CN115851442A - Anaerobic single cell separation rapid screening method for electrochemical active bacteria - Google Patents

Abstract

The invention discloses a rapid screening method for anaerobic single-cell separation of electrochemically active bacteria, which belongs to the field of environmental microbial energy engineering. The present invention obtains the mixed bacterial solution containing anaerobic microorganisms from a bioelectrochemical system, and dilutes the diluted bacterial solution with anaerobic buffer in an anaerobic bottle; the diluted bacterial solution is added dropwise to the Anaerobic culture is carried out in the microwells of the cell plate in the anaerobic LB medium of tungsten oxide particles; the anaerobic single cells of electrochemically active bacteria are obtained after the color of the liquid in the microwells changes from light yellow to blue or dark blue. The present invention carries out cultivation and identification at the single cell level, avoids other electrochemically active bacteria being unable to be screened out due to a large amount of enrichment of fast-growing electrochemically active bacteria, and improves the throughput and efficiency of screening new bacterial strains. The method for screening electrochemically active bacteria provided by the invention can be extended to the screening of other anaerobic environment microorganisms.

Description

A Rapid Screening Method for Anaerobic Single Cell Isolation of Electrochemically Active Bacteria

technical field

The invention belongs to the field of environmental microbial energy engineering, and in particular relates to a rapid screening method for anaerobic single-cell separation of electrochemically active bacteria.

Background technique

Bioelectrochemistry system (BES) is a new type of environmentally friendly renewable energy and resource recovery technology. Its basic working principle is that microorganisms catalyze the oxidation of organic matter at the anode and generate electrons and protons. The electrons and protons pass through the external circuit and The anolyte is conducted to the cathode for reduction reaction. BES can use organic matter in sewage to realize processes such as electricity generation, methane generation, hydrogen generation, and electrochemical desalination. It has advantages in energy saving, reduction of sludge generation, and energy conversion and storage.

Electrochemically active bacteria refer to microorganisms capable of extracellular electron transfer, and the process of extracellular electron transfer usually needs to be carried out under anaerobic conditions. Electrochemically active bacteria are an important part of the bioelectrochemical system, directly affecting the performance of the bioanode and the entire system. However, the current strain resources of electrochemically active bacteria are insufficient, and the mining methods of strain resources are relatively simple. It is of great significance for the theoretical research and practical application of bioelectrochemical systems to develop new and efficient screening methods for electrochemically active bacteria and enrich the strain resources of electrochemically active bacteria.

At present, the main screening method for electroactive microorganisms is the enrichment culture method. This method enriches electroactive microorganisms in BES and obtains electroactive strains after multiple separations and purifications. However, the screening throughput is low and the screening period is long, so screening cannot be avoided. The defects such as the inhibitory effect of the fast-growing electrochemically active bacteria that occupy the growth advantage on other electrochemically active bacteria limit the mining of electrochemically active bacteria. Other screening methods that use high-throughput, high-precision instruments have the problems of relying on large instruments and high costs.

Contents of the invention

Aiming at the problems existing in the current screening methods for electrochemically active bacteria, the present invention provides a simple and high-throughput rapid screening method for anaerobic single-cell separation of electrochemically active bacteria, and specifically provides an anaerobic single-cell separation method for electrochemically active bacteria. Rapid screening method for cell isolation.

The concrete technical scheme that the present invention adopts is as follows:

The present invention provides a rapid screening method for anaerobic single cell separation of electrochemically active bacteria, specifically as follows:

S1: Obtain a mixed bacterial solution containing anaerobic microorganisms from a microbial fuel cell, and dilute the mixed bacterial solution with an anaerobic buffer in an oxygen-free anaerobic bottle to obtain a diluted bacterial solution;

S2: Drop the anaerobic LB medium containing nano-tungsten oxide particles into each microwell of the cell culture plate, and drop the diluted bacterial solution into the microwells in the form of a single droplet for anaerobic culture ; And only one to several bacterial cells are included in the single diluted bacterial liquid droplet added dropwise to the microwell;

S3: During the anaerobic culture, observe whether there is a discoloration reaction of tungsten oxide converted to tungsten bronze in the microwells, and extract electrochemically active strains from the microwells where the discoloration reaction occurs.

Preferably, the above-mentioned anaerobic bottle production process is to pass through nitrogen gas with a purity of 99.999% in the serum bottle for 5 minutes, then seal it and perform high-pressure sterilization treatment.

Preferably, the above-mentioned anaerobic buffer is 1×PBS buffer.

As a preference, the preparation method of the above-mentioned anaerobic LB medium containing nano-tungsten oxide particles is as follows: add LB liquid medium into the three-necked flask and seal it, and heat it with nitrogen on one side to discharge oxygen; cool after heating, and then Add nano-tungsten oxide particles for autoclaving.

