CN115851442A - Anaerobic single cell separation rapid screening method for electrochemical active bacteria - Google Patents
Anaerobic single cell separation rapid screening method for electrochemical active bacteria Download PDFInfo
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Abstract
The invention discloses a method for rapidly screening electrochemical active bacteria by anaerobic single cell separation, belonging to the field of environmental microbial energy engineering. The method comprises the steps of obtaining mixed bacterial liquid containing anaerobic microorganisms from a bioelectrochemical system, and diluting the mixed bacterial liquid with anaerobic buffer liquid in an anaerobic bottle to obtain diluted bacterial liquid; dripping the diluted bacterium liquid into micropores of a cell plate of an anaerobic LB culture medium containing nano tungsten oxide particles in a single-droplet mode to perform anaerobic culture; and obtaining the anaerobic unicells of the electrochemical active bacteria after the color of the liquid in the micropores is changed from light yellow to bluish or dark blue. The invention carries out culture and identification at the single cell level, avoids the situation that other electrochemical active bacteria cannot be screened out due to the massive enrichment of the fast-growing electrochemical active bacteria, and improves the flux and the efficiency of screening new strains. The electrochemical active bacteria screening method provided by the invention can be expanded to screening of other anaerobic environment microorganisms.
Description
Technical Field
The invention belongs to the field of environmental microbial energy engineering, and particularly relates to a method for rapidly screening electrochemical active bacteria by anaerobic single cell separation.
Background
The bioelectrochemical system (BES) is a novel environmentally-friendly renewable energy and resource recovery technology, and the basic working principle of the bioelectrochemical system is that microorganisms catalyze and oxidize organic substances at an anode to generate electrons and protons, and the electrons and the protons are respectively conducted to a cathode through an external circuit and anolyte to generate a reduction reaction. BES can utilize organic matters in sewage to realize processes such as electricity generation, methane generation, hydrogen generation, electrochemical desalination and the like, and has advantages in the aspects of energy conservation, sludge generation reduction, energy conversion and storage and the like.
The electrochemically active bacteria refer to microorganisms capable of extracellular electron transfer, and the process of extracellular electron transfer generally needs to be performed under anaerobic conditions. The electrochemical active bacteria are important components of a bioelectrochemical system and directly influence the performance of a bioanode and the whole system. However, the strain resources of the current electrochemical active bacteria are insufficient, and the excavation means of the strain resources is single. The novel and efficient electrochemical active bacteria screening means is developed, the strain resources of the electrochemical active bacteria are enriched, and the method has important significance for theoretical research and practical application of a bioelectrochemical system.
At present, the main screening method of the electroactive microorganisms is an enrichment culture method, the electroactive microorganisms are enriched in BES, and the electroactive strains are obtained after multiple times of separation and purification, so that the defects of low screening flux, long screening period, incapability of avoiding the inhibition effect of fast-growing electrochemical active bacteria occupying growth dominance in screening on other electrochemical active bacteria and the like exist, and the strain resource excavation of the electrochemical active bacteria is limited. Other screening methods using high-throughput and high-precision instruments have the problems of dependence on large instruments and high cost.
Disclosure of Invention
The invention provides a simple and high-flux method for rapidly screening electrochemical active bacteria by anaerobic single-cell separation, and particularly provides a method for rapidly screening electrochemical active bacteria by anaerobic single-cell separation, aiming at the problems of the current method for screening electrochemical active bacteria.
The invention adopts the following specific technical scheme:
the invention provides a method for rapidly screening electrochemical active bacteria by anaerobic single cell separation, which comprises the following steps:
s1: obtaining mixed bacteria liquid containing anaerobic microorganisms from a microbial fuel cell, and diluting the mixed bacteria liquid by using anaerobic buffer liquid in an anaerobic bottle without oxygen to obtain diluted bacteria liquid;
s2: dropwise adding an anaerobic LB culture medium containing nano tungsten oxide particles into each micropore of a cell culture plate, and dropwise adding the diluted bacterium liquid into the micropore in a single-droplet mode to perform anaerobic culture; and the single diluted bacterium liquid drop dripped into the micropore only contains one to a plurality of thalli;
s3: and observing whether a discoloration reaction of converting tungsten oxide into tungsten bronze occurs in the micropores in the anaerobic culture process, and extracting the electrochemically active strain from the micropores with the discoloration reaction.
Preferably, the anaerobic bottle is prepared by introducing nitrogen gas with a purity of 99.999% into a serum bottle for 5min, sealing, and autoclaving.
Preferably, the anaerobic buffer is 1 × PBS buffer.
Preferably, the preparation method of the anaerobic LB medium containing the nano tungsten oxide particles comprises the following steps: adding LB liquid culture medium into a three-neck flask, sealing, introducing nitrogen into one side of the three-neck flask, and heating to discharge oxygen; cooling after heating is finished, and then adding the nano tungsten oxide particles for autoclaving.
