Elmer AA700), and the nuclide removal rate of the microorganism to the liquid culture medium is calculated.

1.4 screening of combinations of microorganisms for Cesium tolerance and removal Rate

Through screening of tolerance and removal capacity of Cs, bacillus subtilis, deinococcus radiodurans, bacillus cereus and pseudomonas fluorescens are selected as the later-stage research strains of the Cs. At a Cs concentration of 100mg L ^-1 Under the condition, the growth curve and the removal rate of the microbial combined flora are finally obtained by adopting four-factor three-parallel orthogonal experimental research.

TABLE 1 Cs-tolerant microorganism combinatorial screening orthogonal experimental table

In table 1, strains 1 to 4 are respectively bacillus subtilis, deinococcus radiodurans, bacillus cereus and pseudomonas fluorescens; test numbers A-I are combinations of 9 different microorganisms.

And selecting four groups of optimal microorganism combinations according to the tolerance condition of the microorganisms to nuclide and the removal capability of the microorganisms to the nuclide, and carrying out later-stage microorganism-plant combined repair research.

1.5 ability of microorganisms to combine secretion-promoting indices

The ability of the microorganism to secrete a combination of IAA, ACC deaminase and siderophore was determined by different methods.

1.5.1 analysis of the ability of a combination of microorganisms to produce IAA

Adopting IAA to draw a standard curve, preparing two groups of IAA solutions with the concentration of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 and 17.5mg L ^-1 And 25, 50, 75, 100, 125, 150, 175mg L ^-1 Respectively taking 4mL of IAA, adding 4mL of PC colorimetric solution into the first group, and adding S into the second group ₂ 4mL of colorimetric solution, standing in the dark for 0.5h, taking out immediately, and measuring OD by using a spectrophotometer ₅₃₀ And adjusting the value to 0 by adding distilled water of colorimetric solution, repeating the operation for three times to obtain data, and drawing a standard curve.

1.5.2 analysis of the Capacity of the combination of microorganisms to produce ACC deaminase

And (3) determining the activity of the deaminase by using an ACC (ACC) colorimetric method based on ninhydrin. Drawing a standard curve: take 0, 50, 100, 200, 300, 400 and 500ug mL ^-1 To 5mL of each of the ACC solutions in 25mL of a colorimetric tube, 1mL of 0.5% ninhydrin reagent was added, and the mixture was shaken well with a stopper. Placing in water bath at 90 deg.C for 20-25min, cooling to room temperature, and measuring OD with model 721 spectrophotometer ₅₃₀ And (4) optical density value. A calibration curve was prepared using the optical density and concentration of the standard solution.

1.5.3 analysis of the capability of the combination of microorganisms to produce siderophores

0.3640g of cetyltrimethylammonium bromide was dissolved in 100mL of distilled water to obtain 10mmol L ^-1 0.0164g of ferric chloride was dissolved in 100mL of distilled water to obtain 1mmol L of the solution (1) ^-1 The solution (small amount of diluted HCI can be added) is firstly dissolved with ethanol for 0.1210g of CAS, then deionized water is used for constant volume to 100mL, and finally CAS detection solution is prepared.

The dominant microorganism combination is added into MKB culture medium at 28 ℃ for 150r min ^-1 Culturing for 48 h; using a centrifuge for 3500 rpm ^-1 Centrifuging for 15min, taking supernatant for detection, mixing the supernatant with 3mL CAS detection solution, standing for 1h, determining absorbance (A) at 630nm wavelength by using a 722-type spectrophotometer after reaction is completed, and zeroing with double distilled water and contrast; and simultaneously, taking 3mL of CAS detection solution and 3mL of supernatant of uninoculated MKB liquid culture medium, fully and uniformly mixing, and determining the light absorption value by the same method to obtain a reference value (Ar).

The ratio of A to Ar is used as a quantitative index for comparing the secretion of siderophores by various microorganisms. The smaller the ratio of A to Ar, the more the amount of secretion of siderophore.

