CN115181701B - Phosphate-solubilizing bacterium and method for solidifying U (VI) by using same - Google Patents
Phosphate-solubilizing bacterium and method for solidifying U (VI) by using same Download PDFInfo
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- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
The invention provides a phosphate-solubilizing bacterium, the name of which is: pantoea (Pantoea sp.) GRINML12, accession number: china center for type culture Collection, address: the preservation date of the university of Wuhan in Chinese is: 11 months and 20 days in 2019, the preservation number is: cctccc NO: m2019960 the insoluble inorganic phosphorus source is used as free phosphate, U (VI) in uranium pollution system and the free phosphate generate coprecipitation reaction to generate stable uranyl phosphate mineral (HUO) 2 PO 4 ，Ca(UO 2 ) 2 (PO 4 ) 2 ) Etc.). The U (VI) with high toxicity, easy migration, strong bioavailability and radioactivity can be effectively solidified through mineralization, so that the treatment of uranium pollution is realized.
Description
Technical Field
The invention relates to the field of microorganism treatment of heavy metal pollution, in particular to a phosphate solubilizing bacterium and a method for solidifying U (VI) by using the phosphate solubilizing bacterium.
Background
Along with the great development of global nuclear energy, the demand of uranium resources is increasingly increased, a large amount of uranium-containing wastes are generated in the processes of mining, dressing, smelting and processing and utilizing uranium ores, and according to incomplete statistics, the amounts of waste stone and tailings generated in the mining of the uranium ores are respectively about 2.8x10 7 t and 3X 10 7 t. The uranium-containing wastewater is diffused to the surrounding environment to form uranium-containing polluted soil and wastewater due to unreasonable stockpiling and management of the huge amount of uranium-containing pollutants, and meanwhile, the uranium pollution has the characteristics of wide range, long duration, concealment and the like, and can form a long-term potential threat to human health and environment. Uranium mainly exists in the form of U (IV) and U (VI) in nature, wherein U (VI) has the characteristics of higher toxicity (chemical toxicity and radiation toxicity), easy migration, stronger bioavailability and the like compared with U (IV); along with the enrichment of food chains to human bodies, the food chains have different degrees of injury to the livers, kidneys and bones of the human bodies, serious uranium poisoning even induces various diseases or causes gene mutation, organism distortion and even canceration, and the serious uranium poisoning causes great threat to the health of the human bodies. Therefore, the key to the remediation of uranium pollution is the remediation of U (VI), and the remediation of uranium-containing contaminated sites by curing U (VI) in the environment has become one of the major problems faced by environmental protection efforts.
At present, various technologies for treating uranium pollution are reported, wherein microbial solidification is considered to be an ideal uranium pollution adsorption technology, but how to obtain uranium-tolerant microorganisms capable of stably solidifying uranium pollutants is still a problem to be solved.
Disclosure of Invention
In order to solve the above problems, a first object of the present invention is to provide a phosphate-solubilizing bacterium which can secrete a phosphate-solubilizing enzyme during self-metabolism and dissolve a poorly soluble inorganic phosphorus source (ground phosphate rock, calcium phosphate, etc.) into a phosphate in a free state.
A second object of the present invention is to provide a screening medium for screening the above-mentioned phosphate-solubilizing bacteria.
A third object of the present invention is to provide a phosphate solubilizing medium for culturing the above phosphate solubilizing bacteria.
A fourth object of the present invention is to provide a method for solidifying U (VI) by phosphate solubilizing bacteria, which comprises co-precipitating the phosphate dissolved by phosphate solubilizing bacteria in free form with U (VI) to form a stable uranyl phosphate mineral, such as HUO 2 PO 4 ，Ca(UO 2 ) 2 (PO 4 ) 2 ) And so on, thereby reducing the radioactivity of U (VI), preventing the expansion of pollution range caused by the continuous diffusion of U (VI) to the periphery, and preventing the utilization of animals and plants.