Furthermore, the addition amount of LB nutrient broth in the anaerobic LB culture medium containing the nano tungsten oxide particles is 25g/L, and the addition amount of the nano tungsten oxide particles is 3g/L.
Preferably, the temperature of the heating process is 180 ℃, and the heating time is 1h. The cooling temperature is 50-60 ℃.
Preferably, the anaerobic culture process comprises placing the cell culture plate into an anaerobic bag and placing the bag into an anaerobic gas-generating bag, performing constant temperature shaking culture, and then performing constant temperature standing culture.
Further, the constant-temperature shaking culture process is carried out for 1h under the conditions of 30 ℃ and 150 r/min; the constant temperature static culture condition is 30 ℃.
Preferably, the diluted bacterium solution is output in a single-droplet mode through a droplet delivery combination device; the liquid drop conveying combined device comprises a micro-injection pump, an injector and a needle head, wherein the injector is mounted on the micro-injection pump, and the injector and the needle head are connected through a PFA hose.
Preferably, the concentration of the diluted bacterium liquid is 100cells/mL, the needle adopts a 7-gauge needle, and the flow rate of the micro-injection pump is 500 muL/min.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention omits the process of repeated separation and purification, judges whether the culture is a pure culture with extracellular electron transfer capacity through first-generation culture, and improves the screening efficiency;
(2) By single cell culture, the invention eliminates interspecies competition of different electrochemical active bacteria, and particularly avoids the singleness of screening results brought by the inhibition effect of fast-growing electrochemical active bacteria on other electrochemical active bacteria;
(3) The single cell separation rapid screening method provided by the invention also provides a new separation screening thought for screening other anaerobic environment microorganisms.
Drawings
FIG. 1 shows the phylogenetic tree construction results of the strains selected in example 1;
FIG. 2 is a start-up graph of the microbial electrochemical reactor in example 2 after being connected to a circuit. (a) To inoculate strains in the wells corresponding to 1-A5, and (b) to inoculate strains in the wells corresponding to 1-F10.
Detailed Description
The invention will be further elucidated and described with reference to the drawings and the detailed description. The technical features of the embodiments of the present invention can be combined correspondingly without mutual conflict.
Example 1
The embodiment provides a method for rapidly screening electrochemical active bacteria by anaerobic single cell separation, which comprises the following steps:
(1) Introducing nitrogen with purity of 99.999% into a 20mL serum bottle for 5min, rapidly plugging a rubber plug and sealing an aluminum cap, and autoclaving at 121 deg.C for 20min before use to obtain an anaerobic bottle.
(2) And extracting battery liquid in the microbial fuel cell operated in a laboratory by using an injector, and obtaining mixed bacteria liquid contained in the battery liquid and placing the mixed bacteria liquid in an anaerobic bottle. And determining the concentration of bacteria contained in the mixed bacteria liquid by using a bacteria counting plate, and diluting the mixed bacteria liquid in an anaerobic bottle by using a 1 XPBS anaerobic buffer solution to obtain a diluted bacteria liquid with the concentration of 100 cells/mL.
(3) Adding 25g LB nutrient broth and 1L deionized water into a three-neck flask, sealing, introducing high-purity nitrogen gas into one side, heating at 180 deg.C for 1h to remove oxygen, and cooling to 50 deg.C to obtain LB culture medium. Taking 50mL LB culture medium by a 60mL syringe, subpackaging into 100mL serum bottles, sealing, adding 0.15g of nano tungsten oxide particles, and autoclaving at 121 ℃ for 20min to obtain the anaerobic LB culture medium containing the nano tungsten oxide particles.
(4) In an anaerobic glove box, 150 μ L of anaerobic LB medium containing nano tungsten oxide particles was added to each well of a 96-well cell culture plate using an 8-gang pipette.
(5) In an anaerobic glove box, a 20mL syringe is used for sucking diluted bacteria liquid in an anaerobic bottle, the syringe is installed on a micro-injection pump LSP-02-2B, a PFA hose is used for connecting the syringe and a No. 7 needle, the flow rate of the micro-injection pump is set to be 500 muL/min, so that the needle stably outputs single liquid drops, and each liquid drop theoretically contains one to several thalli under the condition. And moving the needle head to enable the single liquid drop to fall into the single micropore of the cell culture plate respectively to obtain the bacteria liquid culture plate. In this example, a total of 192 microwells of 2 96-well plates were seeded.
It should be noted that, the volume of a single liquid drop in each micro-well of the cell culture plate and the concentration of the diluted bacteria liquid are controlled, so that each liquid drop theoretically contains one to several bacteria, preferably only 1 bacteria, so that the pure bacteria can be obtained through one-generation culture in the subsequent culture process. However, since the cells have a polymerization property, there may be no cells per droplet or a plurality of cells may be polymerized in actual operation.