2. Results and analysis

2.1 Cesium tolerant microbial screening

Culturing bacillus subtilis, deinococcus radiodurans, bacillus cereus and pseudomonas fluorescens in a culture medium containing Cs, respectively carrying out solid plate primary screening, liquid secondary screening and removal rate detection, and finally detecting and screening the tolerance and removal rate of four microorganisms.

2.1.1 solid plate prescreening of cesium tolerant microorganisms

Inoculating Bacillus subtilis, Bacillus cereus, deinococcus radiodurans, Citrobacter and Pseudomonas fluorescens to 40 μ L of the mixture with the concentrations of 0, 200 and 500mg L respectively ^-1 Cs ⁺ The strain was cultured for 24 hours in TGY solid medium, and the cesium tolerance of 5 microorganisms was examined, and the results are shown in FIG. 1.

The tolerant growth conditions obtained after primary screening of the four microorganism solid plates are shown in figure 1 (figure 1 is a primary screening graph of the tolerant solid plates of the microorganisms to Cs with different concentrations; in figure 1, a corresponds to bacillus cereus, b corresponds to deinococcus radiodurans, c corresponds to bacillus citreus, d corresponds to bacillus subtilis, and e corresponds to pseudomonas fluorescens).

In the figure, Bacillus cereus, radiation resistant200 and 500mg L of deinococcus kirilowii and bacillus subtilis ^-1 Cs ⁺ Compared with the CK group, the experimental group has no obvious difference, which indicates that 3 microorganisms have Cs ⁺ Has good tolerance. FIG. 1c 500mg L of Citrobacter ^- ¹ Cs ⁺ The colonies on the experimental group were few compared to 200mg L ^-1 Cs ⁺ The experimental group and the CK group show that the citrobacter can treat low-concentration Cs ⁺ Has certain tolerance, and Cs is present at high concentration ⁺ Has strong inhibition on the growth of the citrobacter. The tolerance of the pseudomonas fluorescens to Cs is lower than that of a, b and d, but the pseudomonas fluorescens is found to have good capability of promoting the growth of plants and enhancing the enrichment of nuclides of the plants through consulting documents. Therefore, Pseudomonas fluorescens was selected for further screening experiments. And finally, adopting bacillus cereus, deinococcus radiodurans, bacillus subtilis and fluorescent pseudomonas as the re-screening experimental strains.

2.1.2 liquid rescreening of cesium tolerant microorganisms

Adding four microorganisms obtained by primary screening into a sample containing Cs with concentration gradient of 0, 20, 50, 100, 200, 500mg L ^-1 The liquid TGY medium of (1) was cultured, and the absorbance and the content of nuclides Cs in the medium were measured at 600nm every 24 hours.

The growth of the bacillus subtilis is promoted in liquid culture media containing different cesium concentrations, and the bacterium concentration reaches the highest value at 72 hours. After bacillus subtilis grows for 72 hours, the concentration is 200mg L ^-1 The bacteria concentration is highest in the environment of (2). The growth conditions of the bacillus subtilis in five cesium-containing culture media with different concentrations are better than that of a blank culture medium in 72 hours, and the bacillus subtilis has high tolerance to cesium.

Bacillus cereus in cesium 100mg L ^-1 、200mg L ^-1 The growth was fastest, reaching a maximum at 72 h. The other three concentrations and the blank all began to level and decline at 48 h.

The deinococcus radiodurans can also grow normally in a solution of Cs, wherein the concentration is 100mg L ^-1 And 200mg L ^-1 At a concentration of (A), the growth is best, and at 72h, the growth is sufferedThe bacteria concentration is gradually reduced by inhibition.

The growth of the pseudomonas fluorescens is inhibited to a certain extent under the concentration of Cs, wherein 20mg L ^-1 And 50mg L ^-1 The growth conditions of (2) are relatively good. However, P.fluorescens has a very good plant growth promoting effect, and therefore P.fluorescens is still selected as an alternative strain.