Further, the addition amount of LB nutrient broth in the above-mentioned anaerobic LB medium containing nano-tungsten oxide particles is 25 g/L, and the addition amount of nano-tungsten oxide particles is 3 g/L.

Preferably, the temperature of the above heating process is 180° C., and the heating time is 1 h. The cooling temperature is 50-60°C.

Preferably, the above-mentioned anaerobic culture process is to put the cell culture plate into an anaerobic bag and put it into an anaerobic gas-generating bag, shake and cultivate at a constant temperature, and then culture at a constant temperature.

Further, the constant temperature shaking culture process is 1 hour at 30°C and 150r/min; the constant temperature static culture condition is 30°C.

Preferably, the above-mentioned diluted bacterial solution is output in the form of a single droplet through the droplet delivery combination device; the droplet delivery combination device includes a micro-injection pump, a syringe and a needle, the syringe is installed on the micro-injection pump, and PFA is passed between the syringe and the needle. Hose connections.

Preferably, the concentration of the diluted bacterial solution is 100 cells/mL, the needle is a No. 7 needle, and the flow rate of the micro-injection pump is 500 μL/min.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention saves the process of repeated separation and purification, and judges whether it is a pure culture with extracellular electron transfer ability through first-generation culture, and improves the screening efficiency;

(2) The present invention eliminates the interspecies competition of different electrochemically active bacteria through single-cell culture, especially avoids the singleness of screening results brought about by the inhibition of fast-growing electrochemically active bacteria to other electrochemically active bacteria;

(3) The rapid screening method for single cell separation provided by the present invention also provides a new separation and screening idea for the screening of other anaerobic environment microorganisms.

Description of drawings

Fig. 1 is screened in embodiment 1 to the bacterial strain phylogenetic tree construction result;

Fig. 2 is the start-up graph of the microbial electrochemical reactor in Example 2 after it is connected to the circuit. (a) is inoculating the strain in the microwell corresponding to 1-A5, and (b) is inoculating the strain in the microwell corresponding to 1-F10.

Detailed ways

The present invention will be further elaborated and illustrated below in conjunction with the accompanying drawings and specific embodiments. The technical features of the various implementations in the present invention can be combined accordingly on the premise that there is no conflict with each other.

Example 1

This embodiment provides a rapid screening method for anaerobic single-cell separation of electrochemically active bacteria, specifically as follows:

(1) Introduce nitrogen gas with a purity of 99.999% into the 20mL serum bottle for 5 minutes, quickly plug the rubber stopper and seal the aluminum cap, and autoclave at 121°C for 20 minutes before use to obtain an anaerobic bottle.

(2) Use a syringe to extract the battery liquid in the microbial fuel cell operated in the laboratory, and obtain the mixed bacterial liquid contained in it and place it in an anaerobic bottle. Use a bacterial counting plate to determine the concentration of bacteria contained in the above mixed bacterial solution, and use 1×PBS anaerobic buffer solution to dilute the mixed bacterial solution in the anaerobic bottle to obtain a diluted bacterial solution with a concentration of 100 cells/mL.

(3) Add 25g of LB nutrient broth and 1L of deionized water into the three-necked flask to seal it, pass high-purity nitrogen into one side, heat at 180°C for 1h to remove oxygen, and cool to 50°C to obtain LB medium. Use a 60mL syringe to take 50mL of LB medium and divide it into a 100mL serum bottle to seal it, then add 0.15g of nano-tungsten oxide particles, and autoclave at 121°C for 20 minutes to obtain anaerobic LB medium containing nano-tungsten oxide particles.

(4) In an anaerobic glove box, add 150 μL of anaerobic LB medium containing nano-tungsten oxide particles to each microwell of a 96-well cell culture plate with an 8-row pipette gun.

(5) In the anaerobic glove box, use a 20mL syringe to absorb the diluted bacterial solution in the anaerobic bottle, install the syringe on the micro-syringe pump LSP-02-2B, connect the syringe to the No. 7 needle with a PFA hose, and set The flow rate of the micro-injection pump is 500 μL/min, so that the needle can output a single droplet stably. Under this condition, each droplet theoretically contains one to several bacterial cells. Move the needle so that single droplets fall into individual microwells of the cell culture plate to obtain a bacterial culture plate. In this example, a total of 192 microwells were seeded in two 96-well plates.

It should be noted that the volume of a single droplet in each microwell of the above-mentioned cell culture plate and the concentration of the diluted bacterial solution are controlled so that each droplet theoretically contains one to several bacterial cells, preferably Theoretically, it contains only one bacterium, so that pure bacteria can be obtained through one-generation culture in the subsequent cultivation process. However, due to the aggregation characteristics of bacteria, there may be no bacteria in each droplet or multiple bacteria aggregates in actual operation.

