Method for detecting energy-tending movement of bacteria towards crystal minerals

Abstract

The invention discloses a method for detecting energy-tending movement of bacteria towards crystal minerals, and belongs to the technical field of biology. The method comprises the following steps: firstly, culturing bacteria with energy-taxis movement capability to a logarithmic phase to obtain a bacteria solution; secondly, uniformly mixing the agarose colloid with the crystal mineral suspension to prepare crystal mineral agarose balls coagulated on a polyester line; thirdly, preparing a bacterial suspension based on the bacterial liquid; and finally, suspending the crystal mineral agarose ball in the bacterial suspension, culturing, taking out, measuring the area of a section discoloration region of the crystal mineral agarose ball by using ImageJ software, and detecting the activity strength of the bacteria to the crystal mineral energy taxis movement. On the basis of dissolution and extraction of metal ions in agarose balls, the concentration of the metal ions generated by biologically reducing crystal minerals is effectively reflected by measuring the area of a section discoloration region, so that the activity strength of the energy-tending movement of bacteria to the crystal minerals is accurately detected; compared with a traditional swimming plate method, the method is simpler and more convenient to operate, meanwhile, bacterial cells can better show the swimming capacity in a liquid phase system, and the detection time is shortened.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a novel detection method, namely a suspended agarose ball method, which is used for detecting the kinetic movement of bacteria toward crystalline minerals. Background Art

[0002] In the ever-changing natural environment, many bacteria with mobility and degradation capabilities can effectively sense and locate metabolic substrates through taxis. Among these bacteria, there is a class of bacteria that can perform taxis, which sense and strategically move toward substances that can generate energy for themselves. Through taxis, this type of bacteria finds and uses electron acceptors in the environment to maintain their life activities and metabolic processes. These electron acceptors include organic pollutants, heavy metals, and metal oxides. Bacterial taxis improves the bioavailability and biodegradation efficiency of pollutants, and plays an important role in the microbial degradation of pollutants.

[0003] Harris et al. pointed out in Biochemical Society Transactions Vol. 40 in 2012 that bacteria can metabolize organic matter to produce electrons and transport these electrons across the membrane to the outside of the cell to complete the reduction of insoluble electron acceptors such as metal oxides and electrodes. This process is called extracellular electron transport (EET).

[0004] Minerals in nature are divided into crystalline and amorphous minerals. Microscopically, each crystalline mineral has different crystal forms and crystal faces. In recent years, many studies have shown that minerals with different crystal faces have different chemical properties. Huang Xiaopeng et al. pointed out in Environmental Science: Nano Volume 5 in 2018 that hematite with different crystal faces has different adsorption capacity for Cr(VI), and hematite with {001} crystal face (001-Fe2O3) is more likely to adsorb hexavalent chromium. This is because 001-Fe2O3 can provide more adsorption sites, which enhances the adsorption between it and Cr(VI). Rong Shaopeng et al. pointed out in ACS Catalysis Volume 8 in 2018 that manganese dioxide with different crystal faces has different catalytic oxidation ability for formaldehyde. Manganese dioxide with {310} crystal face (310-MnO2) has higher surface energy, which is conducive to the generation of oxygen vacancies and thus improves the HCHO oxidation activity. Therefore, 310-MnO2 has a stronger catalytic oxidation ability for formaldehyde.

[0005] Shewanella oneidensis MR-1 is a common facultative anaerobic bacterium widely distributed in most mesophilic environments such as soil, sediment, surface water and groundwater. Studies have shown that MR-1 can locate the location of insoluble electron acceptors through EET systems and signal transduction systems, thereby performing autonomous directional movement. This directional movement of MR-1 will affect the transformation and environmental fate of pollutants adsorbed on the surface of solid oxides. Based on this characteristic, MR-1 has broad application prospects in the bioremediation of environmental pollution. Therefore, it is necessary to develop a method for detecting the directional movement of MR-1 toward crystalline minerals.

[0006] The swimming plate method and the capillary method are the main experimental methods for studying the tropism of bacterial cells in recent years. However, when using the swimming plate method, each set of experiments requires multiple sets of swimming plates for comparison and control before a conclusion can be drawn. At the same time, the experimental method usually takes more than 24-48 hours, and the experimental conclusion cannot be obtained quickly. The capillary method is more suitable for studying the tropism activity of bacteria toward non-crystals and weak crystals. For most crystalline minerals, if the capillary is placed vertically, due to the high density and small gap of the crystalline minerals, the uneven dispersion in the liquid will block the capillary; if the capillary is placed horizontally, the crystalline minerals will be deposited on the side wall of the capillary, affecting the detection effect of tropism.