Four kinds of microorganisms were inoculated in L containing Cs at a concentration of 0, 20, 50, 100, 200, 500mg ^-1 The culture is carried out in the liquid culture medium, the light absorption value at 600nm is measured once every 24 hours by using a spectrophotometer, and the influence of the concentration of Cs on the growth of microorganisms is researched; 6mL of bacterial liquid is taken, centrifuged and 5mL of supernatant is added into a digestion tank, 2.5mL of perchloric acid and 5mL of nitric acid are added for digestion at 180 ℃, the digestion solution is uniformly measured by a flame Atomic Absorption Spectrophotometer (AAS), and the result of sample liquid atomic absorption spectrum measurement Cs is shown in FIGS. 6, 7, 8 and 9.

The removal capacity of the four microorganisms to Cs is 20mg L ^-1 Is the best at Cs concentration of (2), since 20mg L ^-1 The concentration base number in the concentration environment of (2) is small, and thus the removal rate is high. However, the pair of Bacillus subtilis, Bacillus cereus and deinococcus radiodurans was 500mg L ^-1 The removal effect of (A) was significantly enhanced, and the preliminary analysis was probably due to the Cs concentration of 500mg L ^-1 In the process, the toxicity to the microorganisms is strong, the threshold value which can be born by the microorganisms is broken through, the cell structure of the microorganisms is damaged, and the Cs enter the cells, so that the removal capacity of the Cs is improved.

2.2 construction of a combination of cesium-tolerant microorganisms

The four Cs-tolerant microorganisms were introduced into Table 1 to obtain an orthogonal combination, the combination was mixed and cultured at a certain ratio, and the growth curve of the microorganism combination and the concentration of the nucleic acid in the medium were measured every 12 hours, starting from 24 hours.

The microbial combination continuously grows under Cs stress, wherein A, B, D combination grows better, the OD value of A combination is still in an ascending stage at 72h, and the OD value of D combination is always at the highest value before 48 h. B. The H combination grows best at 60H, and the C combination is weak in the whole growth process.

FIG. 11 shows the elimination effect of the combination of microorganisms under Cs stress.

As can be seen from FIG. 11, the microbial composition was 100mg L ^-1 The A, B combination has stronger removal capability to Cs in the environment at the Cs concentration of (3), and the D, H combination has higher removal rate at 72 h. In the application, the removal rate of the B combination reaches about 32% at 24h at most. The removal rate of all combinations to Cs is within 35%, and the content of Cs in the culture medium is unstable, which may be caused by death of microorganisms due to high toxicity of Cs to the microorganisms, and the concentration of Cs in the culture solution is increased due to release of enriched Cs by cell disruption.

2.3 ability of the microorganism to secrete IAA, ACC deaminase and siderophore in combination

2.3.1 capability of the microorganism to secrete IAA in combination

According to the method 1.5.1, the concentration of indoleacetic acid (IAA) standard solution is plotted as the abscissa (x), OD ₅₄₀ Drawing a standard curve for the ordinate (y) to obtain a low concentration range regression equation: y is 0.0104x-0.0070, R ² 0.9995; the regression equation for the high concentration range is 0.0205x-0.5516, R ² ＝0.9993。

FIG. 12 shows a standard curve of IAA secretion by microorganisms; in fig. 12, a is a low concentration IAA standard curve, and B is a high concentration standard curve.

Respectively adding A, B, D, H combinations with strong tolerance to Cs obtained by screening into culture medium for culture, respectively reacting with two colorimetric liquids, substituting the measured OD value into the IAA standard curve in figure 12, and measuring the light absorption value in the S interval ₂ In the range of (1), S is selected ₂ And marking the song. The results show that 4 microbial combination flora all have the capability of secreting IAA. Measuring absorbance at S ₂ In the range of (1), so S is also selected ₂ The results are shown in Table 2. The IAA producing capacity of the four microorganism combinations is not greatly different, wherein the combination A has the strongest capacity, and the IAA producing amount is 59.2667mg L ^-1 The rest three groups are 52mg L ^-1 Left and right.