In order to achieve the above object, the present invention provides a phosphate solubilizing bacterium, which has the classification name: pantoea (Pantoea sp.) GRINML12, accession number: china center for type culture Collection, address: the preservation date of the university of Wuhan in Chinese is: 11 months and 20 days in 2019, the preservation number is: cctccc NO: m2019960.
The invention also provides a screening culture medium for screening the phosphate solubilizing bacteria, which comprises the following formula: glucose 5g/L, naCl 1g/L, mgSO 4 1.5g/L，(NH 4 ) 2 SO 4 1.5g/L，MgCl 2 0.5g/L, adding the components in sequence during preparation, adding the next component after each component is uniformly mixed, adding 1g/L of tricalcium phosphate after all the added components are dissolved, uniformly shaking, adding 20g/L of agar powder, supplementing with distilled water after uniformly mixing, regulating the pH value to 6.0, and sterilizing at the high temperature of 121 ℃ for 30min.
Since tricalcium phosphate is insoluble in water, it is added and shaken well.
The invention also provides a phosphate solubilizing culture medium for culturing the phosphate solubilizing bacteria, the formula of the phosphate solubilizing culture medium is 10g/L glucose, 2g/L NaCl, (NH) 4 ) 2 SO 4 1.5g/L，MgCl 2 6g/L, 5g/L of tricalcium phosphate and 3.5g/L of KCl, adding the components in sequence during preparation, adding the next component after each component is completely dissolved, adding 5g/L of tricalcium phosphate after the medicament is dissolved, adding distilled water after shaking uniformly, regulating the pH to 6.0, and sterilizing at the high temperature of 121 ℃ for 30min.
The invention also provides a method for curing U (VI) by the phosphate solubilizing bacteria, which comprises the following steps:
1) Inoculating the phosphate-solubilizing bacteria into LB culture medium for aerobic enrichment culture, and growing until the phosphate-solubilizing bacteria grow to logarithmic phase and OD 600 The value reaches 0.5, and the phosphate-dissolving strain seed bacterial liquid is prepared for standby;
2) Adding the phosphate solubilizing bacteria seed bacterial liquid in the step 1) into the phosphate solubilizing culture medium of the phosphate solubilizing bacteria, and culturing for 72 hours to prepare phosphate solubilizing bacteria bacterial liquid;
wherein, the concentration of free phosphate radical in the phosphate dissolving system is detected every 12 hours, the phosphate concentration in the phosphate dissolving system of the phosphate dissolving bacteria is ensured to be 50-100mg/L, and when the phosphate concentration is lower than 50mg/L, 5g/L of tricalcium phosphate is supplemented.
When the phosphate concentration is ensured to be more than 50mg/L, the curing reaction with U (VI) can be realized.
3) Collecting U (VI) -containing percolate from a water collecting pool at the downstream of uranium-polluted soil, inoculating the phosphate-dissolving bacteria liquid prepared in the step 2) to form a system of U (VI) in phosphate-dissolving bacteria solidification percolate, and setting a reaction period for 72 hours.
Preferably, the inoculum sizes of the phosphate-solubilizing bacteria in the step 1), the phosphate-solubilizing bacteria seed bacterial liquid in the step 2) and the phosphate-solubilizing bacteria bacterial liquid in the step 3) are 1-10% by volume.
Preferably, the culture temperature of the phosphate solubilizing bacteria in the step 1), the preparation of the phosphate solubilizing bacteria seed bacterial liquid in the step 2) and the preparation of the phosphate solubilizing bacteria bacterial liquid in the step 3) is 20-35 ℃.
Preferably, the culture conditions of the phosphate-solubilizing bacteria in the step 1), the phosphate-solubilizing bacteria seed bacterial liquid in the step 2) and the phosphate-solubilizing bacteria bacterial liquid in the step 3) are aerobic environments, and the rotating speed of a shaking table is 120-180r/min.