[0007] In order to circumvent the defects of the above-mentioned detection method, a detection method that is simple to operate and less time-consuming, suitable for the kinetic movement of bacteria to crystalline minerals, is developed. The present invention innovatively proposes a suspended agarose ball method. In a liquid phase system, bacterial cells can autonomously swim to agarose balls containing crystalline minerals, and reduce the crystalline minerals in the agarose balls to generate low-valent metal compounds of different colors, which in turn causes the agarose balls to gradually change color from the edge. This method can compare and analyze the kinetic movement of the same bacteria to different crystalline minerals in the same system, and the operation method is simple; based on the hydrochloric acid dissolution and extraction experiment of metal ions in agarose balls, it is verified that this method can effectively reflect the concentration of metal ions generated by biological reduction of crystalline minerals by measuring the area of ​​the cross-section color change region, thereby accurately detecting the strength of the kinetic movement activity of bacteria to crystalline minerals. In addition, bacterial cells are not restricted by solid phase gels in liquid phase systems, and can better demonstrate the ability to swim, thereby shortening the detection time. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a new method for detecting the kinetic movement of bacteria toward crystalline minerals: the suspended agarose ball method, which effectively reflects the concentration of metal ions generated by biological reduction of crystalline minerals by measuring the area of ​​the color change region of the agarose ball cross section, thereby accurately detecting the strength of the kinetic movement activity of bacteria toward crystalline minerals. At the same time, this method can compare and analyze the kinetic movement of the same bacteria toward different crystalline minerals in the same system, the operation method is simple, and the bacterial cells are not restricted by the solid phase gel in the liquid phase system, and can better show the ability to swim, thereby shortening the detection time. The present invention is intuitive, easy to operate and time-saving.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] A method for detecting bacterial tropism toward a crystalline mineral comprises: first, culturing bacteria with tropism to a logarithmic growth phase to obtain a cell concentration of 2×10 11 cells / mL of bacterial solution. Secondly, agarose colloid is evenly mixed with the crystal mineral suspension to prepare crystal mineral agarose balls condensed on the polyester line. Thirdly, a bacterial suspension is prepared based on the bacterial solution. Finally, the crystal mineral agarose balls are suspended in the bacterial suspension, taken out after cultivation, and the strength of the bacterial kinetic activity toward the crystal mineral is detected based on the area of ​​the color change region of the cross section of the crystal mineral agarose balls. Specifically, the following steps are included:

[0011] Step 1: Culture bacteria;

[0012] Step 1.1, select bacteria that can perform tactic movement as the research object, take out the bacteria from the -80℃ refrigerator, and place them on a sterile operating table that has been sterilized by ultraviolet light for 15 minutes after freezing and thawing for later use;

[0013] Step 1.2, dissolving tryptic soy broth (TSB) in 30 mL of ultrapure water to obtain a TSB medium with a concentration of 30 g / L, sterilizing by high pressure at 121° C. for 20 min, and using it for bacterial activation after cooling;

[0014] Step 1.3, dissolve TSB in 100 mL ultrapure water to obtain a TSB medium with a concentration of 30 g / L, sterilize it by high pressure at 121° C. for 20 min, and use it for bacterial expansion culture after cooling;

[0015] Step 1.4, in a sterile environment, take 100 μL of the strain from the bacterial culture medium obtained in step 1.1 and inoculate it into the TSB medium obtained in step 1.2, and then place the TSB medium in a constant temperature shaker at 30°C and 150 r / min for 12 hours to activate the strain. Add 100 μL of bacterial solution to each 30 mL of TSB medium used for bacterial activation;

[0016] Step 1.5, after the bacterial strain is activated, the bacterial solution is transferred to the TSB medium obtained in step 1.3 for expansion culture. The culture conditions are the same as those in step 1.4. After 12 hours, the bacteria are cultured to the logarithmic growth phase. 1 mL of bacterial solution is added to every 100 mL of TSB medium used for bacterial expansion culture.

[0017] Step 2: preparing crystal mineral agarose spheres;

[0018] Step 2.1, sieve the crystalline mineral through a 200-mesh sieve to obtain crystalline mineral powder, and ensure that the mineral particles can form a stably distributed mineral suspension. The crystalline minerals include hematite, pyrolusite, goethite, etc.;

[0019] Step 2.2, at room temperature, weigh the sieved crystal mineral powder and add it to sterilized ultrapure water to prepare a crystal mineral suspension of appropriate concentration. The concentration of the crystal mineral suspension is 3-4 mM;

[0020] Step 2.3, prepare 10mL 2% agarose colloid. Add 0.0238g HEPES to a beaker, add 0.0084g sodium bicarbonate, 0.001g peptone, 0.002g yeast extract powder after it is completely dissolved, and add 0.032g sodium lactate as an electron donor, shake well to make it a uniform solution, then drop 4M NaOH solution in midair until the solution pH = 7.2, transfer the above solution to a 10mL serum bottle, add 0.2g agarose powder, use high-purity nitrogen aeration for 20min to remove oxygen from the above mixture, sterilize it at 121℃ for 20min, form 2% agarose colloid, cool to an appropriate temperature and set aside, the agarose temperature is 50-60℃;

[0021] Step 2.4, under sterile conditions, uniformly mixing the agarose colloid obtained in step 2.3 and the crystal mineral suspension obtained in step 2.2 at a volume ratio of 1:1, so that the mineral particles are uniformly dispersed in the agarose colloid to obtain a mixed solution;

[0022] Step 2.5, use a pipette to draw 0.5 mL of the mixed solution obtained in step 2.4, and drop it into the mold pre-placed with the polyester line to form a crystalline mineral agarose ball condensed on the polyester line. The size and shape of the agarose ball can be adjusted according to experimental needs, and is generally a sphere with a radius of 5 mm.