TABLE 2 IAA-producing ability of combinations of microorganisms under Cs stress

2.3.2 ability of the microorganism to combine secretion of ACC deaminase

According to the method in 1.5.2, the capability of the microorganism to produce ACC deaminase is detected, and an ACC standard curve graph is drawn, wherein the regression equation is that y is 0.0011x +0.0297, and R ² 0.9993. And (4) measuring a light absorption value of the screened cesium-resistant microorganism combination by using the same method, and substituting the light absorption value into a standard curve to calculate the ACC secretion amount.

FIG. 13 shows the standard curve for ACC deaminase secretion by microorganisms. According to the ACC standard curve of FIG. 13, it can be calculated that the difference of the ACC deaminase-producing ability of the microorganism is large, wherein the ACC content in the culture medium measured by the combination H is minimum, which proves that the ACC deaminase-producing ability is strong, the ACC deaminase-producing ability of the combination A is poor, and the combination is B, D times.

TABLE 3 ACC deaminase producing ability of combinations of microorganisms under Cs stress

2.3.3 ability of microorganisms to secrete siderophores in combination

As the value of the absorbance value (Ar) of the detection liquid is larger, the smaller the A/Ar value is, the stronger the siderophore production capacity of the strain is.

As can be seen from FIG. 14, the four microorganisms all have a strong ability to produce siderophores in combination, wherein combination A has the strongest ability to produce siderophores, and combination B has relatively poor ability to produce siderophores in combination H.

3. Total analysis

3.1 tolerance and removal of nuclides by microorganisms

The main reason why the removal rate of the Cs by the microorganism in the culture medium is low is that Cs and K, Na both belong to the elements of the first group of the periodic table of elements and have similar chemical properties, so that when K, Na ions in the environment are more, the removal capability of the Cs by the microorganism is affected, and when the culture medium is prepared, NaCl is usually added, so that sufficient Na ions may have a certain effect on the removal of the Cs by the microorganism, and the biological toxicity of Cs is stronger than Sr, the toxic action on the microorganism is relatively stronger, and therefore, the removal capability of the Cs by the microorganism is also affected.

3.2 nuclide removal ability of microorganism combination

The growth curve inhibition of the Cs-resistant microbial combination starts from 24h, the growth curve inhibition keeps an ascending trend, the overall trend is stable until 60h, the removal capacity is overall low, and the removal rate is below 40%.

3.3 ability of the microorganism to secrete IAA, ACC deaminase and siderophore in combination

The capability of producing IAA of the microorganism combination obtained by screening of the invention is 50mgL ^-1 Above, obviously higher than the prior art.

In the microbial combination, except that the ACC deaminase secretion capability of the H combination is poor, the ACC deaminase secretion capabilities of other combinations are strong, wherein the A, B combination capability is the strongest and is 497.4058 and 588.1834mg L respectively ^-1 。

The chelate of the iron carrier and the Fe can be directly absorbed and utilized by plants, and the growth of the plants is promoted. The iron carrier can also dissolve cadmium in soil while generating chelation on common metal, which shows the importance of judging the content of the iron carrier. The microorganisms of the invention have strong capability of secreting siderophores, which shows that the microorganisms have good effect.

3.4 summary knot

(1) The combined growth profile from the Cs-resistant microorganisms gives: A. b, D combination grew better, and the OD value of D combination was always at the highest value before 48 h. The removal rate of the microbial combination under Cs stress shows that the removal rate of D, H combination is higher than that of other combinations at 72h, and the removal rate of B combination reaches about 32% at 24 h.