Preferably, the pH in the system of the culture of the phosphate solubilizing bacteria in the step 1), the culture of the phosphate solubilizing bacteria seed bacterial liquid in the step 2) and the solidification U (VI) of the phosphate solubilizing bacteria bacterial liquid in the step 3) is 5-8.
The invention has the advantages that:
1. the phosphate-solubilizing bacteria Pantoea sp.GRINML12 provided by the invention is separated from a uranium tailing pond, can keep activity in a uranium-containing system, has the capability of dissolving insoluble phosphorus sources, and can be used for coprecipitating U (VI) and phosphate to dissociate sufficient free phosphate.
2. The phosphorus source utilized by the phosphate-dissolving bacteria Pantoea sp.GRINML12 can be phosphate rock powder and waste containing indissolvable phosphorus, and can realize the environment-friendly restoration concept of treating waste with waste. Can be used in the fields of soil fattening, ecological improvement and heavy metal pollution treatment.
3. According to the invention, the phosphate-dissolving bacteria GRINML12 is adopted to solidify U (VI), and the co-precipitation effect of free phosphate and U (VI) is dissociated in the self-metabolism process of the phosphate-dissolving bacteria, so that uranyl phosphate mineral is generated. The ability of U (VI) to migrate to the surrounding environment is limited after the uranyl phosphate mineral is precipitated, so that the risk of U (VI) diffusing to the surrounding environment is reduced, and the management and control of uranium pollution areas can be realized. Meanwhile, after the uranyl phosphate mineral is precipitated, the uranyl phosphate mineral is not absorbed and utilized by plants including crops, so that the toxic action on the plants is reduced; the toxic action on human, animal and microorganism using plants is also reduced directly or indirectly, and the bioavailability of U (VI) is reduced.
The invention has the beneficial effects that:
the invention provides a phosphate solubilizing bacterium and a method for solidifying U (VI) by using the phosphate solubilizing bacterium, wherein the phosphate solubilizing bacterium generates stable uranyl phosphate mineral (HUO) through coprecipitation of dissociated free phosphate and U (VI) 2 PO 4 ，Ca(UO 2 ) 2 (PO 4 ) 2 ) Etc.); the phosphate-dissolving bacteria can utilize the waste containing no or low price phosphorus as a phosphorus source, and the dissociated phosphate is not only used for solidifying heavy metals, but also has wide application scenes in the fields of hardening and barren soil improvement, biological fattening, ecological restoration and the like, and can effectively reduce the cost of the fertilizer fields such as heavy metal pollution treatment, barren soil improvement and the like; due to the broad presence of phosphorus sources in the environment, phosphorus-decomposing bacteria are provided with sufficient phosphorus sources, which is advantageous for sustainable curing U (VI). The phosphate-solubilizing bacteria have high uranium toleranceThe phosphate solubilizing bacteria can survive in uranium polluted environment, and solidification of U (VI) in uranium polluted wastewater or soil is realized.
Drawings
Fig. 1 is a morphological diagram of phosphorus-decomposing bacteria GRINML12 provided by the invention.
Fig. 2 is an electron microscope scan of the phosphorus-decomposing bacteria GRINML12 provided by the invention.
FIG. 3 is a graph showing the phosphorus-decomposing efficiency of the phosphorus-decomposing bacteria GRINML12.
FIG. 4 is a graph showing the results of tolerance of the phosphorus-decomposing bacteria GRINML12 to acclimation at different U (VI) concentrations.
FIG. 5 is a graph showing the curing efficiency of the phosphorus-decomposing bacteria GRINML12 provided by the invention on a U (VI) solution with an initial concentration of 5 mg/L.
FIG. 6 is a graph showing the curing efficiency of the phosphorus-decomposing bacteria GRINML12 provided by the invention on a U (VI) solution with an initial concentration of 10 mg/L.
FIG. 7 is a graph showing the curing efficiency of the phosphorus-decomposing bacteria GRINML12 provided by the invention with respect to a solution having an initial concentration of 15mg/L U (VI).