[0023] Step 3: preparing bacterial suspension;

[0024] Step 3.1, prepare buffer solution. Prepare two bottles of 500 mL of 20 mM HEPES buffer solution, adjust the pH to neutral with NaOH, pass high-purity N2 into one bottle to remove O2, and sterilize both bottles at 121°C for 20 min. The one passed with high-purity N2 is defined as anaerobic HEPES buffer solution, and the other bottle is defined as aerobic HEPES buffer solution;

[0025] Step 3.2, collect the bacterial solution obtained in step 1.5 by centrifugation. After shaking the bacterial solution obtained in step 1.5, use a pipette to draw 20 mL into a sterilized centrifuge tube, centrifuge at a low speed to protect the bacterial flagella and ensure that the bacteria have good autonomous movement ability. After centrifugation, remove the supernatant and wash with aerobic HEPES buffer. Repeat the above operation 3 times and set aside. The low speed rotation speed is 5000-6000r, and the time is 3-5min;

[0026] Step 3.3: Resuspend the bacterial cells obtained in step 3.2 in an anaerobic chamber using anaerobic HEPES buffer to adjust the cell concentration to 2 × 10 11 cells / mL, obtain the bacterial suspension, and pipette 50mL of the bacterial suspension into a 50mL anaerobic penicillin bottle for later use.

[0027] Step 4: Suspend the crystal mineral agarose spheres in the bacterial suspension;

[0028] Step 4.1, suspending the agarose spheres containing crystalline minerals with different crystal faces condensed on the polyester thread prepared in step 2.5 in the MR-1 suspension obtained in step 3.3, and pressing them with a rubber stopper and an aluminum cap;

[0029] Step 4.2, place the experimental system obtained in step 4.1 in an anaerobic box at a temperature of 30°C for cultivation. Bacteria will autonomously tend to crystalline minerals in the liquid phase system and reduce them. Therefore, over time, bacteria can reduce crystalline minerals to low-valent compounds, causing the crystalline mineral agarose balls to gradually change color from the outside to the inside. After 12 hours, take out the agarose balls, divide the agarose balls into two hemispheres of equal volume along the diameter, place them on white paper, and take a photo containing two cross-sections. The above operations are all performed in an anaerobic box;

[0030] Step 4.3, import the cross-section photo obtained in step 4.2 into ImageJ version 1.54d, use the "Freehand selections" tool in the toolbar to select the non-discolored area, and then use the Measure tool under the "Analyze" option in the menu bar to measure the non-discolored area S1, and the result is displayed in the Area column; then measure the cross-section area S2, the method is the same as above, S2-S1 can get the discolored area S3. The larger S3 is, the stronger the kinetic activity of the bacteria toward the crystal mineral.

[0031] Beneficial effects of the present invention:

[0032] (1) The present invention intuitively reflects the strength of the bacterial kinetic activity toward crystalline minerals based on the size of the color change region of the agarose sphere cross section.

[0033] (2) The present invention can form a contrast in the same anaerobic penicillin bottle, is easy to operate, and avoids subtle differences in culture medium concentration, bacterial liquid concentration, and crystal mineral concentration between different bottles; at the same time, the bacteria are not restricted by the solid phase gel in the liquid phase system and can better demonstrate the ability to swim, so it takes less time. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a schematic diagram of the suspended agarose sphere method;

[0035] Figure 2 These are the characterization results of hematite with different crystal faces, where (A) is the scanning electron microscope image of 001-Fe2O3; (B) is the scanning electron microscope image of 001-Fe2O3; and (C) is the X-ray diffraction analysis image of hematite with different crystal faces.

[0036] Figure 3 These are the characterization results of pyrolusite with different crystal faces, where (A) is the scanning electron microscope image of 110-MnO2; (B) is the scanning electron microscope image of 310-MnO2; and (C) is the X-ray diffraction analysis image of pyrolusite with different crystal faces.

[0037] Figure 4 (A) is the correlation between the color change area of ​​the agarose sphere cross section and the Fe(II) concentration generated by MR-1 reducing hematite with different crystal faces.