The advantageous combination of Cs is: a (bacillus subtilis: radioresistant deinococcus: bacillus cereus: pseudomonas fluorescens: 1:1:1), B (bacillus subtilis: radioresistant deinococcus: bacillus cereus: pseudomonas fluorescens: 1:2: 2), D (bacillus subtilis: radioresistant deinococcus: bacillus cereus: pseudomonas fluorescens: 2:1:2:0), H (bacillus subtilis: radioresistant deinococcus: bacillus cereus: pseudomonas fluorescens: 0:2:1: 0).

(2) The combined growth profile from the Cs-resistant microorganisms gives: A. b, D combination grew better, and the OD value of D combination was always at the highest value before 48 h. The removal rate of the microbial combination under Cs stress shows that the removal rate of D, H combination is higher than that of other combinations at 72h, and the removal rate of B combination reaches about 32% at 24 h. A. B, D, H all four combinations showed good results in terms of their ability to secrete IAA, ACC deaminase and siderophores.

4. Microbial-forage grass combined remediation of cesium-contaminated soil

On the basis, a plant-microorganism combination is established to carry out the plant-microorganism combined remediation research of the Cs-polluted soil.

4.1 materials and methods

4.1.1 pasture and microorganism combinations

The inventor screens three kinds of pasture with strong tolerance and high enrichment capacity by investigating the tolerance and the enrichment capacity of different pasture to Cs: sudan grass (Sorghum Sudanense (Piper) Stapf.), Proteus (Phleum pratense L.), and Pennisetum hybridum (Pennisetum americanum. times. Pennisetum purpureum cv.).

The Cs-resistant microbial combination is as follows: a (bacillus subtilis: radioresistant deinococcus: bacillus cereus: pseudomonas fluorescens: 1:1:1), B (bacillus subtilis: radioresistant deinococcus: bacillus cereus: pseudomonas fluorescens: 1:2: 2), D (bacillus subtilis: radioresistant deinococcus: bacillus cereus: pseudomonas fluorescens: 2:1:2:0), H (bacillus subtilis: radioresistant deinococcus: bacillus cereus: pseudomonas fluorescens: 0:2:1: 0).

And (3) combining the three pastures with Cs resistance with the four screened microorganism combinations respectively, inoculating the microorganism bacterium liquid to the root of the plant in the growth process of the pastures, and researching the influence of the microorganism combinations on the biomass and the enrichment capacity of the pastures.

4.1.2 preparation of the Material

4.1.1.1 preparation of Cesium contaminated soil

Pass meterThe Cs concentration of the preparation is 20, 50 and 100mg kg ^-1 The soil of (2). The stirring method comprises the following steps: weighing a certain amount of CsCl by calculation, dissolving the solid in deionized water, uniformly stirring the solid and farmland soil together, standing for 1 month, and adding water into the soil every ten days for secondary mixing during the standing period. And after the standing period is finished and the soil is naturally air-dried, putting the soil into flowerpots, wherein 1kg of soil is filled in each flowerpot.

4.1.1.2 culture of microorganism combinations

Respectively culturing Bacillus subtilis, Bacillus cereus, deinococcus radiodurans and Pseudomonas fluorescens in TGY culture medium until bacterial OD ₆₀₀ When the concentration reaches 1.0, the bacterial liquids are mixed and shaken up according to the proportion to respectively obtain A, B, D, H combinations.

TABLE 4 Cs repair dominance microorganism combination table

4.1.2 grass planting and growth management

And (3) selecting full seeds, soaking the seeds in a 5% sodium hypochlorite solvent for 8min, repeatedly washing the seeds with deionized water, and then soaking the seeds in warm water for 24 h. And uniformly sowing the soaked seeds in a flowerpot, controlling the humidity and temperature required by the germination of the seeds, and observing the germination condition. After the seeds germinate, selecting 25 grass seedlings with better growth vigor for fixing plants, pulling out seedlings with poor growth vigor and over dense seedlings, irradiating 12 hours each day by using a fluorescent lamp, and watering each pot of pasture with the same amount of water regularly to keep the most suitable growth conditions of the pasture.

