阅读说明：本技术 一种检测微生物吸附金属离子能力的方法 (Method for detecting metal ion adsorption capacity of microorganisms ) 是由 张燕飞 谭玲 尤晓颜 赵国屏 于 2021-09-06 设计创作，主要内容包括：本发明提供了一种检测微生物吸附金属离子能力的方法,其特征在于,所述方法包括：配置吸附样品的步骤；侧向散射光(SSC)值变化量测试的步骤；根据所述微生物的SSC值的变化量以确定或比较所述微生物的金属离子吸附能力的步骤。本发明提供的检测方法具有以下优点：检测所需测试体积小,上样速度快,反应灵敏,对样品无损,测试和筛选速度快。(The invention provides a method for detecting the capability of microorganisms to adsorb metal ions, which is characterized by comprising the following steps: a step of disposing an adsorption sample; a step of testing the variation of the side scattered light (SSC) value; a step of determining or comparing the metal ion adsorption capacity of the microorganism based on the change amount of the SSC value of the microorganism. The detection method provided by the invention has the following advantages: the volume of the test required by the detection is small, the sample loading speed is high, the reaction is sensitive, the sample is not damaged, and the test and screening speed is high.)

1. A method for detecting the ability of a microorganism to adsorb metal ions, the method comprising:

configuring an adsorption sample, wherein the adsorption sample contains microorganisms and metal ions, the concentration of the microorganisms in the adsorption sample is 2-25g/L (wet weight), and the microorganisms adsorb the metal ions;

a step of measuring a change amount of a side scattered light (SSC) value by measuring a change amount of an SSC value of a microorganism having adsorbed said metal ion relative to an SSC value of a microorganism not having adsorbed said metal ion under the same detection conditions using a flow cytometry,

determining or comparing the metal ion adsorption capacity of the microorganism according to the change amount of the SSC value of the microorganism.

2. The method according to claim 1, wherein in the step of configuring the adsorption samples, N adsorption sample groups are configured to satisfy the following condition:

i) the species and concentration of microorganisms contained in each adsorption sample group are the same, and the metal ion concentrations are different;

ii) the species of the microorganisms are different between each adsorbed sample group, and the concentrations of the microorganisms are the same; and

iii) the microorganism species and concentration are the same and the metal ion species are different between each adsorbed sample group.

3. The method of claim 2, wherein in the step of configuring to adsorb a sample, it further satisfies the following condition:

iv) the same number of samples is present between each adsorbed sample group and the concentration of metal ions in the corresponding samples is the same between each adsorbed sample group.

4. A method according to any one of claims 1 to 3, wherein the metal ions include, but are not limited to, any ionic form of any one or more metals selected from: mg, Al, Si, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Y, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In, Te, Os, Ir, Pt, Au, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm and Yb, wherein the concentration of the metal ions is 0.125-2.0 mM.

5. The method according to any one of claims 1 to 4, wherein the microorganism is activated and cultured with a component medium (CDM) selected from any one of the group consisting of YPD medium, MM medium or SC medium before the adsorption sample is disposed; the microorganism includes but is not limited to yeast, Escherichia coli, Cupriavidus, Bacillus subtilis, fungal spore or microorganism library obtained by genetic recombination engineering.

6. The method of any one of claims 1 to 5, wherein the apparatus used in the flow cytometry technique comprises a flow cytometer or a flow cytometer sorter.

7. The method according to claim 1, characterized in that it comprises:

comparing the change amount of the SSC value of the microorganism with a working curve of the metal adsorption of the microorganism to determine the amount of the metal ion adsorption of the microorganism.

8. A method of screening microorganisms having different abilities to adsorb metal ions, the method comprising:

displaying/expressing artificially synthesized polypeptide and protein libraries with metal ion adsorption function on the cell surface of a microorganism, thereby constructing a microorganism library;

detecting the microorganisms in the microorganism library by using the method according to any one of claims 1 to 6 to compare the metal ion adsorption capacity of each microorganism in the microorganism library.

9. A method for recovering metal ions by using recombinant genetically engineered bacteria, which comprises screening microorganisms having a relatively high adsorption capacity to metal ions from the recombinant genetically engineered bacteria by the method of claim 8, and recovering the metal ions by using the microorganisms.

Technical Field

The invention belongs to the field of microbial adsorption and metal ion recovery, and relates to a method for adsorbing and recovering metal ions by a microbiological method, in particular to a method for detecting the capability of microorganisms for adsorbing metal ions.

Background

The metal has unique performance and use, has wide application in industrial production and is closely related to the life of people. Due to scarcity, non-reproducibility, and non-uniformity of distribution of metal resources in various countries, metal resources have been the focus of world competition. For example, platinum group metals (including six metal elements of platinum (Pt), palladium (Pd), rhodium (Rh), iridium (Ir), osmium (Os), and ruthenium (Ru)) are widely used in the fields of catalysis, pharmacy, microelectronics, electroplating, and the like, and are used as "strategic reserve metals" and "first high-tech metals" worldwide.