[0038] Figure 4 (B) is the correlation between the color change area of ​​the agarose sphere cross section and the Mn(II) concentration generated by MR-1 reducing pyrolusite with different crystal faces. DETAILED DESCRIPTION

[0039] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0040] Example 1

[0041] Agarose sphere method was used to detect the kinetic activity of MR-1 towards hematite on different crystal faces

[0042] Shewanella MR-1 was selected as the research object of tropism;

[0043] Step 1: Cultivating Shewanella MR-1;

[0044] Step 1.1, select MR-1 as the research object, take out the MR-1 bacteria from the -80℃ refrigerator, and place it on a sterile operating table that has been sterilized by UV for 15 minutes after freezing and thawing for later use;

[0045] Step 1.2, dissolve TSB in 30 mL ultrapure water to obtain a TSB medium with a concentration of 30 g / L, sterilize it by high pressure at 121°C for 20 min, and use it for MR-1 activation after cooling;

[0046] Step 1.3, dissolve TSB in 100 mL ultrapure water to obtain a TSB medium with a concentration of 30 g / L, sterilize it by high pressure at 121°C for 20 min, and use it for the expansion culture of MR-1 after cooling;

[0047] Step 1.4, in a sterile environment, take 100 μL of the strain from the bacterial culture medium obtained in step 1.1 and inoculate it into the TSB medium obtained in step 1.2, and then place the TSB medium in a constant temperature shaker at 30°C and 150 r / min for 12 hours to activate MR-1. Add 100 μL of MR-1 bacterial solution to each 30 mL of TSB medium used for MR-1 activation;

[0048] Step 1.5, after the MR-1 activation treatment, the MR-1 bacterial solution is transferred to the TSB medium obtained in step 1.3 for expansion culture. The culture conditions are the same as those in step 1.4. After 12 hours, MR-1 is cultured to the logarithmic growth phase. 1 mL of MR-1 bacterial solution is added to each 100 mL of TSB medium used for MR-1 expansion culture.

[0049] Step 2: preparing two hematite agarose spheres with different crystal faces;

[0050] Step 2.1, synthesize {001} crystal face hematite (001-Fe2O3) and {100} crystal face hematite (100-Fe2O3) by hydrothermal method, characterize their morphology by SEM, identify the mineral species and verify the purity by XRD. Pass the above two different crystal face hematites through a 200 mesh sieve to obtain two different crystal face hematite powders;

[0051] Step 2.2, at room temperature, weigh the sieved two kinds of hematite powders with different crystal faces and add them into sterilized ultrapure water to prepare 3 mM suspensions of the two kinds of hematite with different crystal faces;

[0052] Step 2.3, prepare 10mL 2% agarose colloid. Add 0.0238g HEPES to a beaker, add 0.0084g sodium bicarbonate, 0.001g peptone, 0.002g yeast extract powder after it is completely dissolved, and add 0.032g sodium lactate as an electron donor, shake well to make it a uniform solution, then add 4M NaOH solution dropwise in midair until the solution pH = 7.2, transfer the above solution to a 10mL serum bottle, add 0.2g agarose powder, use high-purity nitrogen aeration for 20min to remove oxygen from the above mixture, sterilize it at 121℃ for 20min under high pressure to form 2% agarose colloid, cool it to 50℃ and set aside;

[0053] Step 2.4, under sterile conditions, uniformly mixing the agarose colloid obtained in step 2.3 and the two hematite suspensions with different crystal faces obtained in step 2.2 at a volume ratio of 1:1, so that the hematite particles are uniformly dispersed in the agarose colloid to obtain a mixed solution;

[0054] Step 2.5, use a pipette to draw 0.5 mL of the mixed solution obtained in step 2.4, and drop it into the mold pre-placed with the polyester thread to form a hematite agarose ball condensed on the polyester thread. The agarose ball is spherical with a radius of 5 mm.

[0055] Step 3: preparing MR-1 suspension;

[0056] Step 3.1, prepare buffer solution. Prepare two bottles of 500 mL of 20 mM HEPES buffer solution, adjust the pH to neutral with NaOH, pass high-purity N2 into one bottle to remove O2, and sterilize both bottles at 121°C for 20 min. The one passed with high-purity N2 is defined as anaerobic HEPES buffer solution, and the other bottle is defined as aerobic HEPES buffer solution;

[0057] Step 3.2, collect the MR-1 bacterial solution obtained in step 1.5 by centrifugation. After shaking the MR-1 bacterial solution obtained in step 1.5, use a pipette to transfer 20 mL to a sterilized centrifuge tube, centrifuge at 6000r for 3 min, remove the supernatant after centrifugation, and then wash with aerobic HEPES buffer. Repeat the above operation 3 times and set aside;

[0058] Step 3.3: Resuspend the MR-1 cells obtained in step 3.2 in an anaerobic chamber using anaerobic HEPES buffer to adjust the cell concentration to 2 × 10 11 cells / mL, and obtain MR-1 suspension. Pipette 50mL of MR-1 suspension into a 50mL anaerobic penicillin bottle for later use.