FIG. 8 is a graph showing the curing efficiency of the phosphorus-decomposing bacteria GRINML12 provided by the invention with respect to a solution with an initial concentration of 20mg/L U (VI).
FIG. 9 shows OD in a uranium solidifying system of phosphorus-decomposing bacteria GRINML12 on U (VI) solution with initial concentration of 5mg/L 600 Value change graph.
FIG. 10 shows OD in a uranium solidifying system of phosphorus-decomposing bacteria GRINML12 versus U (VI) solution with initial concentration of 10mg/L 600 Value change graph.
FIG. 11 shows OD in a uranium solidifying system of the phosphorus-decomposing bacterium GRINML12 solution with initial concentration of 15mg/L U (VI) 600 Value change graph.
FIG. 12 shows OD in a uranium solidifying system of the phosphorus-decomposing bacterium GRINML12 with initial concentration of 20mg/L U (VI) solution 600 Value change graph.
Fig. 13 is a graph showing the effect of the phosphate solubilizing bacteria GRINML12 on solidifying uranium contaminated leachate.
Fig. 14 is an EDS phase characterization diagram of a precipitated product of uranium contaminated leachate solidified by Pantoea sp.grinml12, provided by the present invention.
Detailed Description
The embodiments of the present invention will be described in detail and fully described below to enable those skilled in the art to more readily understand the advantages and features of the present invention and to make a clear and concise description of the scope of the present invention.
The invention provides a phosphate solubilizing bacterium, which has the classification name: pantoea (Pantoea sp.) GRINML12, accession number: china center for type culture Collection, address: the preservation date of the university of Wuhan in Chinese is: 11 months and 20 days in 2019, the preservation number is: cctccc NO: m2019960.
The invention also provides a screening culture medium for screening phosphate-solubilizing bacteria GRINML12 and a phosphate-solubilizing culture medium for phosphate-solubilizing, wherein the components and the formula content of the screening culture medium and the phosphate-solubilizing culture medium are shown in the following table 1.
TABLE 1 Medium composition and content thereof
In the screening culture medium and the phosphate dissolving culture medium, except tricalcium phosphate, one culture medium component is required to be completely dissolved and the other component is required to be added, so that the tricalcium phosphate is added step by step and uniformly mixed, and the tricalcium phosphate is insoluble in water, so that the tricalcium phosphate is added and then uniformly shaken. Finally, the pH was adjusted to 6.0 after adding distilled water, and then autoclaved at 121℃for 30min. The screening culture medium is insoluble in distilled water at room temperature, so the agar powder is added after the previous components are added, the distilled water is added, the pH value is regulated to 6.0, and then the culture medium is autoclaved at 121 ℃ for 30min and is used by pouring into a flat plate.
The LB culture medium comprises the following components: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride were added to 1L deionized water at a time, pH was adjusted to 6, and then autoclaved at 121℃for 30min.
EXAMPLE 1 isolation and identification of phosphate-solubilizing bacteria GRINML12
Taking a percolate sample of uranium contaminated soil around a uranium tailing pond in Jiangxi province, and carrying out gradient dilution 1, 2, 3, 4 and 5 (minutes)Respectively correspond to 10 -1 ，10 -2 ，10 -3 ，10 -4 ，10 -5 ) 100. Mu.L of the dilution was spread on a plate prepared from the phosphate-solubilizing bacteria screening medium, and incubated at 30℃for 72 hours. Then, single colonies were picked and subjected to three-section streaking on a plate, and single colonies were isolated. After single colony is picked, the single colony is transferred into a new screening culture medium plate, and the separation-purification culture is continued by utilizing a plate streak separation method until single colony is obtained, and the single colony is obtained by drawing lines by a three-zone purification method. The purified single colony is inoculated into 100mL of LB culture medium, and is subjected to shaking culture at 30 ℃ and 150rpm for 48 hours, so that bacterial liquid for identifying bacterial is obtained. Finally, the strain is identified by analysis of 16S rDNA clone library technology. 1mL of bacterial liquid is centrifuged to obtain bacterial mud, total DNA is extracted, and a PCR technology is utilized to amplify 16S rDNA fragments by using prokaryotic general primers 27f and 1492 r. The PCR product was purified and ligated with the T-easy vector of Promega to transform E.coli DH 5. Alpha. The picked yellowish colonies were determined to be positive by colony PCR and 4 clones were sequenced by enzymatic cleavage. The homology of the strain with Pantoea sp.T55 is determined to be more than 99%, and the strain is identified as Pantoea (Pantoea) bacteria and is named as: pantoea sp. GRINML12 (Pantoea sp. GRINML12 for short).