(6) Put the cell culture plate containing the bacterial solution into an anaerobic bag, put an anaerobic gas producing bag in it and seal it, take it out from the anaerobic glove box, place it in a constant temperature shaker, and heat it at 30°C, 150r/h Incubate at constant temperature and shake for 1 h under the condition of 1 min. Anaerobic bags can absorb oxygen and release carbon dioxide to ensure an oxygen-free environment.

(7) Observe the color of nano-tungsten oxide particles in each microwell of the cell culture plate every other day. If the color of the liquid in one or more microwells changes from light yellow to cyan or dark blue, it indicates that the microwell The electrochromic reaction in which tungsten oxide turns into tungsten bronze appeared, so electrochemically active strains can be extracted from the micropores where the electrochromic reaction occurs.

Tungsten oxide is an n-type semiconductor material whose main structure is an octahedron composed of tungsten atoms and oxygen atoms connected end to end. There are pores in the interior of this octahedral structure, which can embed small molecular cations such as Na ⁺ and K ⁺ to form tungsten bronze. This structure makes tungsten oxide have electrochromic properties. Specifically, in terms of the principle involved in this example, tungsten oxide can receive electrons transferred from electrochemically active bacteria to the outside of the cell to form tungsten bronze, which is accompanied by light yellow oxidation in appearance. Tungsten turns to blue tungsten bronze, so a color change reaction is used to indicate the occurrence of extracellular electron transfer.

In this example, it was found that 3 micropores had different degrees of color change. In the anaerobic glove box, the bacterial liquid in the micropores with color change was sucked out, and a part was inoculated into the anaerobic LB solution containing nano-tungsten oxide particles for expansion and storage. 3'), 1492R (5'-TACGGTTACCTTGTTACGACTT-3') for PCR, and the genomic DNA of the screened strains was extracted and amplified for sequencing analysis. The strains were initially identified by BLAST sequence alignment of NCBI.

After comparison, the three strains isolated and screened belonged to two species in the genus Bacillus. Microwells 1-A5 and 2-A6 are Bacillus cereus, and 1-F10 is Bacillus pumilus. A phylogenetic tree was constructed according to the comparison results, and the results are shown in Figure 1.

It should be noted that although 3 strains of specific bacteria were isolated and screened in this example, the rapid screening method for anaerobic single-cell separation of electrochemically active bacteria provided by the present invention is a general screening method, which can be obtained from electrochemically active bacteria. The pure bacterial culture with extracellular electron transfer ability is screened out from the battery fluid in which the active bacteria are the dominant species. However, the specific strains obtained by each screening will be different due to the difference in the sampling of the battery fluid and the contingency of the experiment process, and it is not limited to only screen these three specific strains.

Example 2

In this example, in order to verify the electricity production ability of the strains screened in Example 1, a microbial electrochemical reactor was constructed for electrochemical characterization studies, as follows:

(1) A 1.5*1cm graphite electrode is used as the anode, a 1.5*1cm stainless steel mesh is used as the cathode, and titanium wire is used as the conductive material.

(2) Pass the titanium wire wire through the rubber stopper to suspend the graphite electrode and the stainless steel electrode in the reactor. The reactor is a 20mL three-opening glass bottle, which is sealed with an aluminum cover and autoclaved at 121°C for 20min. After the sterilization is completed, the reference electrode is installed on the reactor in the ultra-clean workbench, and sterile high-purity nitrogen is introduced to evacuate the oxygen in the reactor.

(3) Add 1g of glucose, 1g of sodium acetate, 0.31g of NH ₄ Cl, 0.13g of KCl, 12.5mL of trace salt solution, 1L of PBS with a concentration of 50mM into the three-necked flask and seal it, and then pass high-purity nitrogen into it and heat to remove oxygen , then stop heating, use a 60mL syringe to dispense into 100mL serum bottles and seal, fill each bottle with 50mL, autoclave at 121°C for 20 minutes before use, add 0.25mL sterile vitamins to each bottle after sterilization, and obtain the reactor culture solution .

(4) Select the Bacillus cereus (Bacillus cereus) and the Bacillus pumilus (Bacillus pumilus) in 1-F10 that are screened in the microwell 1-A5 in Example 1 to build electrochemical reactors respectively, and in each reactor Add 10 mL of reactor culture fluid, and inoculate the strains screened above at 10% volume of the reactor culture fluid. Connect the reactor into the circuit, add an external voltage of 0.7V to the cathode terminal, and connect a 10Ω external circuit resistance in series in the circuit to detect the current in the circuit. Use a data acquisition instrument to record the voltage data at both ends of the external circuit resistance every 20 minutes. The entire example was run in a 30°C constant temperature room.