(6) And (3) putting the cell culture plate containing the bacterial liquid into an anaerobic bag, putting the anaerobic bag into the anaerobic bag, sealing the bag, taking the bag out of an anaerobic glove box, putting the bag into a constant-temperature shaking table, and carrying out constant-temperature shaking culture for 1h at the temperature of 30 ℃ and at the speed of 150 r/min. The anaerobic gas generating bag can absorb oxygen and release carbon dioxide to ensure an anaerobic environment.
(7) Observing the color of the nano tungsten oxide particles in each micropore of the cell culture plate every other day, and if the color of the liquid in one or more micropores is changed from light yellow to bluish or dark blue, indicating that the electrochromism reaction of converting tungsten oxide into tungsten bronze occurs in the micropore, thus obtaining the electrochemically active strain by extracting from the micropore in which the electrochromism reaction occurs.
Tungsten oxide is an n-type semiconductor material, and the main structure of the tungsten oxide is an octahedron formed by tungsten atoms and oxygen atoms in an end-to-end mode. The octahedral structure has pores for embedding Na + 、K + And the like to form tungsten bronze. The structure enables the tungsten oxide to have electrochromic characteristics, and particularly, according to the principle related to the example, the tungsten oxide can receive electrons transmitted to the outside of the cell by electrochemical active bacteria to form tungsten bronze, and the tungsten bronze apparently changes along with light yellow tungsten oxide into blue tungsten bronze, so that the occurrence of electron transmission outside the cell is indicated by a color change reaction.
In this example, it was found that 3 microwells exhibited color changes to different degrees. The microporous bacterial solution with color change was aspirated from the inside of an anaerobic glove box, one part of the solution was inoculated into an anaerobic LB solution containing nano tungsten oxide particles for amplification culture and preservation, and the other part was subjected to PCR using bacterial universal primers 27F (5. Strains were initially identified by BLAST sequence alignment at NCBI.
Through comparison, 3 strains of bacteria separated and screened belong to 2 species in Bacillus (Bacillus). Wherein the micropores 1-A5 and 2-A6 are Bacillus cereus and 1-F10 are Bacillus pumilus, and the phylogenetic tree is constructed according to the comparison result, and the result is shown in FIG. 1.
Although 3 specific strains were isolated and screened in this example, the method for rapidly screening electrochemically active bacteria by anaerobic single cell isolation provided by the present invention is a general screening method, and can screen a pure bacterial culture having an extracellular electron transfer ability from a cell fluid in which electrochemically active bacteria are dominant. However, the specific strains obtained by each screening are different depending on the sampling difference of the battery fluid and the chance in the experimental process, and it is not limited to screening only the 3 specific strains.
Example 2
In this example, in order to verify the electricity generating capacity of the strain screened in example 1, a microbial electrochemical reactor was constructed for electrochemical characterization studies, as follows:
(1) The graphite electrode of 1.5 x 1cm is used as an anode, the stainless steel mesh of 1.5 x 1cm is used as a cathode, and the titanium wire is used as a conductive material.
(2) And (3) penetrating a titanium wire through a rubber plug to suspend the graphite electrode and the stainless steel electrode in the air in a reactor, wherein the reactor is a 20mL glass bottle with three openings, sealing an aluminum cover, and then sterilizing at 121 ℃ for 20min under high pressure. After sterilization, the reference electrode was attached to the reactor in a clean bench and sterile high purity nitrogen was introduced to evacuate the reactor of oxygen.
(3) Into a three-necked flask were placed 1g of glucose, 1g of sodium acetate, and 0.31g of NH 4 Cl,0.13g KCl,12.5mL of a trace amount of saline solution, 1L of PBS with a concentration of 50mM and sealed, after introducing high-purity nitrogen and heating to remove oxygen, the heating was stopped, and the mixture was dispensed into a 100mL serum bottle by a 60mL syringe and sealedSealing, filling 50mL of the culture medium into each bottle, autoclaving at 121 ℃ for 20min before use, and adding 0.25mL of sterile vitamin into each bottle after sterilization to obtain the reactor culture medium.
(4) Bacillus cereus (Bacillus cereus) obtained by screening in the micropores 1-A5 in the example 1 and Bacillus pumilus (Bacillus pumilus) in the micropores 1-F10 are respectively selected to build electrochemical reactors, 10mL of reactor culture solution is added into each reactor, and the screened strains are inoculated according to the volume of 10% of the reactor culture solution. The reactor is connected into a circuit, an external voltage of 0.7V is added to the cathode end, and a 10 omega external circuit resistor is connected in series in the circuit to detect the current in the circuit. And recording voltage data at two ends of the external circuit resistor every 20min by using a data acquisition instrument. The whole example was run in a thermostatic chamber at 30 ℃.