4.1.3 inoculation of microorganisms

When the pasture grass plants grow to a seedling stage (the average plant height of the plants is 3cm), the screened microorganism combination is injected to the root layers of the plants by an injector, the five-point inoculation method is adopted, bacteria liquid is injected uniformly in holes, the injection depth is 5cm, the bacteria liquid is injected once every 5 days, 20mL of the bacteria liquid is injected each time, the total injection amount is 4 times, and the total amount is 80 mL. Wherein, the schematic diagram of the five-point inoculation method is shown in FIG. 15.

4.1.4 harvesting and processing of samples

Harvesting the pasture before heading when the pasture grows until the overground part is not higher any more, slightly kneading the soil at the root of the plant by hands, completely taking out the plant together with the underground part, washing the soil at the root by water, separating the underground part from the overground part, carrying out enzyme deactivation treatment in a drying oven at 105 ℃ respectively, crushing the sample into powder by using a mortar after the completion, weighing 0.3g of the powder, and digesting in a microwave digestion instrument (Anton Paar Multiwave PRO). The digested sample was measured for Cs content by flame atomic absorption spectrophotometer (Perkin Elmer AA 700).

Calculating each index of the measured data through a formula, wherein the calculation formula of each index is as follows:

plant Nuclide content (NC, mg/kg) ═ dry weight above ground x Nuclide content above ground + dry weight below ground x Nuclide content below ground)/(dry weight above ground + dry weight below ground;

enrichment (biocontrol yield, BCQ, mg/pot) i.e. dry weight per pot of plants x phytonuclide content;

enrichment factor (BCF) plant nuclide content/actual soil nuclide content;

transfer coefficients (TF) are the amount of aboveground nuclides enriched in a plant/amount of underground nuclides enriched in a plant.

4.2 analysis of results

4.2.1 Effect of the combination of microorganisms on the Dry weight of grass

The dry weight data of the pasture which is obtained by deactivating enzymes after harvesting the pasture, removing water in the pasture and respectively weighing different parts of different pastures is shown in figures 16, 17 and 18.

FIG. 19 shows a diagram of pasture growth; wherein, a: growth conditions under different concentrations after combinations of the sumicio inoculations D; b: the pennisetum alopecuroides is inoculated with the combination A and then grows under different concentrations; c: the dosage of the drug is 100mg kg ^-1 Differences (CK) after inoculation of different combinations of microorganisms.

It was found that different Cs concentrations had a greater effect on the biomass of the upper part of three pastures, of which three pastures had a Cs concentration of 20mg kg ^-1 The biomass is lowest, and the Cs concentration of Sudan grass and Timmoxi is 100mg kg ^-1 Also showed strong growth ability, as shown in FIG. 19: (a) As shown. Sudan grass in 20mg kg ^-1 And 50mg kg ^-1 When the growth is inhibited, the Cs concentration reaches 100mg kg ^-1 The biomass increased again and approached the 0 concentration control group; simexi has Cs concentration of 50 and 100mg kg ^-1 The biomass is obviously higher than 20mg kg ^-1 And exceeds the 0 concentration control group, which shows that the medium-high concentration Cs has a certain promotion effect on the growth of the temmoxy; as shown in fig. 19(b), the biomass of pennisetum alopecuroides under stress of three concentrations was lower than that of the 0 control group, indicating that pennisetum alopecuroides is poor in tolerance to Cs, resulting in inhibition of pennisetum alopecuroides growth under Cs stress.