In addition, with the increasing demand of people for electronic products and other metal products, the smelting and production of metals in various countries are expanding, and a large amount of heavy metal waste liquid and heavy metal pollution are accompanied. Therefore, the adsorption and removal of heavy metals from environmental and industrial waste water is also one of the major issues in the world today. At present, the secondary resource recovery technology of platinum group metal mainly comprises pyrometallurgical, hydrometallurgical and biological treatment technologies. However, the traditional pyrometallurgical and hydrometallurgical recovery methods have the problems of high energy consumption, corrosive and toxic leachate, easy secondary pollution and the like.

The microbial adsorption method which is researched and developed in the 80 th of the 20 th century is to utilize microbial cells and metabolites thereof to enrich metal ions in an aqueous solution through physical and chemical actions (including complexation, precipitation, oxidation reduction, ion exchange and the like), and then realize the purpose of recovering the metal ions in the aqueous phase through solid-liquid two-phase separation. The microbial adsorption method has the advantages of mild adsorption conditions, green and environment-friendly adsorption process and low cost.

At present, the main methods for determining the adsorption capacity of microorganisms on metal ions are as follows: an indirect method, which utilizes an inductively coupled plasma emission spectrometer (ICP-OES) and an inductively coupled plasma mass spectrometer (ICP-MS) to determine the change of the metal ion concentration of the solution before and after adsorption; the direct method is that the microorganisms adsorbing the metal ions are digested by using strong acid such as aqua regia, hydrofluoric acid and the like, and the concentration of the metal ions in the digestion solution is measured by using ICP-OES or ICP-MS.

The two methods are used for accurately measuring the concentration of metal ions inHas wide application in the field of measurement. However, there are some drawbacks to these two approaches: (1) the indirect method requires a large volume of test sample, and the ICP-OES or ICP-MS test usually requires 5-10mL of solution for determination, and although the volume can be obtained by dilution, the measurement error is increased; (2) the direct method has large amount of bacteria to be tested, the strong acid digestion method usually needs about 0.1g of dry weight of bacteria to carry out experiments, and the adsorption experiment cost of rare metals or noble metals is indirectly increased; (3) ICP-OES or ICP-MS has long testing time and large labor intensity, most of the current two machines need manual sample loading, and the testing time of a single sample is 2-5min according to the proficiency of operators; (4) the ICP-OES or ICP-MS test cost is high, and particularly the experimental expense requirement for measuring the content of the metal ions in a large batch is high. Therefore, the conventional ICP-OES or ICP-MS methods cannot screen a complex system or a microorganism library (abundance of microorganism) due to the limitation of the above technical characteristics>10⁴) Strains with medium and high metal adsorption capacity.

Disclosure of Invention

Problems to be solved by the invention

In view of the problems in the prior art, the present invention provides a method for rapidly screening a strain having a high metal ion adsorption capacity from a complex system or a microorganism library. Furthermore, the present invention also provides a method for screening a microorganism having a relatively strong adsorption capacity from a biological library obtained by genetic engineering using the screening method of the present invention, and a method for adsorbing or recovering metal ions using the microorganism.

Means for solving the problems

The present invention has been completed based on the following findings and findings through the studies of the inventors:

after the microorganisms have adsorbed certain metal ions, the metal ions are reduced to 0-valent or metal oxide nanoparticles. Furthermore, flow cytometry enables the analysis of the Side Scatter (SSC) of individual cells. Side scattered light (SSC) is sensitive to the refractive indices of cell membranes, cytoplasm, nuclear membranes and also gives a sensitive response to larger particles in the cytoplasm. Therefore, the metal ion adsorption capacity of the microorganism can be indirectly determined qualitatively or semi-quantitatively by analyzing the change of the side scattered light after the metal ion is adsorbed by the microorganism cell by using the flow cytometry.

More specifically, it was found that the above technical problem can be solved by implementing the following technical solution:

[1] the invention firstly provides a method for detecting the capability of microorganisms to adsorb metal ions, wherein the method comprises the following steps:

configuring an adsorption sample, wherein the adsorption sample contains microorganisms and metal ions, the concentration of the microorganisms in the adsorption sample is 2-20g/L (wet weight), and the microorganisms adsorb the metal ions;

a step of measuring a change amount of a side scattered light (SSC) value by measuring a change amount of an SSC value of a microorganism having adsorbed said metal ion relative to an SSC value of a microorganism not having adsorbed said metal ion under the same detection conditions using a flow cytometry,

determining or comparing the metal ion adsorption capacity of the microorganism according to the change amount of the SSC value of the microorganism.

[2] The method according to [1], in the step of configuring adsorption samples, N adsorption sample groups are configured to satisfy the following conditions:

i) the species and concentration of microorganisms contained in each adsorption sample group are the same, and the metal ion concentrations are different;

ii) the species of the microorganisms are different between each adsorbed sample group, and the concentrations of the microorganisms are the same; and

iii) the microorganism species and concentration are the same and the metal ion species are different between each adsorbed sample group.

