Quantitative counting method for biochar-loaded microorganisms

Abstract

The invention discloses a quantitative counting method for biochar-loaded microorganisms. The method comprises the following steps: 1) centrifuging a to-be-detected bacterial liquid of biochar-loaded microorganisms, and determining the free bacterial concentration of the supernatant bacterial liquid by adopting a plate counting method; (2) cleaning and drying the biochar loaded with microorganisms, measuring the mass of the biochar, adding normal saline for the first time, oscillating, centrifuging, taking supernatant liquid, counting by adopting a plate counting method, and recording a counting result as A1; adding normal saline into the centrifugal tube without the supernatant for the second time, shaking, centrifuging, taking the supernatant, counting, and recording the counting result as A2; and performing result verification according to the following a)-c) to obtain the quantitative count of the to-be-detected bacterial liquid of the biochar-loaded microorganisms: when A2lt is greater than or equal to 0 and smaller than 5% A1, the count is A1, when A2 is greater than or equal to 5% A1 and smaller than or equal to 20% A2, the count is A1 + A2, when A2gt is greater than or equal to 5% A1 and smaller than or equal to 20% A2, the experimental result is inaccurate, and redetermination is needed. The method effectively solves the problems of bacterial loss, free bacterial interference, low desorption and adsorption efficiency and the like in the traditional technology.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial detection and biochar application, and relates to a quantitative counting method for biochar loaded microorganisms. BACKGROUND

[0002] Biochar, as a carbon-based material with high specific surface area and porous structure, is widely used in environmental remediation, agricultural microbial preparations and industrial fermentation due to its excellent adsorption performance and stability. By loading beneficial microorganisms (such as Bacillus subtilis), biochar can enhance the environmental resistance of bacterial cells, prolong the survival time and improve the colonization efficiency, thereby optimizing its application effect in pollution degradation, soil improvement and biological fertilizer. However, the loading efficiency and survival number of microorganisms on the surface of biochar directly affect its actual performance, so there is an urgent need for a precise and reliable counting method to evaluate the activity and concentration of loaded bacteria and provide a scientific basis for the optimization of microbial-carrier composite systems.

[0003] At present, there is no unified standard for the quantitative counting method of biochar loaded microorganisms, and the common techniques have significant defects: (1) loss of bacterial cells: high-speed centrifugation or ultrasonic treatment may damage the structure of microbial cells, leading to underestimation of the number of viable bacteria; (2) interference of free bacteria: free bacteria on the surface of biochar that are not completely washed off are easily miscounted as loaded bacteria, resulting in an overestimation of the results; (3) low desorption efficiency: traditional shaking or washing methods are difficult to completely remove the attached bacteria, affecting the accuracy of counting. In addition, the existing methods generally lack verification steps, which cannot determine whether the desorption is sufficient or whether there are residual bacteria that have not been detected, resulting in insufficient data reliability.

[0004] Therefore, it is of great significance to develop a standardized counting method that combines efficient desorption, gradient separation and multi-step verification to promote the industrial application of microbial-biochar technology. SUMMARY

[0005] The purpose of the present application is to provide a quantitative counting method for biochar loaded microorganisms.

[0006] The quantitative counting method for biochar loaded microorganisms provided by the present application comprises the following steps: 1) centrifuging the biochar loaded microorganism sample solution to separate the supernatant and the precipitate, and obtaining the bacterial solution and the biochar loaded microorganisms, respectively; 2) determining the concentration of free bacteria in the sample solution by plate counting method; 3) washing the biochar loaded microorganisms with a buffer solution to remove the free bacteria; 4) repeating steps 2) and 3) to obtain the concentration of loaded bacteria; and 5) calculating the total number of loaded bacteria in the biochar by multiplying the concentration of loaded bacteria by the volume of biochar. 2) washing and drying the microorganism-loaded biochar, determining the mass of the microorganism-loaded biochar, then centrifuging after adding physiological saline for the first time, taking the supernatant, and counting the number of microorganisms in the supernatant by plate counting, and recording the counting result as A1; centrifuging after adding physiological saline for the second time, taking the supernatant, and counting the number of microorganisms in the supernatant by plate counting, and recording the counting result as A2; then verifying the result according to a)-c) as follows, that is, obtaining the quantitative counting of the microorganism-loaded biochar to be tested: When 0≤A2<5%A1, the counting of the microorganism-loaded biochar to be tested is A1; When 5%A1≤A2≤20%A2, the counting of the microorganism-loaded biochar to be tested is A1+A2; When A2>20%A1, the experimental result is inaccurate, and the microorganism-loaded biochar to be tested needs to be taken again to determine the above steps 1)-2).