[0059] Step 4: Suspend hematite agarose spheres in MR-1 suspension;

[0060] Step 4.1, suspend the two different crystal face hematite agarose spheres condensed on the polyester line prepared in step 2.5 in the MR-1 suspension obtained in step 3.3, and press them tightly with a rubber stopper and an aluminum cap;

[0061] Step 4.2, place the experimental system obtained in step 4.1 in an anaerobic box at a temperature of 30°C for cultivation, and take out the agarose balls after 12 hours. Since MR-1 will autonomously move toward hematite in the liquid phase system and reduce it, as time goes by, MR-1 reduces the red hematite to yellow iron carbonate, making the agarose balls containing hematite gradually lighter in color from the outside to the inside. MR-1 has different energy-movement abilities toward hematite with different crystal faces, so the agarose balls containing hematite with different crystal faces have different sizes of lighter areas. Figure 1 As shown, the agarose sphere is divided into two hemispheres of equal volume along the diameter, placed on white paper, and a photo containing two cross sections is taken. The above operations are all carried out in an anaerobic box;

[0062] Step 4.3, import the cross-section photo obtained in step 4.2 into ImageJ version 1.54d, use the "Freehand selections" tool in the toolbar to select the undiscolored area, and then use the Measure tool under the "Analyze" option in the menu bar to measure the area S1 of the undiscolored area, and the result is displayed in the Area column; then measure the cross-section area S2, the method is the same as above, S2-S1 can get the discolored area S3. The larger S3 is, the stronger the energy-movement activity of MR-1 toward the hematite on the crystal surface.

[0063] To further verify the accuracy of this method, the agarose sphere was taken out every 4 hours, divided into two hemispheres of equal volume along the diameter, and the area of ​​the discoloration region was measured using ImageJ software, and the sphere was placed in 5mM hydrochloric acid for dissolution and extraction of Fe(II). After 24 hours of dissolution and extraction, 1 mL was taken to pass through a water filter membrane with a pore size of 0.22um. 0.2 mL of the sample after passing through the membrane was added with 1.8 mL of phenanthroline colorimetric agent, and the color was developed for 20 minutes. The above operations were all performed in an anaerobic box. After color development, the solution was taken out, the absorbance at 562 nm was measured, and the Fe(II) concentration generated by MR-1 reducing hematite with different crystal faces was calculated. Fitting the above two sets of data found that the area of ​​the discoloration region of the profile was positively correlated with the concentration of Fe(II) generated by MR-1 reduction, that is, the larger the area of ​​the discoloration region, the greater the concentration of Fe(II) generated by MR-1 reduction, indicating that the energy-movement activity of MR-1 to the crystal face hematite is stronger, and the method is accurate.

[0064] In Example 1, the suspended agarose ball method can detect the strength of MR-1's tactic movement activity toward hematite on different crystal faces. Among them, at 12 hours, the color change of agarose balls in the MR-1 bacterial solution and the results of the cross-sectional photos of agarose balls can show that in the bacterial solution, the edge color of the agarose balls containing hematite gradually becomes lighter, indicating that MR-1 can sense the presence of hematite on different crystal faces and perform tactic movement.

[0065] Figure 4 (A) is the correlation between the area of ​​the color change region of the agarose sphere section and the Fe(II) concentration generated by MR-1 reducing hematite with different crystal faces after taking out the samples every 4 hours in Example 1. The results show that the area of ​​the color change region of the agarose sphere section is positively correlated with the Fe(II) concentration generated by MR-1 reducing hematite, and the area of ​​the color change region of the 001-Fe2O3 agarose sphere section is larger at the same time, indicating that the energy-movement activity of MR-1 to 001-Fe2O3 is greater than the energy-movement activity to 100-Fe2O3. At the same time, it is verified that the Fe(II) concentration generated by MR-1 reducing 001-Fe2O3 is higher, and the method is accurate.

[0066] Example 2

[0067] Using agarose sphere method to investigate the difference in MR-1's energetic movement toward different crystal faces of pyrolusite

[0068] Shewanella MR-1 was selected as the research object of tropism;

[0069] Step 1: as step 1 in Example 1;

[0070] Step 2: preparing two pyrolusite agarose balls with different crystal faces;

[0071] Step 2.1, synthesize {110} crystal face pyrolusite (110-MnO2) and {310} crystal face pyrolusite (310-MnO2) by hydrothermal method, characterize their morphological characteristics by SEM, identify the mineral species and verify the purity by XRD. Pass the above two different crystal face pyrolusites through a 200 mesh sieve to obtain two different crystal face pyrolusite powders;

[0072] Step 2.2, at room temperature, weigh two sieved pyrolusite powders with different crystal faces and add them into sterilized ultrapure water to prepare 4 mM suspensions of two different crystal faces of pyrolusite;

