Method for rapidly detecting mineralization ability of microorganisms

Abstract

The invention relates to the technical field of biological cement detection, in particular to a method for rapidly detecting the mineralization capacity of microorganisms. Comprising the following steps: 1, putting 100ml of a working solution into a 250ml triangular flask, adding 1ml of a to-be-detected bacterial solution, and replacing with 1ml of purified water for contrast; step 2, carrying out oscillation reaction for 6 hours at 150 RPM in an environment of 30 DEG C; step 3, under the state of 3000RPM, centrifuging for 10 minutes, collecting CaCO3 precipitate, and cleaning twice with purified water; 4, drying to constant weight, and measuring dry weight m1 (g); step 5, soaking and washing in a 0.1 M hydrochloric acid solution for several times until no obvious bubbles are generated, washing with deionized water, re-drying in the drying oven, and weighing and recording as m2 (g); 6, the difference value of the weight of m1 and the weight of m2 is the weight of CaCO3 generated by the MICP, the calcium carbonate generation rate of the microorganisms is calculated through a formula, and the calculation formula is that the calcium carbonate generation rate of the microorganisms is V (g / h) = (m1-m2) / t. And the working solution is a mixed solution which is prepared by using 0.01 to 0.5 M of PBS (Phosphate Buffer Solution) (with the pH value of 8.0 to 9.0) and contains 0.3 to 0.7 M of urea and 0.3 to 0.7 M of calcium acetate.

Description

Technical Field

[0001] The invention relates to the technical field of biocement detection, and in particular to a method for rapidly detecting the mineralization ability of microorganisms. Background Art

[0002] Microbial induced calcium carbonate precipitation (MICP) is a widely existing biomineralization in nature. The biocement developed based on this can significantly improve the compression, shear and deformation resistance of rock and soil, and is green and environmentally friendly, becoming a hot research direction in the fields of civil engineering and environment. However, due to the very low mineralization rate, it is impossible to quickly determine the mineralization effect of the strain, which brings a lot of trouble to the screening and breeding of strains. Therefore, a method that can quickly predict the mineralization ability of microorganisms is urgently needed.

[0003] At present, no relevant methods for rapid detection of microbial mineralization ability have been found in the retrieved patents and papers. There is only the traditional grouting method. The detection process of this method is mainly divided into three steps: first, put sand in the mold; then, pour the bacterial solution to be tested into the sand; finally, pour the mixed solution that promotes biomineralization on the sand at regular intervals, and achieve mineralization and cementation of the sand by pouring the mixed solution of biomineralization for a long time and multiple times. After a certain period of pouring, the loose sand in the mold will be mineralized and cemented, and have a certain mechanical strength. The mineralization ability of the microorganism can be evaluated by measuring its hardness.

[0004] The existing methods for detecting the mineralization ability of microorganisms have the following problems:

[0005] 1. The detection cycle is long and high-throughput screening is not possible.

[0006] Usually the watering time needs to last for 14-21 days or even longer, and this method cannot be used for high-throughput screening.

[0007] 2. There are many uncontrollable factors that affect the accuracy of the test.

[0008] There are many uncontrollable factors in the testing process, including the adhesion ability of microorganisms on the sand surface, retention time, flow rate and dispersion of the mixed solution that promotes biomineralization in the sand gaps, etc., which will have a significant impact on the hardness of the final mineralized cement body and affect the accuracy and stability of the test results.

[0009] 3. The testing workload is large and the cost is high.

[0010] During the irrigation period, samples need to be added at least twice a day, which is labor-intensive and has high testing costs. In addition, in order to eliminate the above uncontrollable factors, many parallel tests need to be added, which further increases the testing costs. Summary of the invention

[0011] Microbial mineralization is the process of producing calcium carbonate, and its production rate is directly related to the efficiency and intensity of mineralization. However, in the existing testing process using sand as a carrier, the interaction time between microorganisms and the substrate is limited, which seriously affects its mineralization rate, and therefore requires a very long testing cycle.

[0012] In addition, there are many uncontrollable factors in the test process, including the ability of microorganisms to adhere to the sand surface, retention time, the flow rate and dispersion of the mixed solution that promotes biomineralization in the cracks of the sand, etc., which will affect the effect of microbial mineralization and have a significant impact on the hardness of the final mineralized cementation body, thereby affecting the accuracy and stability of the test results.

