CN113416647A - Method for rapidly screening heavy metal Cd/Cr-resistant microorganisms - Google Patents
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
本发明提供了一种快速筛选耐重金属Cd/Cr微生物的方法，包括：(1)将重金属污染的土壤样品配制成土壤悬浊液涂布于含Cd/Cr的LB固体培养基平板上，得到耐Cd/Cr的菌株；(2)将形态差异明显的菌株分别接种在LB固体培养基平板上进行纯化培养；(3)纯化后得到的单一菌株分别接种于加有含Cd/Cr的LB液体培养基的96孔板中，利用酶标仪在不同时刻监测菌株的OD600。本发明采用Tecan infinite 200酶标仪和96孔板联合使用的方法，使用微量的实验药品快速分离培养对Cd/Cr具有高效去除作用的菌种，并能实时监测菌株对Cd/Cr去除能力的大小，减少了不必要的浪费以及污染问题，大大提高实验效率。
The present invention provides a method for rapidly screening heavy metal-resistant Cd/Cr microorganisms, comprising: (1) preparing a soil sample contaminated by heavy metals into a soil suspension and coating it on a Cd/Cr-containing LB solid medium plate to obtain Cd/Cr-resistant strains; (2) The strains with obvious morphological differences were inoculated on LB solid medium plates for purification and culture; (3) The single strains obtained after purification were respectively inoculated into LB liquid containing Cd/Cr In the 96-well plate of the medium, the OD 600 of the strain was monitored at different times by a microplate reader. The invention adopts the method of combined use of Tecan infinite 200 microplate reader and 96-well plate, uses a small amount of experimental drugs to quickly separate and cultivate the bacterial species with efficient removal of Cd/Cr, and can monitor the ability of the bacterial strain to remove Cd/Cr in real time. size, reduce unnecessary waste and pollution problems, and greatly improve experimental efficiency.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a method for rapidly screening heavy metal resistant Cd/Cr microorganisms.
Background
In recent years, with rapid development of agriculture and industry, the problem of environmental pollution is becoming more serious. Wherein, the influence in the aspects of sewage, atmosphere, solid waste and agricultural materials can cause the heavy metal content of soil to continuously rise, finally leads to the serious heavy metal pollution problem of soil. In 2014, the national soil pollution condition survey bulletin published by the national environmental protection department and the national soil resources department in combination: the total point exceeding rate of soil in China is 16.1%, the point exceeding rate of soil in cultivated land in China is higher to 19.4%, the polluted cultivated land area is estimated to be about 3.5 hundred million mu, and the red line of 18 hundred million mu cultivated land is protected in China. The content of heavy metal in the soil exceeds the standard, so that the agricultural products are greatly damaged, and finally, the life health and safety of human beings are harmed. Among various heavy metals in polluted soil, Cd and Cr have great harm to the ecological environment of soil, and are called five-toxic elements, especially Cd, which is called the head of five-toxicity because of its high mobility, high toxicity and wide pollution area.
At present, methods for repairing heavy metal contaminated soil include physical methods, chemical methods, biological methods and the like, but most of the physical methods and the chemical methods have the defects of high cost, large workload, irreversibility of soil change, damage to soil indigenous flora, possible generation of secondary pollution and the like, and the bioremediation technology has the advantages of high efficiency, ecological environment friendliness, low cost and the like. In recent years, plant-microorganism combined remediation technology for heavy metal contaminated soil is widely concerned. Among them, indigenous microorganisms in the contaminated soil often have higher heavy metal resistance, and the microorganisms separated from the indigenous microorganisms can effectively restore heavy metal pollution due to adaptation to local natural environment, so that the method has higher economic and ecological benefits. The method screens out microorganisms which have strong adaptability to climatic environment and soil condition, are coordinated with phytoremediation and have good effect, provides abundant and selectable microbial materials for the bioremediation of the heavy metal contaminated soil, and has great significance for the development of future bioremediation technology.