The increase in grass biomass is evident by the addition of the combination of microorganisms as shown in fig. 19 (c). For the 0 concentration, the biomass of the experimental groups was significantly increased after the addition of the microbial combination. CK group of Simexi is 20, 50, 100mg kg ^-1 The biomass was lower than that of the 0 concentration control group, but 50 and 100mg kg of the microorganism were inoculated after the combination of four microorganisms ^-1 The biomass of the experimental group exceeded that of the 0 control group. After four combinations are added to the root of Sudan grass, the combination B is 0, 50 and 100mg kg ^-1 Are higher than the other four groups, thus showing that the combination B has obvious positive effect in promoting the increase of the biomass of the sudan grass. In tomorsis, the A, B, D combination has a strong effect on increasing tomorsis biomass at four concentrations, so the three microorganism combinations are the dominant combination for increasing tomorsis overground dry weight. The Chinese pennisetum is 20, 50 and 100mg kg ^-1 The lower dry weight was inhibited, but overall the A, D combination had better promoting effect in the four concentrations.

FIG. 20, FIG. 21, FIG. 22 are sequential graphs showing the effect of combinations of microorganisms on the lower dry weight of Sudan grass, Thimocksia, and hybrid pennisetum under Cs stress.

The influence of Cs on the underground dry weight is small, and the dry weight average of the lower part of Sudan grass under four concentrations is stable and is not promoted or inhibited; the dry weight of underground part of the Simmonxi is influenced by Cs and is consistent with that of the overground part, and the dry weight of underground part is inhibited by three concentrations of Cs and 20mg kg ^-1 When receivingThe inhibition is strongest at 50 and 100mg kg ^-1 The method can obtain a tiny promoting effect in time, but the overall change is not large; the biomass of the lower part of the pennisetum setosum is smaller compared with the Sudan grass and the Timmosis, and the experiment group shows a slight inhibition effect compared with the 0-concentration control group under the stress of Cs.

The effect of the microbial combination on the underground dry weight is also significant, and the A, B, D combinations in the Sudan grass 0 concentration control group all promote the increase of the lower biomass of the pasture, wherein the B combination has the best effect and is 0, 20 and 50mg kg ^-1 The promoting effect on the underground biomass of the sudan grass is strongest when the concentration reaches 100mg kg ^-1 Then, the four combinations all have the promotion effect on the underground biomass, wherein A, D, E has the strongest promotion capability. In the Tmochi, the capacity of the combination of four microorganisms is more prominent, and the combination of microorganisms produces a significant promoting effect on the dry weight of the lower part of the pasture in all concentrations, at 0 and 20mg kg ^-1 The promoting effect of the A combination is most obvious, and is 50 to 100mg kg ^-1 The effect of the combination was most pronounced at B, D, where the D combination was 100mg kg ^-1 The dry weight of the subsurface in Kyoxi is 3 times that of the CK group. The weight of the pennisetum is 0 to 20mg kg ^-1 The composition has good curative effect of B, D, and the dosage is 50mg kg or 100mg kg ^-1 The combination of D, E showed a better calming effect.

4.2.2 Effect of microorganism combinations on pasture enrichment efficiency

FIG. 23, FIG. 24, and FIG. 25 are the effect of combinations of microorganisms on the enrichment of cesium in sudan grass, Thimocksia, and hybrid pennisetum grasslands, respectively, under Cs stress.

The microbial combination has obvious promoting capacity on the increase of the dry weight of the pasture, but has certain inhibiting effect on the capacity of enriching Cs on the upper part of the pasture, except for 100mg kg of pennisetum alopecuroides ^-1 The enrichment capacity of the four combinations is approximately equal to that of CK, the B combination is slightly stronger than that of CK, and the enrichment capacity of the other experimental groups is inhibited by the microbial combination.

As shown in FIG. 26, FIG. 27 and FIG. 28, the amount of Sudan grass is 20mg kg ^-1 At Cs concentration of grass, none of the four combinations showed any promotion of the grass's ability to enrich CsThe action is carried out. At 50mg kg ^-1 The A, B combination showed an accelerating effect on the sudan grass enrichment capacity, while the D, H combination produced a relative inhibiting effect. At 100mg kg ^-1 The combination A, D had the effect of promoting enrichment capacity, while the combination B, H produced an inhibitory effect. In temmoxy, the four combinations do not contribute to the positive grass-enriching capacity. In the field of hybrid pennisetum 100mg kg ^-1 The enrichment capacity of the pasture to the Cs is enhanced after the combination A is inoculated under the Cs concentration.