[3] The method according to [2], characterized in that in the step of configuring to adsorb a sample, it further satisfies the following condition:

iv) the same number of samples is present between each adsorbed sample group and the concentration of metal ions in the corresponding samples is the same between each adsorbed sample group.

[4] The method according to any one of [1] to [3], wherein the metal ions include, but are not limited to, any ionic form of any one or more metals selected from the group consisting of: mg, Al, Si, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Y, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In, Te, Os, Ir, Pt, Au, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm and Yb, wherein the concentration of the metal ions is 0.125-2.0 mM.

[5] The method according to any one of [1] to [4], wherein the microorganism is activated and cultured with a component medium (CDM) selected from any one of the group consisting of YPD medium, MM medium or SC medium before the adsorption sample is disposed; the microorganism includes but is not limited to yeast, Escherichia coli, Cupriavidus, Bacillus subtilis, fungal spore or microorganism library obtained by genetic recombination engineering.

[6] The method according to any one of [1] to [5], wherein the apparatus for flow cytometry comprises a flow cytometer or a flow cytometer sorter.

[7] The method according to [1], wherein the method comprises:

comparing the change amount of the SSC value of the microorganism with a working curve of the metal adsorption of the microorganism to determine the amount of the metal ion adsorption of the microorganism.

[8] Further the present invention provides a method for screening microorganisms having different abilities to adsorb metal ions, wherein the method comprises:

displaying/expressing artificially synthesized polypeptide and protein libraries with metal ion adsorption function on the cell surface of a microorganism, thereby constructing a microorganism library;

the microorganisms in the microorganism library are detected by the method according to any one of [1] to [6] above to compare the metal ion adsorption capacities of the respective microorganisms in the microorganism library.

[9] The invention also provides a method for recovering metal ions by using the recombinant genetically engineered bacteria, wherein the method comprises the steps of screening out microorganisms with relatively high adsorption capacity to the metal ions from the recombinant genetically engineered bacteria by using the method [8], and recovering the metal ions by using the microorganisms.

ADVANTAGEOUS EFFECTS OF INVENTION

Through the implementation of the technical scheme, the invention can obtain the following technical effects:

compared with the prior art, the method for measuring the adsorption (reduction) capacity of the microorganism with high flux metal ions based on the flow cytometry has the following advantages in particular compared with the ICP-OES or ICP-MS measuring method:

(1) the detection method based on the flow cytometry is sensitive in response and belongs to a nondestructive detection method: the change of the adsorption capacity of the microorganism can be detected in a range of extremely low metal ion concentration (for example <0.5mM), and the microorganism is in a buffer solution in the whole process, so that a single strain with high adsorption capacity can be obtained without damage;

(2) the test volume required for detection is small: generally, the test volume of 20 muL can meet the requirement of cell flow analysis, so that the adsorption system can be within 1 mL;

(3) the sample loading speed is high: according to instruments of different models, the flow cytometry analyzer can realize automatic sample loading of 96-hole or even 384-hole plates, thereby greatly saving the working time of scientific research personnel and reducing the labor intensity;

(4) the testing speed is fast: the flow cytometry can even achieve the capability of testing 10 ten thousand cells per second by controlling parameters such as pressure, liquid flow velocity, thallus concentration and the like;

(5) the screening speed is high: by delineating the range of the target flora, the flow cytometer is able to sort the desired cells at high speed into well plates or plates of up to 96 wells for expanded culture.

Drawings

FIG. 1: shows the change of Pd ion adsorption capacity of yeast cells cultured in different culture media and the change of SSC value in a flow cytometer, wherein:

(a) FIG. shows the adsorption amounts of YPD, MM and SC medium for culturing 10g/L microbial cells (wet weight) under different initial Pd ion concentrations (0.125-2.0 mM);

(b) the graph shows the relationship between the adsorption amount of microbial cells and the increase in the median value of SSC at different initial concentrations of Pd (0.25-1.5 mM). Where the square icon represents YPD medium, the circular icon represents MM medium, the triangle represents SC medium, and SSC represents the side scattering light coefficient.

FIG. 2: shows the changes of the adsorption capacity of the thallus and the median value of SSC under different initial Pd ion concentrations (0.125-2.0mM) and different microbial bacterium concentrations (2.5-20g/L), wherein:

(a) the change of the microbial thallus adsorption capacity under the conditions of different initial Pd ion concentrations (0.125-2.0mM) and different microbial thallus concentrations (2.5-20 g/L);

(b) the relationship between the increment of the median of SSC and the initial Pd ion concentration under different microbial concentrations (2.5-20 g/L);

(c) FIG. 2(b) shows the relationship between the amount of adsorbed cells and the increase in the median of SSC at the initial Pd concentration in the relevant interval.

FIG. 3: and (3) a schematic diagram showing the change of the adsorption capacity of the high adsorption strain screened by the flow cytometry under different initial Pd ion concentrations.