[0073] Step 2.3, prepare 10mL 2% agarose colloid. Add 0.0238g HEPES to a beaker, add 0.0084g sodium bicarbonate, 0.001g peptone, 0.002g yeast extract powder after it is completely dissolved, and add 0.032g sodium lactate as an electron donor, shake well to make it a uniform solution, then add 4M NaOH solution dropwise in midair until the solution pH = 7.2, transfer the above solution to a 10mL serum bottle, add 0.2g agarose powder, use high-purity nitrogen aeration for 20min to remove oxygen from the above mixture, sterilize it at 121℃ for 20min, form 2% agarose colloid, cool to 60℃ and set aside;

[0074] Step 2.4, under sterile conditions, uniformly mixing the agarose colloid obtained in step 2.3 and the two pyrolusite suspensions with different crystal faces obtained in step 2.2 at a volume ratio of 1:1, so that the pyrolusite particles are uniformly dispersed in the agarose colloid to obtain a mixed solution;

[0075] Step 2.5, use a pipette to draw 0.5 mL of the mixed solution obtained in step 2.4, and drop it into the mold pre-placed with the polyester thread to form a pyrolusite agarose ball condensed on the polyester thread. The agarose ball is spherical with a radius of 5 mm.

[0076] Step 3: preparing MR-1 suspension;

[0077] Step 3.1, as in step 3.1 of Example 1;

[0078] Step 3.2, collect the MR-1 bacterial solution obtained in step 1.5 by centrifugation. After shaking the MR-1 bacterial solution obtained in step 1.5, use a pipette to transfer 20 mL to a sterilized centrifuge tube, centrifuge at 5000r for 5 minutes, remove the supernatant after centrifugation, and then wash with aerobic HEPES buffer. Repeat the above operation 3 times and set aside;

[0079] Step 3.3, as step 3.3 in Example 1.

[0080] Step 4: Suspend the pyrolusite agarose spheres in the MR-1 suspension;

[0081] Step 4.1, suspend the two different crystal surface pyrolusite agarose balls condensed on the polyester line prepared in step 2.5 in the MR-1 suspension obtained in step 3.3, and press them tightly with a rubber stopper and an aluminum cap;

[0082] Step 4.2, place the experimental system obtained in step 4.1 in an anaerobic box at a temperature of 30°C for cultivation, and take out the agarose ball after 12 hours. Since MR-1 will autonomously move toward pyrolusite in the liquid phase system and reduce it, as time goes by, MR-1 reduces the black pyrolusite to white manganese carbonate, making the agarose ball containing pyrolusite gradually lighter in color from the outside to the inside. MR-1 has different energy-movement abilities toward pyrolusite with different crystal faces, so the agarose ball containing pyrolusite with different crystal faces has different sizes of lighter areas. Figure 1 As shown, the agarose sphere is divided into two hemispheres of equal volume along the diameter, placed on white paper, and a photo containing two cross sections is taken. The above operations are all carried out in an anaerobic box;

[0083] Step 4.3, import the cross-section photo obtained in step 4.2 into ImageJ version 1.54d, use the "Freehand selections" tool in the toolbar to select the non-discolored area, and then use the Measure tool under the "Analyze" option in the menu bar to measure the area S1 of the non-discolored area, and the result is displayed in the Area column; then measure the cross-section area S2, the method is the same as above, S2-S1 can get the discolored area S3. The larger S3 is, the stronger the energy-movement activity of MR-1 toward the crystal surface pyrolusite is.

[0084] To further verify the accuracy of this method, the agarose sphere was taken out every 4 hours, divided into two hemispheres of equal volume along the diameter, and the area of ​​the discoloration region was measured using ImageJ software, and the sphere was placed in 5mM hydrochloric acid for dissolution and extraction of Mn(II). After 24 hours of dissolution and extraction, 5mL was taken to pass through a water filter membrane with a pore size of 0.22um, and 1mL of the sample after passing through the membrane was taken. 5mL of sodium pyrophosphate-sodium acetate buffer solution and 2mL of potassium periodate solution were added in sequence, and the reaction was carried out for 15min. The above operations were all carried out in an anaerobic box. After color development, the solution was taken out, the absorbance at 525nm was measured, and the concentration of Mn(II) generated by MR-1 reducing different crystal face pyrolusite was calculated. Fitting the above two sets of data found that the area of ​​the section discoloration region was positively correlated with the concentration of Mn(II) generated by MR-1 reduction, that is, the larger the area of ​​the discoloration region, the greater the concentration of Mn(II) generated by MR-1 reduction, indicating that the energy-movement activity of MR-1 to the crystal face pyrolusite is stronger, and this method is accurate.

[0085] In Example 2, the suspended agarose ball method can be used to detect the strength of the kinetic activity of MR-1 toward pyrolusite with different crystal faces. Among them, at 12 hours, the results of the color change of the agarose ball in the MR-1 bacterial solution and the cross-sectional photo of the agarose ball can show that in the bacterial solution, the edge color of the agarose ball containing pyrolusite gradually becomes lighter, while the agarose ball without pyrolusite does not change color, indicating that MR-1 can sense the presence of pyrolusite with different crystal faces and perform kinetic movement.