As shown in Figure 2, Figure 2 (a) is a graph showing the output voltage of the electrochemical reactor Bacillus cereus (Bacillus cereus) in the inoculation microwell 1-A5 as a function of time, and a stable peak value appears at about 420h, at which time the current Density is about 30mA/m ² ; Fig. 2 (b) is the curve graph of the output voltage of the electrochemical reactor of Bacillus pumilus (Bacillus pumilus) in inoculation microwell 1-F10 with time, and a stable peak appears at about 400h, at this time The current density is about 20 mA/m ² .

The effectiveness of the separation and rapid screening method provided by the invention in screening electrochemically active bacteria is illustrated. Through single-cell culture, multiple separation and purification steps are omitted, screening time is saved, and interspecies competition between different electrochemically active bacteria is eliminated, especially the inhibition of fast-growing electrochemically active bacteria on other electrochemically active bacteria is avoided. The electrochemically active bacteria that grow relatively weakly in the separation system can also be screened out, increasing the diversity of strain resources. And the invention provides a new screening method for electrochemically active bacteria, which can be extended to the screening of other anaerobic environment microorganisms.

The above-mentioned embodiments are only preferred solutions of the present invention, but they are not intended to limit the present invention. Various changes and modifications can be made by those skilled in the relevant technical fields without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims (10)

1. an anaerobic single-cell separation rapid screening method of electrochemically active bacteria, is characterized in that, specifically as follows: S1: Obtain a mixed bacterial solution containing anaerobic microorganisms from a bioelectrochemical system, and dilute the mixed bacterial solution with an anaerobic buffer in an oxygen-free anaerobic bottle to obtain a diluted bacterial solution; S2: Add the anaerobic Luria-Bertani (LB) medium containing nano-tungsten oxide particles dropwise to each microwell of the cell culture plate, and drop the diluted bacterial solution to the microwell in the form of a single droplet Anaerobic culture is carried out in the microwell; and only one to several bacterial cells are included in the single diluted bacterial solution droplet added to the microwell; S3: During the anaerobic culture, observe whether there is a discoloration reaction of tungsten oxide converted to tungsten bronze in the microwells, and extract electrochemically active strains from the microwells where the discoloration reaction occurs. 2. the rapid screening method of anaerobic single cell separation of a kind of electrochemically active bacteria according to claim 1, it is characterized in that, described anaerobic bottle is to pass into the serum bottle and be 99.999% nitrogen gas 5min after sealing and obtained by autoclaving. 3 . The rapid screening method for anaerobic single cell separation of electrochemically active bacteria according to claim 1 , wherein the anaerobic buffer is 1×PBS buffer. 4 . 4. the rapid screening method of anaerobic single cell separation of a kind of electrochemically active bacteria according to claim 1, it is characterized in that, the preparation method of the anaerobic LB medium containing nano tungsten oxide particles is as follows: Add LB liquid medium to the flask and seal it, and heat it with nitrogen on one side to discharge oxygen; cool it after heating, and then add nano-tungsten oxide particles for high-pressure sterilization. 5. the rapid screening method of anaerobic single cell separation of a kind of electrochemically active bacterium according to claim 4, it is characterized in that, the addition amount of LB nutrient broth in the anaerobic LB culture medium containing nano tungsten oxide particles is 25g/L, and the addition amount of nano-tungsten oxide particles is 3g/L. 6. The rapid screening method for anaerobic single-cell separation of electrochemically active bacteria according to claim 4, wherein the temperature of the heating process is 180° C., and the heating duration is 1 h; the cooling temperature It is 50-60°C. 7. the rapid screening method of anaerobic single cell separation of a kind of electrochemically active bacteria according to claim 1, is characterized in that, described anaerobic culture process is that cell culture plate is packed in anaerobic bag and put into Anaerobic air bag, constant temperature shaking culture, and then constant temperature static culture. 8. The rapid screening method for anaerobic single-cell separation of electrochemically active bacteria according to claim 7, wherein the constant temperature shaking culture process is 30 ° C, 150 r/min conditions for 1 h; The constant temperature static culture condition is 30°C. 9. The rapid screening method of anaerobic single-cell separation of electrochemically active bacteria according to claim 1, wherein the diluted bacterial solution is output in the form of a single droplet by a droplet delivery combination device; The drop delivery combination device includes a micro-injection pump, a syringe and a needle, the syringe is installed on the micro-injection pump, and the syringe and the needle are connected by a PFA hose. 10. The rapid screening method of anaerobic single-cell separation of electrochemically active bacteria according to claim 9, wherein the concentration of the diluted bacterial solution is 100cells/mL, and the needle adopts a No. 7 needle. The flow rate of the micro-injection pump is 500 μL/min.

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