Detailed Description

The present invention will be described in detail below. The technical features described below are explained based on typical embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that:

in the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, the numerical ranges indicated by "above" or "below" mean the numerical ranges including the numbers.

In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

As used herein, the term "optional" or "optional" is used to indicate that certain substances, components, performance steps, application conditions, and the like are used or not used.

In the present specification, unless otherwise specified, the external or environmental conditions relating to the model member in the present specification are all obtained under room temperature conditions, and the "room temperature" used herein means an indoor environmental temperature of "25 ℃.

In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

The invention firstly provides a method for detecting the metal ion adsorption capacity of microorganisms, and the detection method is a detection method based on a flow cytometry technology. In some specific embodiments, the method for detecting the adsorption capacity of a metal ion of a microorganism of the present invention is a qualitative detection method; in other embodiments, the method for detecting the metal ion adsorption capacity of a microorganism of the present invention may be a quantitative or semi-quantitative method.

(microorganisms)

As to the kind of the microorganism to be used in the present invention, there is no particular limitation as long as it is a microorganism capable of adsorbing a metal ion.

As such microorganisms, there may be enumerated, but not limited to, yeasts, Escherichia coli, Cupriavidus, Bacillus subtilis, fungal spores or microorganism libraries. The microorganism library may be a kind of microorganism obtained by a method such as gene editing, modification, or artificial synthesis.

The microorganisms described above may be transformed into other microorganisms by conventional microorganism transformation methods (for example, plasmid amplification). In some embodiments of the present invention, various microorganisms with pre-designed gene segments can be obtained by a controllable plasmid amplification method, and then these microorganisms can be combined into a microorganism library to further detect the metal ion adsorption capacity or screen out microorganisms with relatively strong metal adsorption capacity.

The culture of the microorganism is not particularly limited in the present invention, and the culture can be carried out using a medium commonly used in the art, and examples of the medium include YPD (Yeast peptide dextrose) medium, MM (minor medium) medium, and SC (synthetic complex) medium, but from the viewpoint of detection accuracy of the present invention, MM or SC is preferably used, and SC is more preferably used as the medium.

(Metal ion)

For the purposes of the present invention, metal ions are those which can be present in ionic form in water or acid solutions.

In some particular embodiments of the invention, these metal ions may include, but are not limited to, any ionic form of any one or more metals selected from the group consisting of: mg, Al, Si, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Y, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In, Te, Os, Ir, Pt, Au, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, and Yb.

(adsorption of Metal ions by microorganisms)

The microorganism of the present invention may adsorb or absorb metal ions by mixing the microorganism with the metal ions in an aqueous solution or an acid solution, and in some specific embodiments, the microorganism may reduce the metal ions to elemental metal or may convert the metal ions to metal oxides by biochemical action.

(flow cytometry method)

The present invention is based on the detection of the amount of change in the side scattered light (SSC) value of a microorganism having metal ions adsorbed thereon by a conventional flow cytometry method, and can detect the adsorption capacity of the microorganism to the metal ions in a qualitative, quantitative or semi-quantitative manner. In the present invention, the side scattered light (SSC) value refers to the median value of SSC.

As previously mentioned, the present invention recognizes that after microorganisms adsorb certain metal ions, the metal ions are reduced to 0 valent or metal oxide nanoparticles. Further, existing flow cytometry techniques are capable of analyzing the side scattered light (SSC) values of individual cells. The properties of side scattered light (SSC) are sensitive to the refractive indices of cell membranes, cytoplasm, nuclear membrane and also to the larger particles in the cytoplasm. Therefore, the metal ion adsorption capacity of the microorganism can be indirectly determined qualitatively or semi-quantitatively by analyzing the amount of change in the lateral scattered light value after the metal ion is adsorbed by the microorganism cell by the flow cytometry. And, in some preferred embodiments, the amount of change in the side scattered light value is an amount of increase in the side scattered light value of the microorganism after the microorganism absorbs the metal ion.

Generally, for lateral astigmatism values, several factors may be affected, such as: the kind of microorganism, the kind of culture medium, the influencing components of the microorganism, the culture time of the microorganism, the concentration of the microorganism, the kind and content of metal ions adsorbed by the microorganism, the composition or pH value of the buffer system, and the like may all have an influence on the SSC value in the flow cell test. Therefore, in some specific embodiments, for the convenience of quantitative or comparative studies, it is necessary to perform adsorption of metal ions in the same buffer solution system, usually using microorganisms cultured under the same conditions, so as to perform quantitative analysis or mutual comparison by the change value of SSC values therebetween.

In the present invention, the determination of the metal adsorption capacity of microorganisms as a qualitative, quantitative or semi-quantitative determination using the variation value of the side scattered light (SSC) value is mainly based on the following two points:

in view of the fact that the concentration of microorganisms is in a proper range when the microorganisms adsorb metal ions, so that SSC changes obviously during flow cytometry test, the concentration of the microorganisms is 2-25g/L, preferably 2.5-20g/L when the microorganisms are used for adsorbing the metal ions.