At present, the screening method of Cd/Cr-resistant microorganisms is generally divided into the following steps: (1) preparing a heavy metal contaminated soil sample into a soil suspension, uniformly coating a supernatant on a solid medium flat plate containing Cd/Cr with a certain concentration, and placing the flat plate in a biochemical incubator for constant-temperature culture; (2) separating and purifying the strains with larger morphological difference; (3) preparing culture media with different Cd/Cr concentration gradients (both higher than the heavy metal concentration in the step (1)), inoculating the purified strains on the culture media with different Cd/Cr concentration gradients, and comparing the resistance strength of different strains to Cd/Cr; (4) and inoculating the screened strains into a liquid culture medium with a certain concentration of Cd/Cr, and extracting culture medium supernatant at different culture times to detect the concentration of the remaining Cd/Cr, thereby comparing the removal capacity of different strains on Cd/Cr.
However, the current screening method for Cd/Cr-resistant microorganisms has the following problems:
1. in the experiment, in order to extract the supernatant of the liquid culture medium as many times as possible under different culture times to detect the concentration of the residual Cd/Cr, a large amount of the liquid culture medium and heavy metal solutions are often needed, the cost is wasted, unnecessary pollution is generated, and the heavy metal solutions have toxicity and are very harmful to the body health of research personnel;
2. the number of cultured strains is large, the time for extracting the culture medium supernatant is required, all the culture medium supernatants are difficult to extract at the same time, and the difference of the extraction time can cause that the detection results can not accurately compare the removal capacities of different strains on heavy metals;
3. the digestion of the extracted culture medium supernatant requires a large amount of acid, which not only wastes the cost, but also volatilizes acid steam in the heating digestion process to cause a large amount of pollution, and the digestion steps are complicated and time and labor are wasted;
4. a precise instrument (such as ICP-MS and the like) is required for measuring the concentration of the heavy metal in the culture medium supernatant, the analysis of the precise instrument has higher requirements on the precision of the instrument, the preparation of a standard solution and the operation of researchers, and a great deal of time, labor and financial resources are consumed in the analysis process.
In order to scientifically recognize the action of microorganisms in the environment of heavy metal contaminated soil, the existing method needs to be optimized, and then strains with high-efficiency removal effect on Cd and Cr are quickly separated from the heavy metal contaminated soil.
Disclosure of Invention
Aiming at the defects or improvement requirements of the existing method, the invention provides a method for rapidly screening heavy metal resistant Cd/Cr microorganisms, which adopts a method of combining a Tecan infinite 200 enzyme-labeling instrument and a 96-pore plate, uses a trace amount of experimental drugs to rapidly separate and culture strains with high-efficiency removal effect on Cd/Cr, can monitor the removal capacity of the strains on Cd/Cr in real time, reduces the unnecessary waste and pollution problems in the experimental process, and greatly improves the experimental efficiency.
In order to realize the technical problem, the technical scheme adopted by the invention is as follows:
the invention provides a method for rapidly screening heavy metal Cd/Cr resistant microorganisms, which comprises the following steps:
(1) preparing a soil suspension from a soil sample polluted by heavy metal, and coating the soil suspension on a Cd/Cr-containing LB solid culture medium flat plate to obtain a Cd/Cr-resistant strain;
(2) respectively inoculating the strains with obvious morphological difference on an LB solid medium flat plate for purification culture;
(3) respectively inoculating the single strains obtained after purification into 96-well plates added with LB liquid culture medium containing Cd/Cr, and monitoring OD of the strains at different moments by using an enzyme-labeling instrument600The value is obtained.
Preferably, the soil suspension is prepared by the following method: mixing a soil sample with sterile water according to a weight ratio of 1:10, oscillating at the temperature of 28 ℃ at 150r/min for 30min, and standing for 10 min.
Preferably, the LB solid medium specifically comprises the following components: tryptone: 10g/L, yeast extract: 5g/L, NaCl: 10g/L and 15-20g/L agar.
More preferably, the concentration gradient of Cd/Cr in the LB solid medium containing Cd/Cr in the step (1) is 5/100mg/L, 10/150mg/L and 15/200 mg/L.
Preferably, the LB liquid medium containing Cd/Cr in the step (3) specifically comprises the following components: tryptone: 10g/L, yeast extract: 5g/L, NaCl: 10g/L, and the concentration of Cd/Cr is 15/200 mg/L.
Preferably, the step (3) further comprises preparing the strain into a bacterial suspension by using physiological saline and measuring the concentration of the bacterial suspension by using an ultraviolet spectrophotometer by using the physiological saline as a reference.
More preferably, 20 μ L of bacterial suspension is added to each sample well of the 96-well plate.