As shown in FIG. 2, FIG. 2 (a) is a graph showing the output voltage of the Bacillus cereus electrochemical reactor in seeded microwells 1-A5 as a function of time, showing a steady peak at about 420 hours when the current density is about 30mA/m 2 (ii) a FIG. 2 (b) is a graph showing the output voltage of a Bacillus pumilus electrochemical reactor seeded in microwells 1 to F10 as a function of time, showing a steady peak at a current density of about 20mA/m for about 400 hours 2 。
The effectiveness of the separation and rapid screening method provided by the invention in screening electrochemical active bacteria is demonstrated. Through the unicellular culture, the step of separating and purifying for many times is saved, the screening time is saved, the interspecific competition of different electrochemical active bacteria is eliminated, particularly the inhibiting effect of fast-growing electrochemical active bacteria on other electrochemical active bacteria is avoided, so that the electrochemical active bacteria which grow relatively weakly in a separation system can be screened out, and the diversity of strain resources is improved. The invention also provides a novel method for screening the electrochemical active bacteria, and can be expanded to screening of other microorganisms in anaerobic environment.
The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.
Claims (10)
1. An anaerobic single cell separation rapid screening method of electrochemical active bacteria is characterized by comprising the following steps:
s1: obtaining mixed bacterial liquid containing anaerobic microorganisms from a bioelectrochemical system, and diluting the mixed bacterial liquid by using anaerobic buffer liquid in an anaerobic bottle without oxygen to obtain diluted bacterial liquid;
s2: dropwise adding an anaerobic Luria-Bertani (LB) culture medium containing nano tungsten oxide particles into each micropore of a cell culture plate, and dropwise adding the diluted bacterium liquid into the micropores in a single-droplet mode to perform anaerobic culture; and the single diluted bacterium liquid drop dripped into the micropore only contains one to a plurality of thalli;
s3: and observing whether a discoloration reaction of converting tungsten oxide into tungsten bronze occurs in the micropores in the anaerobic culture process, and extracting the electrochemically active strain from the micropores with the discoloration reaction.
2. The method for rapidly screening electrochemical active bacteria by anaerobic single-cell separation as claimed in claim 1, wherein the anaerobic bottle is obtained by introducing nitrogen with a purity of 99.999% into a serum bottle for 5min, sealing and autoclaving.
3. The method for rapid screening of electrochemical active bacteria by anaerobic single cell separation as claimed in claim 1, wherein the anaerobic buffer is 1 x PBS buffer.
4. The method for rapidly screening electrochemical active bacteria by anaerobic single cell separation as claimed in claim 1, wherein the preparation method of the anaerobic LB medium containing nano tungsten oxide particles is as follows: adding LB liquid culture medium into a three-neck flask, sealing, introducing nitrogen into one side of the three-neck flask, and heating to discharge oxygen; cooling after heating is finished, and then adding the nano tungsten oxide particles for autoclaving.
5. The method for rapidly screening the anaerobic single cell separation of the electrochemically active bacteria according to claim 4, wherein the addition amount of LB nutrient broth in the anaerobic LB medium containing the nano tungsten oxide particles is 25g/L, and the addition amount of the nano tungsten oxide particles is 3g/L.
6. The method for rapidly screening electrochemical active bacteria by anaerobic single cell separation as claimed in claim 4, wherein the temperature of the heating process is 180 ℃, and the heating time is 1h; the cooling temperature is 50-60 ℃.
7. The method for rapidly screening electrochemical active bacteria by anaerobic single cell separation as claimed in claim 1, wherein the anaerobic culture process comprises placing the cell culture plate into an anaerobic bag and placing the bag into an anaerobic gas-generating bag, shaking culture at constant temperature, and then standing culture at constant temperature.
8. The method for rapidly screening electrochemical active bacteria by anaerobic single cell separation according to claim 7, wherein the constant temperature shaking culture process is a culture at 30 ℃ for 1 hour at 150 r/min; the constant temperature static culture condition is 30 ℃.
9. The method for rapidly screening electrochemical active bacteria by anaerobic single-cell separation according to claim 1, wherein the diluted bacterial liquid is output in a single-droplet form through a droplet delivery combination device; the liquid drop conveying combined device comprises a micro-injection pump, an injector and a needle head, wherein the injector is mounted on the micro-injection pump, and the injector and the needle head are connected through a PFA hose.
10. The method for rapidly screening electrochemical active bacteria by anaerobic single cell separation as claimed in claim 9, wherein the concentration of the diluted bacteria liquid is 100cells/mL, the needle is a 7-gauge needle, and the flow rate of the micro-injection pump is 500 μ L/min.
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