② the change quantity of the side scattered light (SSC) value measured by the flow cytometry method and the adsorption quantity of the microorganism (metal adsorption quantity per unit mass) can show a high linear relationship. For example, linear regression processing can give a linear relationship in which the linear correlation coefficient is greater than 0.9, preferably 0.93 or more.

The following verification shows that the test method of the present invention can satisfy the above two conditions, thereby providing the following two tests:

i) qualitatively comparing or screening microorganisms having relatively stronger metal adsorption capacity. Specifically, various microorganisms were obtained under the same culture conditions, and these microorganisms were contacted with metal ions under the same conditions, respectively (the same conditions including the buffer system, the microorganism concentration, the metal ion species and the concentration are the same). The amount of change in the lateral astigmatism (SSC) values of these microorganisms was then measured using a flow cytometry assay. Furthermore, by changing the concentration or type of the metal ion, the metal ion adsorption of the microorganism can be made stronger than that of the microorganism having the same metal ion concentration at different concentrations, or the metal ion adsorption of the microorganism having the same metal ion concentration can be made stronger than that of the microorganism having the same metal ion concentration at different types.

ii) as a quantitative or semi-quantitative method, a microorganism cultured in a defined medium can be selected and a standard curve or working curve of the adsorption amount to the SSC value at a specific microorganism concentration (2 to 25g/L) can be prepared. Then, the adsorption amount of the microorganism obtained under the similar medium conditions was measured using the operation curve (the concentration of the metal ion adsorbed by the microorganism to be measured was the same as that in the case of the operation curve).

Further, by using the method of the present invention, a microorganism having a high ability to adsorb metal ions can be screened from a naturally occurring microorganism group or a library obtained by artificial synthesis or the like, and the metal ions remaining in a substance such as sewage or wastewater can be adsorbed or recovered by using the microorganism.

Examples

The invention will be further illustrated by the following examples and figures. The substances used in the examples except for the microorganisms are commercially available.

Example 1 (influence of Medium species on the side scattering light coefficient in Pd adsorption)

(1) Preparation of a culture medium:

YPD liquid medium (1L): yeast extract 10.0g, peptone 20.0g, glucose 20.0g, and balance H₂O。

Minimum Medium (MM) liquid medium (1L): yeast basic nitrogen source (YNB)1.5g, ammonium sulfate 5g, D-glucose 20g, inositol 0.002mg, adenine hemisulfate 0.1g, aminobenzoic acid 0.1g, tryptophan 0.0224g, leucine 0.2g, and the balance H₂O。

Synthetic Complete (SC) liquid medium (1L): yeast basic nitrogen source (YNB)1.5g, ammonium sulfate 5g, D-glucose 20g, inositol 0.002mg, tryptophan 0.0224g, uracil 0.1g, leucine 0.2g, adenine hemisulfate 0.1g, glutamic acid 0.1g, phenylalanine 0.1g, alanine 0.1g, proline 0.1g, serine 0.1g, aspartic acid 0.1g, isoleucine 0.1g, threonine 0.1g, asparagine 0.1g, leucine 0.1g, tyrosine 0.1g, cysteine 0.1g, lysine 0.1g, valine 0.1g, glutamine 0.1g, methionine 0.1g, glycine 0.1g, arginine 0.1g, histidine 0.1g, aminobenzoic acid 0.1g, and the balance H₂O。

And adding 2% agar powder into the liquid culture medium to prepare a solid culture medium corresponding to the liquid culture medium.

(2) Activation and culture of bacterial species

The strain S.cerevisiae EBY100(Invitrogen, Carlsbad, Calif.) deposited in glycerol tubes was streaked on SC plates and cultured at 30 ℃ for 72 hours. A single colony was picked from the plate and cultured in 5mL of YPD, SC and MM liquid media at 30 ℃ and 200rpm for 12 hours, respectively. Adding 1mL of the bacterial suspension into 100mL of YPD, SC and MM liquid culture medium, culturing at 30 ℃ and 200rpm with shaking for 12h respectively. The cells were collected by centrifugation at 4000rpm for 3min, washed once with 25mM HEPES buffer, and centrifuged again to collect the cells.

(3) Pd ion adsorption experiment

Accurately weighing wet weight of the thalli, preparing 20g/L of bacterial suspension mother liquor by using 25mM HEPES suspension cells, wherein the wet weight of the thalli: the dry weight of the cells was 10: 1.777. In a 24-well plate, adsorption systems having Pd ion concentrations of 0.125, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0mM and cell concentrations (hereinafter referred to simply as "cell concentrations") of 10.0g/L were prepared in an adsorption volume of 1mL, and the experiment was repeated in duplicate. Adsorbing at 30 deg.C and 220rpm for 1h, and centrifuging at 4000rpm for 3min to separate thallus.