4.2.3 Effect of microbial combinations on Individual Pot enrichment of pasture

The single-pot enrichment refers to the mass of all Cs which can be enriched in unit mass of polluted soil by pasture plants at one time. The method is the most direct embodiment of the grass on the Cs pollution capacity through the sum of the dry weight of each part of the plant and the product of the enrichment concentration of each part of the plant. The results of the study are shown in FIGS. 29, 30 and 31, and the combinations of the four microorganisms used in the study were 0mg kg and 50mg kg at low concentrations ^-1 When Cs is present, the grass growth inhibitor has an inhibiting effect on the enrichment capacity of pasture, and the amount of Cs is 100mg kg ^-1 In this case, the combination D has a promoting effect on the enriching ability of all three kinds of pasture. In 100mg kg of Sudan grass ^-1 After the combination D is inoculated under the concentration, the enrichment capacity of the sudan grass is improved to a certain degree, and the enrichment amount of the Sudan grass to Cs can reach 1.2mg kg ^-1 . In the prescription of 10mg kg ^-1 After the root is inoculated with the microorganism combination, D, H combination can improve the capability of grass to enrich Cs, and A, B, D, H four combinations in hybrid pennisetum can be 100mg kg ^-1 The enrichment capacity of the Cs is improved. Where the promoting ability of the combination D is strongest. Thus the combination D is the optimal combination to promote Cs in pasture rich soil.

Research shows that the inoculated microorganism combination has great promotion effect on the dry weight of the pasture, can obviously increase the dry weight of the overground part and the underground part of the pasture, but is between 20 and 50mg kg ^-1 The Cs in the environment has an inhibiting effect on the capability of pasture to enrich Cs, and the content of the Cs is 100mg kg ^-1 Different pasture grasses and different combinations thereof can generate certain promoting effect after being combined. Overall sudan-D combination is the optimal combination to remove Cs.

TABLE 5 enrichment parameters for various plant-microorganism combinations Cs

Note: CK is control with corresponding concentration of no microorganisms; GCC is the cesium content (mg kg) of the above-ground plant ^-1 ) (ii) a UCC is underground plant cesium content (mg kg) ^-1 ) (ii) a CC represents plant cesium content (mg kg) ^-1 ) (ii) a BCQ is the cumulative amount per basin (mg pot) ^-1 ) (ii) a TF is the transfer coefficient; BCF is the enrichment factor.

4.3 summary of

In the application, the dry weight of the overground part and the underground part of the experimental group added with the microorganism combination is obviously higher than that of the CK group without the microorganism, so that the dry weight of the pasture can be obviously improved after the microorganism combination acts on the roots of the plants.

In the application, the transfer coefficient and the enrichment coefficient of the pennisetum alopecuroides under the same concentration are respectively 2.047 +/-0.298 and 5.817 +/-0.469, the transfer coefficient and the enrichment coefficient are respectively 3.272 +/-0.291 and 6.071 +/-0.188 after the combination of the added H, and the transfer coefficient and the enrichment coefficient are improved after the microorganism is added.

The invention is not limited to the foregoing embodiments.

Claims

1. The method for repairing the cesium-polluted soil is characterized in that a microbial compound inoculant is used for repairing the cesium-polluted soil;

in the growth process of the pasture, the microbial compound inoculant is inoculated to the root of the pasture, and the remediation of the cesium-polluted soil is realized through the combined action of the microbial compound inoculant and the pasture;

the pasture is sudan grass; the microbial compound inoculant consists of bacillus subtilis: deinococcus radiodurans: bacillus cereus: the pseudomonas fluorescens is composed of 2:1:2: 0;

the Cs concentration in the cesium-polluted soil is 100mg kg ^-1 。