Preferably, the microplate reader is a Tecan infinite 200 microplate reader.
Preferably, the method further comprises the step of drawing a growth curve of the strain by taking time as an abscissa and taking an absorbance at 600nm as an ordinate, wherein the corresponding slope represents the growth trend of the strain in the time period.
The invention also provides application of the method, which comprises the application in screening microorganisms with tolerance to heavy metal Cd/Cr or screening microorganisms with remediation effect on heavy metal contaminated soil.
The invention has the following beneficial effects:
1. the invention designs a 96-well plate to carry out liquid culture on the screened resistant strains, and a large amount of strains can be cultured by using a small amount of culture medium and heavy metal solution.
2. The invention utilizes a Tecan infinite 200 enzyme-linked analyzer to measure the OD of the strain600And carrying out real-time monitoring so as to compare the removal capacity of different strains on Cd/Cr.
Drawings
FIG. 1 is a graph showing the growth of strains in examples of the present invention.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Examples
First, experimental reagent
In this embodiment, the experimental reagent used includes CdCl2Solution (Cd)2+Concentration 1000mg/L) and K2Cr2O7Solution (Cr)6+Concentration 10000mg/L), wherein CdCl2The solution can also be replaced by Cd (NO)3)2Or CdSO4And (3) solution.
Second, preparation of culture medium
Luria-Bertani (LB) solid Medium: tryptone: 10g/L, yeast extract: 5g/L, NaCl: 10g/L, 15-20g/L agar, pH value: sterilizing at 121 deg.C for 30min at 7.0-7.4.
Luria-Bertani (LB) liquid Medium: tryptone: 10g/L, yeast extract: 5g/L, NaCl: 10g/L, pH value: sterilizing at 121 deg.C for 30min at 7.0-7.4.
The above culture medium can be replaced by other culture medium, such as PDA culture medium, Martin culture medium, etc.
Thirdly, the main flow of the screening method is as follows:
1. culture and isolation and purification of resistant strains
Removing foreign matters such as stones and branches in the soil sample, uniformly weighing 2g of soil sample in a super clean bench, adding sterile water to 20ml, placing in a shaking table at 28 deg.C, and oscillating at 150r/min for 30min to obtain 10-1The soil suspension of (1). Diluting the soil suspension to 10-2、10-3、10-4Uniformly coating the diluted soil suspension on LB solid culture medium plates containing heavy metals Cd/Cr with the concentrations of 5/100mg/L, 10/150mg/L and 15/200mg/L, inversely culturing in a biochemical incubator at 28-30 ℃ for 3-5 days, and observing the color and the form of the cultured strains. Respectively inoculating the strains with obvious differences to an LB solid medium plate for purification culture, and placing the strains in a biochemical incubator at 28-30 ℃ for inverted culture for 2 d. Inoculating the strain to slant solid culture medium, and storing in refrigerator at 4 deg.C. It is activated in liquid culture medium before application.
2. Preparation of bacterial suspension
20mL of the enhanced culture seed was placed in a 50mL sterilized centrifuge tube in a clean bench. Centrifuging for 10min with a high-speed centrifuge at 4000 rpm, discarding the supernatant, suspending the precipitate with 50mL of sterilized normal saline, centrifuging again, washing for 3 times as above, and measuring the concentration of the bacterial suspension with an ultraviolet spectrophotometer with normal saline as reference.
3. Detection of Cd/Cr removal capability of strain
Firstly, using 8-channel pipettor or 12-channel pipettor to take 2.7 mu L of CdCl2Solution and 3.6. mu.L of K2Cr2O7Placing the solution in a sterile 96-well plate, continuously transferring 173.7 μ L of LB liquid culture medium into the 96-well plate to make the Cd/Cr concentration of the culture medium in the 96-well plate be 15/200mg/L (namely Cd2+The concentration is 15mg/L, Cr6+Concentration of 200mg/L), then 20 mu L of the bacterial suspension prepared in the previous step is extracted and respectively added into the bacterial suspensionInto the above 96-well plate, a contaminated microenvironment was simulated.
Putting the 96-well plate into a sterilized plastic sealing box, and laying two layers of gauze which are fully soaked with water at the bottom of the box to ensure that the water surface is lower than the opening of the 96-well plate. Culturing in 28-30 deg.C incubator.