(4) ICP-OES and flow cytometry analysis

The centrifuged supernatant was used for ICP-OES measurement. The cells were suspended in 1000. mu.L of 25mM HEPES, centrifuged at 4000rpm for 3min, and the supernatant was removed, and then suspended in 1000. mu.L of 25mM HEPES to be analyzed for changes in the Side Scattering Coefficient (SSC) by a flow cytometer (Agilent Novocyte). Wherein, when the flow cytometry is used for analysis, the single cell gate is circled out for SSC value analysis.

As shown in fig. 1(a), the s.cerevisiae EBY100 cells cultured in different media (YPD, MM, and SC) had different adsorption capacities for Pd ions at a cell concentration of 10 g/L. Among them, the cells cultured in MM medium had the lowest adsorption capacity for Pd ions (adsorption amount of 127.6. mu.M/g at 2mM Pd concentration), the cells cultured in YPD had the intermediate adsorption capacity (adsorption amount of 148.4. mu.M/g at 2mM Pd concentration), the cells cultured in SC medium had the highest adsorption capacity for Pd ions (adsorption amount of 214.6. mu.M/g at 2mM Pd concentration), and the difference between the highest adsorption amount and the lowest adsorption amount was 1.68 times.

The above experimental results show that: the characteristics of the cell surface are related to the components of the culture medium (growth condition of the cells), and the complete culture medium (SC culture medium) can provide all nutrients for cell growth compared with a minimal culture medium (MM culture medium), thereby being beneficial to the formation of a healthy cell surface.

FIG. 1(b) is a graph showing the relationship between the adsorption amount per cell and the increase in SSC value in flow cytometry at a Pd ion concentration of 0.25 to 1.5mM (data between Pd ion concentrations of 0.125 to 0.25mM and 1.5 to 2.0mM are not shown in FIG. 1(b) because there is no correlation). As is clear from FIG. 1(b), the correlation between the adsorption amount of Pd ions adsorbed on cells cultured in different media and the SSC value was different, and the correlation coefficient of the cells cultured in YPD was the lowest (R)²0.70), and MMThe correlation coefficients of the medium and SC medium reached 0.93 and 0.95, respectively. The reason for this is mainly that YPD medium components are complicated, the uniformity of cultured cells is insufficient compared with synthetic media such as SC and MM, and SSC values of individual cells are measured by flow cytometry, so that the correlation coefficient between the adsorption capacity of individual cells and the increase in SSC values is low. This example demonstrates that, when cultured in a suitable medium, the adsorption capacity of s.cerevisiae EBY100 cells and the increase in SSC value have a strong correlation coefficient, and that the amount of Pd ions adsorbed by the cells can be indirectly characterized by measuring the SSC value.

Example 2 (influence of Yeast cell concentration on side Scattering coefficient in Pd adsorption)

(1) Activation and culture of bacterial species

The strain S.cerevisiae EBY100 deposited in the glycerin tube was streaked on the SC plate and cultured at 30 ℃ for 72 hours. A single colony was picked from the plate and cultured in 5mL SC broth at 30 ℃ for 12h with shaking at 200 rpm. 1mL of the bacterial suspension was added to 100mL of SC liquid medium and cultured at 30 ℃ with shaking at 200rpm for 12 hours. The cells were collected by centrifugation at 4000rpm for 3min, washed once with 25mM HEPES buffer, and centrifuged again to collect the cells.

(2) Pd ion adsorption experiment

Accurately weighing wet weight of thalli, and preparing 40, 20, 10 and 5g/L of bacterial suspension mother liquor by using 25mM HEPES suspension cells respectively, wherein the wet weight of thalli: the dry weight of the cells was 10: 1.777. In a 24-well plate, adsorption systems with Pd ion concentrations of 0.125, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0mM and microbial concentrations of 2.5, 5.0, 10.0 and 20.0g/L were prepared in an adsorption volume of 1mL, and the experiment was repeated in duplicate. Adsorbing at 30 deg.C and 220rpm for 1h, and centrifuging at 4000rpm for 3min to separate thallus.

(3) ICP-OES and flow cytometry analysis

The centrifuged supernatant was used for ICP-OES measurement. The cells were suspended in 1000. mu.L of 25mM HEPES and analyzed for changes in the side scattering light coefficient (SSC) by a flow cytometer (Agilent NovoCyte). Wherein, when the flow cytometry is used for analysis, the single cell gate is circled out for SSC value analysis.

As shown in FIG. 2(a), the adsorption capacity per cell decreased with the increase in the cell concentration and the difference in adsorption capacity between cells at different concentrations increased with the increase in the Pd ion concentration under the condition of 0.125-2.0mM Pd ion concentration.

Under the condition of 0.125mM Pd ions, the bacteria concentration is 2.5g/L and 20g/L, and the bacteria adsorption capacity is 25.7 and 6.2 mu M/g (dry bacteria) respectively; the adsorption capacities of the cells were 336.5. mu.M/g (2.5g/L) and 160. mu.M/g (20g/L) at a Pd ion concentration of 2.0 mM. The reason is mainly that: the ratio of the number of cells to the number of metal ions was lower in the high-concentration cells (20g/L) than in the low-concentration cells (2.5g/L) at the same initial Pd ion concentration, and the amount of adsorbed metal ions per cell was therefore lower.