[0007] In the above method, in step 1), the speed of centrifugation is 1300r / min-1500r / min, and specifically can be 1500r / min, and the time is 3-4 min, and specifically can be 3 min; specifically, the speed can be adjusted according to different bacteria, and the speed and time are adapted to each other, and the higher the speed, the shorter the time required. After gradient dilution, the number of microorganisms in the bacterial solution is counted by plate counting, and specifically, the bacterial solution can be diluted by 10 times.

[0008] In the above method, in step 2), the washing process is as follows: the microorganism-loaded biochar is added to physiological saline, and after oscillation, centrifugation is performed at a speed of 4000-5000r / min (specifically, 5000r / min) for 2-3min (specifically, 3min), The drying temperature is 38℃-40℃, and specifically can be 38℃.

[0009] In the above method, in step 2), the mass of the microorganism-loaded biochar is m c , and the amount of physiological saline added for the first time is 10xm c .

[0010] In the present application, in step 2), the mass of the microorganism-loaded biochar is determined as follows: the empty centrifuge tube is weighed, and the mass is m, the total mass of the microorganism-loaded biochar after washing and drying in the centrifuge tube is m1, and the mass of the microorganism-loaded biochar after drying is m c , m c =m1-m.

[0011] In the method, in step 2), after the first addition of physiological saline, the shaking condition is as follows: the speed can be 3000-4000 r / min, specifically 3000 r / min, and the time can be 20-30 min, specifically 30 min; the shaking is carried out in a vortex mixer to desorb the biochar-loaded microorganisms. The speed of the centrifugation can be 1300-1500 r / min, specifically 1500 r / min, and the time is 3-4 min, specifically 3 min, and after the centrifugation, the layers are separated.

[0012] In the method, in step 2), after the second addition of physiological saline, the speed of the centrifugation can be 3000-5000 r / min, specifically 5000 r / min, and the time can be 2-3 min, specifically 3 min.

[0013] In the method, in step 2), the upper clear liquid is gradiently diluted and then counted by the plate counting method.

[0014] The present application has the following beneficial effects: 1. The present application effectively solves the problems of cell loss, free bacteria interference and low desorption efficiency in the traditional technology by gradient separation, high-efficiency desorption and multi-step verification method.

[0015] 2. The low-speed centrifugation combined with physiological saline washing is used to accurately separate the loaded bacteria and free bacteria; the high-intensity shaking and repeated verification are used to ensure complete desorption and significantly improve the counting accuracy.

[0016] 3. The method has high standardization and simple operation, and provides reliable technical support for the application of the microorganism-biochar composite system. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The flowchart of the quantitative counting method of the biochar-loaded microorganisms of the present application Figure 2 The gradient dilution diagram of the plate counting method. DETAILED DESCRIPTION

[0018] In the following examples, the experimental methods used are conventional methods unless otherwise specified.

[0019] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.