The morphological characteristics of the strains are observed in the flat plate by Pantoea sp.GRINML12, the shape of the bacterial colony is a disc, the color is yellowish, the surface of the bacterial colony is glossy, the edge of the bacterial colony is regular, the diameter of the bacterial colony is about 1.5mm, the result is shown in FIG. 1, and then the Pantoea sp.GRINML12 is observed by a scanning electron microscope: pantoea sp.GRINML12 cells were short rods, approximately 2-3 μm long and 0.5-0.8 μm wide, and the results are shown in FIG. 2.
Example 2 Pantoea sp.GRINML12 dephosphorization Effect
1. Inoculating Pantoea sp.GRINML12 obtained by screening into LB culture medium for activation enrichment culture, wherein the inoculum size is 2% by volume, the culture temperature is 30deg.C, the shaking table rotation speed is 150r/min, microorganism is in logarithmic phase and OD 600 The value reaches 0.5, and the bacterial liquid is used as a phosphate-dissolving bacterial seed bacterial liquid for standby;
2. subpackaging the phosphate solubilizing culture medium into 300mL triangular flasks, and setting a control group (control) and an experimental group (grimn-12), wherein each group is provided with three parallel tests, and the experimental group is inoculated with the phosphate solubilizing strain seed bacterial liquid in the step 1; the adding amount of the phosphate-solubilizing bacteria seed bacterial liquid is 10% according to the volume ratio, and the phosphate-solubilizing bacteria seed bacterial liquid is not inoculated in the control group; the temperature was 30℃and the pH was 6, the shaking table rotation speed was 150rpm/min, and the culture period was 190 hours.
Wherein the effect of phosphate dissolution in the control and experimental systems was measured every 24 hours, and the results are shown in fig. 3. The concentration of the soluble phosphorus in the control group system is slowly increased along with time due to the addition of the phosphate solubilizing bacteria, and compared with the control group, the concentration of the soluble phosphorus in the experimental group system inoculated with the phosphate solubilizing bacteria GRINML12 is obviously increased along with time, and the concentration of the soluble phosphorus in the system reaches 250mg/L within 2-96 hours and is exponentially increased within 190 hours. It was demonstrated that inoculation of Pantoea sp.grinml12 rapidly dephosphorylates poorly soluble phosphorus to soluble phosphorus, also known as phosphate solubilizing bacteria GRINML12. The control group was of little fluctuation and within error.
EXAMPLE 3 tolerance domestication of phosphate-solubilizing bacteria GRINML12 on U (VI)
U (VI) standard solution (U (VI) solution for short) is purchased from standard substance mall, product number: GBW (E) 0080173, gauge concentration: 100. Mu.g/ml.