FIG. 2(b) shows the change in the side scattering light coefficient (SSC value) of the cells at different initial concentrations of Pd ions. In general, the SSC value increases with an increase in the initial concentration, and after reaching an increase of about 0.7, the SSC value cannot be increased by increasing the initial Pd ion concentration. In addition, the sensitivity of the SSC values to the initial Pd concentration decreases with increasing microbial concentration. The SSC value approaches the maximum value at a Pd concentration of 0.5mM under the condition of 2.5g/L bacteria concentration, and has a strong correlation with the initial Pd concentration (R) in a Pd concentration range of 0.125-0.5mM²0.95). Then, the interval of the SSC value and the initial Pd concentration is gradually increased, and the SSC value keeps excellent correlation (R) within 2.0mM Pd ion concentration under the bacteria concentration of 20g/L²＝0.99)。

In the relevant section in FIG. 2(b), the adsorption amount per cell (i.e., specific adsorption capacity) and the median increase in SSC of the cells are shown in FIG. 2 (c). The Pd ion adsorption amount of the unit thallus is in direct proportion to the increment of an SSC value (namely SSC median increment), the correlation coefficient of the unit thallus to the Pd ion adsorption amount is more than 0.9 in the concentration of 2.5-20g/L, and the correlation coefficient of the unit thallus to the Pd ion adsorption amount reaches 0.96 in the concentration of 20 g/L. This example demonstrates that, within a certain range, changes in SSC values in flow cytometry can reflect the amount of Pd ions adsorbed on a unit cell.

Example 3 (application of flow cytometric sorting method in high throughput screening of high metal adsorption strains) (1) establishment of surface display library of s

The saccharomyces cerevisiae surface display plasmid pYD1(addge 73447) was amplified using primers 1-2 and 3-4 (see table 1 specifically) (PCR reaction system and PCR amplification conditions are shown in table 2 and table 3, respectively) shown in table 1, and PCR products were recovered by gel.

The two fragments were ligated by One-Step Cloning (Clonexpress II One Step Cloning Kit) according to the system shown in Table 4 at 37 ℃ for 30 min.

TABLE 1 plasmid cloning primer Table

TABLE 2 PCR reaction System

Reagent	Volume (μ L)
		Buffer(2×)	25
dd H2O	17
		Template (pYD1 plasmid)	2
Primer 1	2
		Primer 2	2
dNTP mix	1
		Taq DNA polymerase	1

TABLE 3 PCR amplification conditions

Temperature (. degree.C.)	Time
		95 (Pre-denaturation)	03:00min
95 (modified)	00:15s
		61.5 (annealing, primer binding)	00:15s (enzyme binding upper sequence)
72 (extension, polymerase temperature)	1:30min(2000bp/min)
		Number of cycles	30 times (twice)
72 (extended intact)	5:00min
		16	0～+oo

TABLE 4 one-step cloning system (ligation system)

Components	Recombination reactions
		Amplification of fragments with primers 1 and 2	1μL
Amplification of fragments with primers 3 and 4	1μL
		5×CEII Buffer	4μL
Recombinase Exnase II	2μL
		dd H2O	12μL

Wherein primers 1-2 of Table 1 are used to amplify metal-binding polypeptide fragments and portions of the plasmid backbone; primers 3-4 in Table 1 were used to amplify a portion of the plasmid backbone. N in the primer 1 represents A, G, C or T; the length of the metal binding polypeptide fragment and part of the plasmid skeleton amplified by the primer 1-2 is close to that of the part of the plasmid skeleton amplified by the primer 3-4. The PCR reaction system for primers 3-4 was the same as that shown in Table 2.

The specific operation is as follows:

melting 100. mu.L E.coli DH 5. alpha. competent cells on ice, adding 10. mu.L of the ligated product, mixing gently, standing on ice for 30min,quickly putting the mixture on ice for 2-3min at 42 ℃ for 45s, adding 900 mu L of non-resistant LB culture medium, performing shake culture at 37 ℃ for 1h, and completely coating the mixture on a Kan resistant plate. After collecting e.coli single colonies on the plate with a cell spatula, the plasmids were extracted. The plasmid was transformed into s.cerevisiae EBY100 as follows: collect 50mL OD₆₀₀The amount of S.cerevisiae EBY100 cells reached 1.0, after the supernatant was removed, 10mL of 0.1M lithium acetate solution was added to suspend the cells, and the cells were collected by centrifugation at 3000rpm for 3 min; adding 300 mu L of 0.1M lithium acetate solution to suspend the thalli, uniformly mixing, and then taking 60 mu L of bacterial suspension and subpackaging into 2.0mL centrifuge tubes; mu.L of the extracted plasmid, 300. mu.L of a transformation premix (comprising 300. mu.L of 50% (w/v) PEG3350, 40.6. mu.L of 1M lithium acetate solution, 3.8. mu.L of 1M Tris (pH 7.6), 0.75. mu.L of 0.5M EDTA and 10. mu.L of salmon sperm) were each placed in each tube, and they were subjected to rotary culture at 30 ℃ for 30min, followed by heat shock at 42 ℃ for 20min, centrifugation at 1500rpm for 30sec, removal of part of the supernatant, and plating of the whole cells with an SC medium (SC-Trp) containing no tryptophan and culture at 30 ℃ for 2 to 3 days. And collecting the bacterial colonies on the plate by using a cell shovel, and subpackaging to obtain the S.cerevisiae EBY100 surface display library.