[0086] Figure 4 (B) is in Example 2, after taking out the sample every 4h, the correlation between the color change area of ​​the agarose sphere section and the Mn(II) concentration generated by MR-1 reducing pyrolusite with different crystal faces is measured. The results show that the color change area of ​​the agarose sphere section is positively correlated with the Mn(II) concentration generated by MR-1 reducing pyrolusite, and the color change area of ​​the 310-MnO2 agarose sphere section is larger at the same time, indicating that the energy movement activity of MR-1 to 310-MnO2 is greater than the energy movement activity to 110-MnO2. At the same time, it is verified that the Mn(II) concentration generated by MR-1 reducing 310-MnO2 is higher, and the method is accurate.

[0087] Comparative Example 1

[0088] The agarose spheres containing hematite with different crystal faces in Example 1 were replaced with agarose spheres without hematite, and other parameters, variables and operations were consistent with those in Example 1.

[0089] In Comparative Example 1, the agarose spheres without hematite did not change color after being cultured in the bacterial solution for 12 h, indicating that MR-1 can sense the presence of hematite with different crystal faces and perform energy-tracing movement.

[0090] Comparative Example 2

[0091] The MR-1 bacterial suspension in Example 1 was replaced with sterilized ultrapure water, and other parameters, variables and operations were consistent with those in Example 1.

[0092] In Comparative Example 2, the agarose spheres containing two different crystal faces of hematite did not change color after being cultured in ultrapure water for 12 hours, indicating that this method can effectively detect the strength of the tropism activity of MR-1 toward hematite with different crystal faces.

[0093] Comparative Example 3

[0094] The agarose spheres containing pyrolusite with different crystal faces in Example 2 were replaced with agarose spheres without pyrolusite, and other parameters, variables and operations were consistent with those in Example 2.

[0095] In Comparative Example 3, the agarose spheres without pyrolusite did not change color after being cultured in the bacterial solution for 12 h, indicating that MR-1 can sense the presence of pyrolusite with different crystal faces and perform energetic movement.

[0096] Comparative Example 4

[0097] The MR-1 bacterial suspension in Example 2 was replaced with sterilized ultrapure water, and other parameters, variables and operations were consistent with those in Example 2.

[0098] In Comparative Example 4, the agarose spheres containing two different crystal faces of pyrolusite did not change color after being cultured in ultrapure water for 12 h, indicating that this method can effectively detect the strength of the kinetic activity of MR-1 toward pyrolusite with different crystal faces.

[0099] The above-described embodiments merely express the implementation methods of the present invention, but they should not be understood as limiting the scope of the present invention. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for detecting the kinetic movement of bacteria toward crystalline minerals, characterized in that: The detection method comprises the following steps: Step 1, culturing bacteria with tropism to a logarithmic growth phase to obtain a bacterial solution; Step 2, uniformly mixing agarose colloid and crystal mineral suspension to prepare crystal mineral agarose balls condensed on the polyester thread; Step 3, preparing a bacterial suspension based on the bacterial solution; Step 4, suspending the crystal mineral agarose spheres in the bacterial suspension, taking them out after culturing, and detecting the strength of the bacterial tropism toward the crystal mineral based on the area of ​​the color change region of the cross section of the crystal mineral agarose spheres.

2. The method for detecting the kinetic movement of bacteria toward crystalline minerals according to claim 1, characterized in that: The step 1 is specifically as follows: Step 1.1, select bacteria that can perform tactic movement as the research object, take out the bacteria from the -80℃ refrigerator, and place them on a sterile operating table for later use after freezing and thawing; Step 1.2, dissolving tryptic soy bean liquid medium TSB in ultrapure water to obtain TSB medium with a concentration of 30 g / L, sterilizing by high pressure at 121° C. for 20 min, and using it for bacterial activation after cooling; Step 1.3, dissolving TSB in ultrapure water to obtain a TSB medium with a concentration of 30 g / L, sterilizing by high pressure at 121° C. for 20 min, and using it for bacterial expansion culture after cooling; Step 1.4, in a sterile environment, extract the strain from the bacterial culture medium in step 1.1 and inoculate it into the TSB medium in step 1.2, and place the TSB medium in a constant temperature shaker at 30°C and 150 rpm for 12 hours to activate the strain; Step 1.5, after the bacterial strain is activated, the bacterial solution is transferred to the TSB medium obtained in step 1.3 for expansion culture. The culture conditions are the same as those in step 1.

4. After 12 hours, the bacteria are cultured to the logarithmic growth phase.

3. The method for detecting the kinetic movement of bacteria toward crystalline minerals according to claim 2, characterized in that: The aseptic operating table in step 1.1 is subjected to ultraviolet sterilization treatment; In step 1.4, 100 μL of the strain was added to every 30 mL of TSB medium; In the step 1.5, 1 mL of bacterial solution is added to every 100 mL of TSB medium used for bacterial expansion culture.