1. Inoculating selected phosphorus-decomposing bacteria GRINML12 into LB culture medium for activation enrichment culture, wherein the inoculum size is 2% by volume, the culture temperature is 30deg.C, the rotation speed of shaking table is 150r/min, microorganism is in logarithmic phase and OD 600 The value reaches 0.5, and the bacterial liquid is used as a phosphate-dissolving bacterial seed bacterial liquid for standby;
2. the U (VI) solution is added into a phosphate solubilizing culture medium, the initial concentration of the U (VI) in the phosphate solubilizing culture medium is 2mg/L, then 10mL of phosphate solubilizing bacteria seed bacteria are added into 90mL of phosphate solubilizing culture medium containing the U (VI), and the phosphate solubilizing culture medium is cultured for 7 days under aerobic conditions at 30 ℃ and 150rpm/min of a shaking table.
After 3.7 days, the bacterial liquid in the step 2 is transferred as mother liquid into a phosphate solubilizing culture medium prepared by the same method as the step 2 but with the initial concentration of U (VI) of 4mg/L, and the inoculum size is 10 percent, and the culture is carried out for 7 days under aerobic conditions at 30 ℃ and with a shaking table of 150 rpm/min. Subsequently, the cells were sequentially transferred to a phosphate solubilizing medium having a higher U (VI) concentration (up to 55 mg/L) and cultured under the same conditions.
The removal rate of U (VI) and the bacterial quantity in the culture solution are detected at regular time in the domestication process until the concentration of U (VI) in the culture makes the bacteria unable to grow, and the concentration is the highest concentration of the phosphate-solubilizing bacteria tolerant to U (VI), and is called the tolerance of microorganisms to U (VI). The U (VI) concentration, the number of times of transfer, and the time (d) required for the OD600 value of the phosphate-solubilizing bacteria in the system to reach 0.2 are shown in Table 2.
TABLE 2 continuous passage acclimatization results of phosphate-solubilizing bacteria
As can be seen from Table 2, by 13 different U (VI) concentration gradient domestication, when the concentration of U (VI) in the culture medium is as high as 55mg/L, the bacteria can also adapt to the environment with high concentration of U (VI) in the system, and the bacteria can also adapt to the microorganism OD 600 The bacterial growth time was 28 days, calculated to reach 0.2 time to achieve transfer.
As shown in FIG. 4, in the acclimation process by the pure chemical reagent method under the same experimental conditions, the OD of the microorganism increases with the increase of the concentration of U (VI) 600 The time for the value to reach 0.2 is correspondingly prolonged, and the microorganism OD is shortened by increasing the transfer times 600 The tolerance degree of the phosphate-solubilizing bacteria GRINML12 to the concentration of U (VI) after 58 times of transfer can reach 55mg/L within the time of reaching 0.2, which is obviously higher than the concentration of U (VI) in the environment (0.05-10 mg/L).
The bacteria are preserved, and the preservation units are: china center for type culture Collection, address: the preservation date of the university of Wuhan in Chinese is: 11 months and 20 days in 2019, the preservation number is: cctccc NO: m2019960.
EXAMPLE 4 effect of phosphate-solubilizing bacterium GRINML12 on uranium solidification
Because the concentration of U (VI) in the natural environment hardly reaches the tolerance limit concentration of 55mg/L of the phosphate-solubilizing bacteria GRINML12, the concentration of U (VI) is only 0.05-10mg/L generally, and the concentration of U (VI) is increased to 20mg/L at the highest to enlarge the range, so that the curing effect of the phosphate-solubilizing bacteria GRINML12 on U (VI) is determined.
1. Inoculating the selected phosphate-solubilizing bacteria GRINML12 into LB culture medium for activating and enrichingCollecting and culturing, wherein the inoculum size is 2% by volume, the culture temperature is 30deg.C, the rotation speed of shaking table is 150r/min, microorganism is in logarithmic growth phase and OD 600 The value reaches 0.5, and the bacterial liquid is used as a phosphate-dissolving bacterial seed bacterial liquid for standby;
2. split charging phosphate solubilizing medium into 300mL triangular flasks, setting experiment group 1 to experiment group 4 and control group 1 to control group 4, each group setting three parallel experiments, and adding U (VI) solution to experiment group 1 to experiment group 4 to make initial concentration of U (VI) in the system 5mg/L (experiment group 1 and control group 1), 10mg/L (experiment group 2 and control group 2), 15mg/L (experiment group 3 and control group 3) and 20mg/L (experiment group 4 and control group 4), respectively.