[0020] Example 1 The quantitative counting method of the biochar-loaded microorganisms is carried out according to the flowchart shown in the figure. Figure 1

[0021] ​Before sampling, the centrifuge tube used in the experiment was first weighed, and the mass was recorded as m, and the result was 12.84 g; (1) Concentration of free Bacillus subtilis in bacterial solution: The biochar used in the example is corn straw biochar. The corn straw was collected from a farm in Lianyungang City, Jiangsu Province. After drying, it was cut into 3~5 cm pieces, crushed by a grinder, and passed through a 150-mesh sieve. It was pyrolyzed at 500℃ for 2 hours in a tubular furnace under N2 environment. The strain used is Bacillus subtilis (CGMCC 1.12939). After the biochar-loaded Bacillus subtilis was fermented in a fermenter for 24 hours, 30mL of fermentation liquid was taken with a centrifuge tube and centrifuged at a low speed of 1500r / min for 3 minutes. The purpose was to settle the biochar in the fermentation liquid to the bottom and retain the free bacteria in the fermentation liquid in the upper layer. Pour the upper bacterial liquid into another centrifuge tube and shake it to mix well. Take 1ml of the bacterial liquid for gradient dilution (the specific dilution gradient is as follows) Figure 2 The plate count method was used to count the number of cells, which was recorded as A0. The result was 3.5×10 10 CFU / mL .

[0022] (2) Concentration of Bacillus subtilis loaded on biochar: The first step is to determine the mass of biochar: add physiological saline to the centrifuge tube containing biochar to 30mL, shake it and centrifuge it at 5000r / min for 3min, and discard the supernatant to remove the free bacteria on the surface of the biochar. Then place the centrifuge tube in a hot air blower and dry it at 38℃. Weigh the centrifuge tube and record the mass as m1, m1=13.86g. At this time, the mass of biochar in the centrifuge tube is m c = m1-m, that is, m c =13.86g-12.84g=1.02g.

[0023] Step 2: Desorption of Bacillus subtilis: Add 10×m c (i.e. 10.2 g ) mass of physiological saline, using a vortex shaker at 3000r / min for 30 minutes to remove the bacteria loaded on the biochar. Then place the centrifuge tube in a centrifuge and centrifuge at 1500r / min for 3 minutes to settle the biochar particles to the bottom of the centrifuge tube and retain the bacteria in the supernatant. Take 1mL of the supernatant and perform gradient dilution. The specific gradient dilution method is as follows: Figure 2The procedure is as follows: 1 mL of well-mixed sample is taken in a sterile manner, injected into a test tube containing 9 mL of sterile NaCl, and mixed to form a 1:10 dilution sample; 1 mL of the 1:10 dilution sample is taken and injected into a test tube containing 9 mL of sterile NaCl, and mixed to form a 1:100 dilution sample, and so on, and so forth, and gradient dilution is performed to a suitable concentration (the average number of colonies is between 30 and 300). After mixing the test tube with the suitable concentration, 0.1 mL is taken and applied to an LB plate, and a sterile coating rod is used to uniformly coat the bacterial liquid. The uniformly coated culture dish is placed in a 37°C constant temperature incubator and incubated for 24 h-48 h, and the number of colonies on the plate is recorded. The bacterial concentration calculation formula is: N x 10 x 10 x 10 x The plate count result is 3.6 x 10 10 CFU / mL , recorded as A1.

[0024] Step 3, verification step: the supernatant in the centrifuge tube in step 2 is discarded, and 30 mL of physiological saline is added again, and after oscillation, centrifugation is performed at a speed of 5000 r / min for 3 min, and the supernatant is discarded. And repeat step 2 (the number of colonies after 6 gradient dilutions is 29) to get the result 2.9 x 10 9 CFU / mL , recorded as A2. A2 is between 5% A1 and 20% A1 (i.e. 5% A1≤ A2≤ 20% A2), so the final quantitative count result of the biochar loaded Bacillus subtilis is A1+ A2=4.0 x 10 10 CFU / mL .

[0025] Example 2 The quantitative count of biochar loaded microorganisms is performed according to the flowchart shown. Figure 1

[0026] The specific operation is the same as that in Example 1, except that in step 2, the number of colonies after 6 gradient dilutions is 233, and the number of colonies after 7 gradient dilutions is 36, and the plate count result is 2.3 x 10 10 CFU / mL , recorded as A1.