4. The method for detecting the kinetic movement of bacteria toward crystalline minerals according to claim 1, characterized in that: The step 2 is specifically as follows: Step 2.1, sieving the crystalline mineral to obtain crystalline mineral powder; Step 2.2, adding the crystalline mineral powder into sterilized ultrapure water to prepare a crystalline mineral suspension, wherein the concentration of the crystalline mineral suspension is 3 to 4 mM; Step 2.3, preparing agarose colloid; Step 2.4, under sterile conditions, uniformly mixing the agarose colloid obtained in step 2.3 and the crystal mineral suspension obtained in step 2.2 at a volume ratio of 1:1 to obtain a mixed solution; Step 2.5, add the mixed solution of step 2.4 dropwise to the mold pre-placed with the polyester thread to form crystalline mineral agarose balls condensed on the polyester thread. The size and shape of the agarose balls are adjusted as needed.

5. The method for detecting the kinetic movement of bacteria toward crystalline minerals according to claim 3, characterized in that: In the step 2.1, the crystalline mineral is sieved through a 200-mesh sieve to obtain crystalline mineral powder; the crystalline mineral includes hematite, pyrolusite, and goethite; In the step 2.3, the process of preparing agarose colloid is as follows: after 0.0238g HEPES is completely dissolved, 0.0084g sodium bicarbonate, 0.001g peptone, 0.002g yeast extract powder, and 0.032g sodium lactate are added, and the mixture is shaken and mixed to form a uniform solution; 4M NaOH solution is added dropwise in midair until the solution pH is 7.2, the above solution is transferred to a serum bottle, 0.2g agarose powder is added, aerated with high-purity nitrogen for 20min, and sterilized at 121°C for 20min to form 2% agarose colloid, and cooled to 50-60°C for use; In step 2.5, the agarose sphere is a sphere with a radius of 5 mm.

6. The method for detecting the kinetic movement of bacteria toward crystalline minerals according to claim 2, characterized in that: The step 3 of preparing the bacterial suspension is specifically as follows: Step 3.1, prepare two bottles of HEPES buffer solution, one bottle is an anaerobic HEPES buffer solution, and the other bottle is an aerobic HEPES buffer solution; Step 3.2, collect the bacterial solution obtained in step 1.5 by centrifugation; after shaking the bacterial solution in step 1.5, centrifuge at a low speed to remove the supernatant, wash with aerobic HEPES buffer, repeat the above operation several times and set aside; Step 3.3: Resuspend the bacterial cells obtained in step 3.2 in an anaerobic chamber using anaerobic HEPES buffer to adjust the cell concentration to 2 × 10 11 cells / mL to obtain bacterial suspension.

7. The method for detecting the kinetic movement of bacteria toward crystalline minerals according to claim 1, characterized in that: The step 3.1 is specifically as follows: prepare two bottles of 20 mM HEPES buffer solution, adjust the pH to neutral using NaOH, introduce high-purity N2 into one bottle to remove O2, and sterilize both bottles by high pressure at 121° C. for 20 min for standby use. The one introduced with high-purity N2 is defined as the anaerobic HEPES buffer solution, and the other bottle is defined as the aerobic HEPES buffer solution; In step 3.2, the low speed is 5000-6000r, and the time is 3-5min.

8. The method for detecting the kinetic movement of bacteria toward crystalline minerals according to claim 1, characterized in that: Step 4: Step 4.1, suspending the agarose beads containing crystalline minerals with different crystal faces condensed on the polyester thread in the bacterial suspension obtained in step 3.3; Step 4.2, placing the system in step 4.1 in an anaerobic box at a temperature of 30°C for cultivation, and the bacteria cause the crystal mineral agarose sphere to gradually change color from the outside to the inside; after the cultivation, the agarose sphere is taken out, and the agarose sphere is divided into two hemispheres of equal volume along the diameter, and placed on white paper, and a photo containing two cross-sections is taken; Step 4.3, based on the cross-sectional photograph obtained in step 4.2, measure the area S1 of the non-discolored region, measure the cross-sectional area S2, and then obtain the discolored region area S3 by S2-S1; the larger S3 is, the stronger the kinetic activity of the bacteria toward the crystalline mineral.

9. The method for detecting the kinetic movement of bacteria toward crystalline minerals according to claim 8, characterized in that: The operations in step 4.2 are all carried out in an anaerobic chamber; the culture time is 12 hours.

10. The method for detecting the kinetic movement of bacteria toward crystalline minerals according to claim 8, characterized in that: The step 4.3 is specifically as follows: import the cross-sectional photograph obtained in step 4.2 into ImageJ version 1.54d, use the "Freehand selections" tool in the toolbar to select the non-discolored area, and then use the Measure tool under the "Analyze" option in the menu bar to measure the area S1 of the non-discolored area, and the result is displayed in the Area column; then measure the cross-sectional area S2, using the same method as above, and obtain the discolored area S3 by S2-S1.

Images