3. According to the volume ratio of 10 percent, the phosphorus-decomposing bacteria GRINML12 is inoculated into the experimental groups 1-4, the temperature is 30 ℃, the pH is 6, the rotation speed of a shaking table is 150rpm/min, and the culture period is 72 hours. The control group was identical to the experimental group except that no phosphate-solubilizing bacteria GRINML12 was inoculated.
Wherein U (VI) curing amount and microorganism OD in the system are measured every 12 hours 600 The results are shown in fig. 5 to 12.
As can be seen from fig. 5 to 12, when the initial concentration of the phosphate-solubilizing bacteria tolerant U (VI) is 5-20mg/L, the uranium removal shows a uniform rule under the condition of different uranium concentrations, and is mainly divided into three stages: (1) A stage of 0-12h, namely a uranium rapid removal stage, wherein biomass is low, but medium components in a system are high, and culture-based uranium has a strong adsorption effect, so that the process is determined to be a chemical substance adsorption process; (2) 12-36h is uranium concentration rising stage, and due to rapid growth of microorganism, yeast in the environment is consumed in large quantity, so that UO adsorbed by yeast in the previous stage is obtained 2 2+ Is released to rapidly increase the uranium concentration in the environment; (3) After 36 hours, the biomass and the concentration of soluble phosphorus reach peak values, uranium in the environment is removed through solidification and precipitation, and the solidification rate of U (VI) in each system reaches more than 90% after 72 hours.
EXAMPLE 4 effect of phosphate-solubilizing bacterium GRINML12 on uranium solidification
A certain uranium tailing pond is piled up with a large amount of uranium tailing slag and uranium-containing waste, and in the long-term storage process, the seepage prevention facility is damaged through wind blowing and insolation, so that uranium-containing seepage liquid seeping from the tailing pond is caused to diffuse to surrounding soil, and the U (VI) in the seepage liquid leached from the surface layer of the collected soil is detected to exceed the groundwater emission standard (0.05 mg/L) by more than 200 times, so that the U (VI) can reach 10mg/L or more, and serious pollution is caused to the surrounding environment.
Collecting the percolate, and treating free uranium in the uranium-polluted surface soil percolate of the phosphate-dissolving bacteria GRINML 12:
1. uniformly subpackaging percolate into 300mL triangular bottles, wherein the amount of the percolate in each bottle is 150mL, the initial U (VI) concentration is 10mg/L, a control group and an experimental group are arranged, and three groups are arranged in parallel;
2. inoculating phosphorus-decomposing bacteria GRINML12 bacterial liquid after the expanded culture and activation into an experimental group, wherein the inoculation volume ratio is 10%, and adding distilled water with the same volume as the phosphorus-decomposing bacteria GRINML12 bacterial liquid into a control group;
3. setting culture conditions: the temperature was 30℃and the rotational speed was 150rpm/min, and the incubation period was 60 hours, in which samples were taken every 12 hours to detect the change in uranium concentration, and the results are shown in FIG. 13.
As can be seen from fig. 13, the difference of uranium concentration change is remarkable in the control group and the experimental group, wherein the uranium concentration in the experimental group shows a decreasing trend, the U (VI) solidification rate of the initial concentration of the system of 10mg/L can be up to more than 99% in 60 hours, and the U (VI) concentration in the control group is almost unchanged. The phase analysis of the precipitated products of the experimental group was collected, and the diffraction peaks of uranium in the detected precipitates were shown in fig. 14. As can be seen from FIG. 14, the free, readily soluble U (VI) of the experimental group gradually co-precipitated into the system upon solidification of the phosphorus-solubilizing bacteria GRINML12.