[0027] Step 3, verification step. The number of colonies after 6 gradient dilutions is 8, and the plate count result is 16 x 10 8 CFU / mL , recorded as A2.

[0028] A2 is much smaller than A1, and A2 is less than 5% A1 (i.e. A2≤ 5% A1), so the final quantitative count result of the biochar loaded Bacillus subtilis is A1, 2.3 x 10 10 CFU / mL ​ Example 3 Quantitative counting of biochar loaded microorganisms was performed according to the flow chart shown in Figure 1. Figure 1 Quantitative counting of biochar loaded microorganisms was performed according to the flow chart shown in Figure 1.

[0029] The same procedure as Example 1 was followed. In the second step, the number of colonies for 6-fold dilution was 386 and for 7-fold dilution was 40, so the plate count result was 3.8 x 10 10 CFU / mL , denoted as A1.

[0030] In the third step, the verification step, the number of colonies for 6-fold dilution and for 6-fold dilution and above were both 0, so it was proved that the second step had completely desorbed the target bacteria, and the final quantitative counting result of biochar loaded Bacillus subtilis was A1, 3.8 x 10 10 CFU / mL .

Claims

1. A method for quantitatively counting biochar-loaded microorganisms, comprising the following steps: 1) centrifuging a test bacterial solution of biochar-loaded microorganisms to separate the supernatant and the precipitate to obtain a bacterial solution and biochar loaded with microorganisms; determining the bacterial solution using a plate count method to obtain a free bacterial concentration in the test sample solution; 2) The microorganism-loaded biochar is washed and dried, and its mass is measured. Then, physiological saline is added to the centrifuge tube for the first time, shaken, and centrifuged. The supernatant is counted using the plate count method, and the count result is recorded as A1. The centrifuge tube from which the supernatant has been discarded is added with physiological saline for a second time, shaken, and centrifuged. The supernatant is counted using the plate count method, and the count result is recorded as A2. The results are then verified according to the following a)-c) to obtain a quantitative count of the test bacterial solution of the biochar-loaded microorganisms: When 0≤A2<5%A1, the count of the test bacterial solution of the biochar-loaded microorganisms is A1; When 5%A1≤A2≤20%A2, the count of the test bacterial solution of the biochar-loaded microorganisms is A1+A2; When A2>20%A1, the experimental result is inaccurate, and the test bacterial solution of the biochar-loaded microorganisms needs to be taken again to perform the above steps 1)-2) determination.

2. The method according to claim 1, wherein In step 1), the centrifugal speed is 1300 rpm to 1500 rpm, and the time is 3 to 4 min; The bacterial solution was diluted serially and then counted using the plate counting method.

3. The method according to claim 1 or 2, characterized in that In step 2), the cleaning process is as follows: adding the biochar loaded with microorganisms to physiological saline, shaking, and centrifuging at a speed of 4000-5000 r / min for 2-3 minutes, The drying temperature is 38°C to 40°C.

4. The method according to any one of claims 1 to 3, characterized in that In step 2), the mass of the biochar loaded with microorganisms is recorded as m c , then the amount of physiological saline added for the first time is 10×m c .

5. The method according to any one of claims 1 to 4, characterized in that In step 2), after the first addition of physiological saline, the shaking conditions are as follows: a rate of 3000-4000 r / min and a time of 20-30 min; The centrifugal speed is 1300 r / min to 1500 r / min, and the time is 3 to 4 minutes.

6. The method according to any one of claims 1 to 4, characterized in that In step 2), after adding physiological saline for the second time, the centrifugation speed is 3000-5000 r / min and the time is 2-3 min.

7. The method according to any one of claims 1 to 6, characterized in that In step 2), the supernatant is diluted stepwise and counted using a plate counting method.

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