OD was measured at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 36h, 48h, 60h and 72h using a Tecan infinite 200 microplate reader600Recording the OD in the 96-well plate at the corresponding time600The value is obtained.
4. Plotting growth curves of the strains
The growth curve of the strain was plotted with time as abscissa and absorbance at 600nm as ordinate, and the corresponding slope indicates the growth tendency of the strain in this time period, as shown in FIG. 1, in which H1, H2, H7, H13 are OD after 72H of cultivation600The 4 strains with the highest growth rate. OD of H7600The value growth rate is highest in the 4 strains, 0-24h, OD of 4 strains600The value increase slopes are all relatively slow, and after 24h, the increase slopes increase rapidly. The reason may be that the strain is just inoculated into the liquid culture medium containing heavy metal, the culture condition changes, and the heavy metal in the culture medium can also inhibit the growth of the strain, so that the strain enters the growth lag phase, and after 24 hours, the strain adapts to the culture environment and can grow and reproduce, so that OD (origin-to-destination) is achieved600The value increases rapidly.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all the embodiments of the present invention are not exhaustive, and all the obvious variations or modifications which are introduced in the technical scheme of the present invention are within the scope of the present invention.
Claims (10)
1. A method for rapidly screening heavy metal Cd/Cr resistant microorganisms comprises the following steps:
(1) preparing a soil suspension from a soil sample polluted by heavy metal, and coating the soil suspension on a Cd/Cr-containing LB solid culture medium flat plate to obtain a Cd/Cr-resistant strain;
(2) respectively inoculating the strains with obvious morphological difference on an LB solid medium flat plate for purification culture;
(3) respectively inoculating the single strains obtained after purification into 96-well plates added with LB liquid culture medium containing Cd/Cr, and monitoring OD of the strains at different moments by using an enzyme-labeling instrument600The value is obtained.
2. The method for rapidly screening heavy metal resistant Cd/Cr microorganisms according to claim 1, wherein the soil suspension is prepared by the following method: mixing a soil sample with sterile water according to a weight ratio of 1:10, oscillating at the temperature of 28 ℃ at 150r/min for 30min, and standing for 10 min.
3. The method for rapidly screening heavy metal-resistant Cd/Cr microorganisms according to claim 1, wherein the LB solid medium specifically comprises the following components: tryptone: 10g/L, yeast extract: 5g/L, NaCl: 10g/L and 15-20g/L agar.
4. The method for rapidly screening heavy metal resistant Cd/Cr microorganisms according to claim 1, wherein the concentration gradient of Cd/Cr in the LB solid medium containing Cd/Cr in the step (1) is 5/100mg/L, 10/150mg/L and 15/200 mg/L.
5. The method for rapidly screening heavy metal resistant Cd/Cr microorganisms according to claim 1, wherein the LB liquid medium containing Cd/Cr in the step (3) comprises the following components: tryptone: 10g/L, yeast extract: 5g/L, NaCl: 10g/L, and the concentration of Cd/Cr is 15/200 mg/L.
6. The method for rapidly screening the heavy metal resistant Cd/Cr microorganisms according to claim 1, wherein the step (3) further comprises preparing the strains into a bacterial suspension by using normal saline, and measuring the concentration of the bacterial suspension by using an ultraviolet spectrophotometer by using the normal saline as a reference.
7. The method for rapidly screening heavy metal resistant Cd/Cr microorganisms according to claim 1, wherein in step (3), 20 μ L of bacterial suspension is added to each sample well of the 96-well plate.
8. The method for rapidly screening heavy metal resistant Cd/Cr microorganisms according to claim 1, wherein the microplate reader is a Tecan infinite 200 microplate reader.
9. The method for rapidly screening heavy metal-resistant Cd/Cr microorganisms according to claim 1, further comprising the step of drawing a growth curve of the strain by taking time as an abscissa and an absorbance at 600nm as an ordinate, wherein a corresponding slope represents a growth trend of the strain in the time period.
10. The use of the method of any one of claims 1 to 9 for the rapid screening of heavy metal Cd/Cr-tolerant microorganisms, including the use in screening of microorganisms that are tolerant to heavy metal Cd/Cr and in screening of microorganisms that have a remediation effect on heavy metal contaminated soil.
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