(2) Screening of high Metal adsorption Capacity Strain in Cerevisiae EBY100 surface display library

Cerevisiae EBY100 surface display library was pipetted and inoculated into 5mL of SC-Trp medium with glucose as a carbon source and cultured overnight (30 ℃ C., 18 h). Then, 200. mu.L of the above bacterial suspension was pipetted and inoculated into 5mL of SC-Trp induction medium containing galactose as a carbon source, and cultured at 30 ℃ for 24 hours. The cells were collected by centrifugation at 4000rpm for 3min, washed once with 25mM HEPES (pH 5.5), and added with an appropriate amount of 25mM HEPES buffer to prepare a 20g/L cell suspension. 1mL of the adsorption system was added with 25. mu.L of 5mM Pd ion mother liquor, 475. mu.L of 25mM HEPES buffer and 500. mu.L of 20g/L bacterial suspension, and adsorbed at 30 ℃ for 1 hour at 220 rpm. The cells were collected by centrifugation at 4000rpm for 3min, washed once with 25mM HEPES and diluted to the appropriate concentration for flow cytometry sorting. And (3) taking out the single cell gate for SSC value analysis, sorting out 0.5% cell group with the largest SSC value, sorting out 25700 cells in total, fully coating in SC-Trp culture medium, and culturing at 30 ℃ for 2-3 days to grow single colonies.

(3) Identification of metal ion adsorption capability of single colony of Cerevisiae EBY100

380 single colonies were picked from the plate and cultured in a 24-well plate at 30 ℃ for 24 hours at 350rpm in 1.0mL of SC-Trp medium with glucose as a carbon source. Then transferring 10 mu L of the strain to a fresh 1mL SC-Trp induction culture medium using galactose as a carbon source, carrying out induction expression for 36h, centrifuging at 3000rpm for 3min, pouring off the supernatant, adding 1mL HEPES buffer solution for washing once, centrifuging, pouring off the supernatant, and adding a proper amount of HEPES suspended bacteria to make the concentration of the bacteria be 10 g/L. To a new 24-well plate, 100. mu.L of 5mM Pd solution, 400. mu.L of 25mM HEPES and 500. mu.L of 10g/L diluted cell suspension were added. Adsorbing at 220rpm for 1h, centrifuging at 3000rpm for 3min, and using the supernatant for ICP-OES to determine Pd concentration. The adsorption capacity of the cells with increased adsorption was verified twice according to the above method under the conditions of final Pd ion concentrations of 0.5, 1.0, 1.5 and 2.0mM, and the experiment was repeated 2 times.

Experimental results as shown in table 5 and fig. 3, the adsorption capacity of 5 single strains (specifically named strain 1, strain 2, strain 3, strain 4 and strain 5, respectively) was improved relative to that of the control strain (s. cerevisiae EBY100 not displaying metal binding polypeptide fragment) in single colonies sorted by SSC value, wherein the adsorption capacity of No. 2 and No. 3 was improved by 32.2% and 47.8% compared to the control at a Pd ion concentration of 2.0mM, reaching 414.3 μ M/g and 463.0 μ M/g. Meanwhile, the metal ion adsorption amount of the single cell is calculated (Table 5), and the average adsorbed Pd ion number of the single cell of 5 strains screened under the initial Pd ion concentration of 2.0mM is respectively increased by 0.93, 2.23, 3.31, 1.38 and 1.24 × 10 compared with that of the control cell⁹And (4) respectively.

The results show that: (1) the polypeptide for surface display of specific adsorption of metal ions by means of genetic engineering can effectively improve the metal adsorption capacity of yeast cells; (2) the adsorption capacity of single cells is analyzed by the flow cytometry technology, and strains with high adsorption capacity can be effectively screened from the library.

TABLE 5 determination of the adsorption Capacity of the high adsorbing Strain

It should be noted that, although the technical solutions of the present invention are described by specific examples, those skilled in the art can understand that the present disclosure should not be limited thereto.

Having described embodiments of the present disclosure, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen in order to best explain the principles of the embodiments, the practical application, or improvements made to the technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Industrial applicability

The microorganism screening method having a strong metal adsorption ability of the present invention can be industrially utilized.

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