As can be seen from the above examples, the present invention provides a phosphate solubilizing bacterium having high tolerance to U (VI) and capable of surviving at a U (VI) concentration of 55mg/L at the highest, and excellent in phosphate solubilizing effect, which can be converted into soluble free phosphate by adding an inorganic or low-valent insoluble phosphorus source to uranium-contaminated environment, and a method for solidifying U (VI) using the same, which can produce a stable uranyl phosphate mineral such as HUO by coprecipitation of free phosphate with U (VI) 2 PO 4 ，Ca(UO 2 ) 2 (PO 4 ) 2 And the like, solidifying the soluble U (VI) into precipitate, reducing the migration effect of the soluble U (VI), reducing the availability of organisms to the soluble U (VI), and realizing the management and control of uranium polluted environment.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (6)
1. The phosphate-solubilizing bacterium is characterized by having the classification name: pantoea genusPantoeasp.) GRINML12, deposit unit: china center for type culture Collection, address: the preservation date of the university of Wuhan in Chinese is: 11 months and 20 days in 2019, the preservation number is: cctccc NO: m2019960.
2. A method for curing U (VI) by phosphate solubilizing bacteria, which is characterized by comprising the following steps:
1) Inoculating the phosphate-solubilizing bacteria according to claim 1 into LB medium for aerobic enrichment culture, and growing until the phosphate-solubilizing bacteria grow to logarithmic phase and OD 600 The value reaches 0.5, and the phosphate-dissolving strain seed bacterial liquid is prepared for standby;
2) Adding the phosphate solubilizing bacterial strain bacterial liquid in the step 1) into a phosphate solubilizing culture medium of phosphate solubilizing bacteria, and culturing for 72 hours to prepare phosphate solubilizing bacterial strain liquid;
3) Collecting U (VI) -containing percolate from a water collecting pool at the downstream of uranium-polluted soil, inoculating the phosphate-dissolving bacteria liquid prepared in the step 2) to form a system of U (VI) in phosphate-dissolving bacteria solidification percolate, and setting a reaction period for 72 hours;
wherein, the formula of the phosphate solubilizing culture medium of the phosphate solubilizing bacteria is glucose 10g/L and NaCl 2g/L, (NH) 4 ) 2 SO 4 1.5 g/L，MgCl 2 6 g/L, KCl 3.5. 3.5g/L, adding the above components in sequence, adding the next component after each component is completely dissolved, adding tricalcium phosphate 5g/L after the above components are completely dissolved, shaking uniformly, supplementing with distilled water, regulating pH to 6.0, and sterilizing at 121deg.C for 30min.
3. The method for curing U (VI) by using the phosphate solubilizing bacteria according to claim 2, wherein the inoculation amount of the phosphate solubilizing bacteria in the step 1), the phosphate solubilizing bacteria seed bacterial liquid in the step 2) and the phosphate solubilizing bacteria bacterial liquid in the step 3) is 1-10% by volume.
4. The method for curing U (VI) by the phosphate solubilizing bacteria according to claim 2, wherein the culturing temperature in the step 1) is 20-35 ℃ when the phosphate solubilizing bacteria seed bacterial liquid is prepared in the step 2) and the phosphate solubilizing bacteria bacterial liquid is prepared in the step 3).
5. The method for curing U (VI) by the phosphate solubilizing bacteria according to claim 2, wherein the culture conditions of the phosphate solubilizing bacteria in the step 1), the phosphate solubilizing bacteria seed bacterial liquid in the step 2) and the phosphate solubilizing bacteria bacterial liquid in the step 3) are aerobic environments, and the rotation speed of a shaking table is 120-180r/min.
6. The method for curing U (VI) by using the phosphate solubilizing bacteria according to claim 2, wherein the pH in the system of the phosphate solubilizing bacteria curing U (VI) in the step 1) culture of the phosphate solubilizing bacteria, the step 2) culture of the phosphate solubilizing bacteria seed bacterial liquid and the step 3) culture is 5-8.
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