[0013] Therefore, based on the principle, the present invention provides a uniform, stable, and timely flow reaction system for the mineralization of microorganisms, which can effectively solve the shortcomings of the above existing testing methods, provide an effective testing method for the mineralization ability of microorganisms, and has the advantages of rapid detection, stability, and low cost.

[0014] The main purpose of the present invention is to provide a method for rapidly detecting the mineralization ability of microorganisms, so as to effectively solve the problems existing in the prior art.

[0015] To achieve the above object, the present invention adopts the following technical solution: a method for rapidly detecting the mineralization ability of microorganisms, comprising the following steps:

[0016] Step 1: Place 100 ml of 0.5 M working solution in a 250 ml Erlenmeyer flask and add 1 ml of the test bacterial solution (OD 600 nm=1.00±0.01), and 1 ml of pure water was used as the control;

[0017] Step 2: Incubate at 30°C with 150 RPM oscillation for 6 hours;

[0018] Step 3: Centrifuge at 3000 RPM for 10 min to collect the CaCO3 precipitate, and wash it twice with pure water;

[0019] Step 4: Dry to constant weight and measure the dry weight m1 (g);

[0020] Step 5: Soak and wash in 0.1M hydrochloric acid solution for several times until no obvious bubbles are generated, then rinse with deionized water, put it back into the oven to dry, and weigh it and record it as m2 (g);

[0021] Step 6: The difference between the weights of m1 and m2 is the weight of CaCO3 generated by MICP. The rate at which microorganisms produce calcium carbonate is calculated using the formula:

[0022] The rate at which microorganisms produce calcium carbonate: V (g / h) = (m1-m2) / t

[0023] Preferably, the working solution in step 1 is: a mixed solution containing 0.3M-0.7M urea and 0.3M-0.7M calcium acetate prepared using 0.01M-0.5M PBS buffer (pH 8.0-9.0).

[0024] Test reaction principle: The mineralization of microorganisms is a process of producing calcium carbonate. The present invention provides a uniform, stable, and timely flowing reaction system for the mineralization of microorganisms, which can realize rapid, stable, and low-cost detection of the mineralization ability of microorganisms.

[0025] The above steps (1) and (2) provide a suitable temperature and sufficient substrate environment for the microorganisms with mineralization ability, which is conducive to the microorganisms producing calcium carbonate. Steps (3)-(5) are to measure the amount of calcium carbonate produced by the microorganisms within a certain period of time and eliminate the influence of the mass of the microorganisms themselves. Step (6) calculates the rate at which the microorganisms produce calcium carbonate.

[0026] The beneficial effects of the present invention are: the mineralization capacity of microorganisms can be quickly predicted. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Attached Figure 1 Rapid determination of calcium carbonate production rate histograms for different microorganisms.

[0028] Attached Figure 2 Histogram showing the coagulation strength of different microbial sand columns. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] Example

[0031] Step 1: Place 100 ml of working solution in a 250 ml conical flask, and add 1 ml of the test bacterial solution 1, 2, 3, 4 (OD 600 nm=1.00±0.01), and 1 ml of purified water was used as a control.

[0032] Step 2: Shake the reaction at 30°C and 150 RPM for 6 h.

[0033] Step 3: Centrifuge at 3000 RPM for 10 min to collect the CaCO3 precipitate, and wash twice with pure water.

[0034] Step 4: Dry to constant weight and measure the dry weight m1 (g).

[0035] Step 5: Soak and wash in 0.1M hydrochloric acid solution for several times until no obvious bubbles are generated, then rinse with deionized water, put back into the oven to dry, and weigh and record as m2 (g).

[0036] Step 6. The difference between the weights of m1 and m2 is the weight of CaCO3 generated by MICP. The rate at which microorganisms produce calcium carbonate is calculated using the formula.

[0037] The working solution in step 1 is: a mixed solution containing 0.4 M urea and 0.6 M calcium acetate is prepared using 0.4 M PBS buffer (pH 8.0).

[0038] Each group was repeated 3 times to obtain the average value, and the data were recorded as shown in Table 1 and Figure 1 shown.

[0039] Table 1 Rapid determination of calcium carbonate production rates by different microorganisms

[0040] sample m1 / g m2 / g V / g / h CK 0.163 0.161 0.0003 1 1.657 0.013 0.2740 2 1.994 0.006 0.3313 3 1.644 0.017 0.2712 4 2.544 0.019 0.4208

[0041] The sand column condensation effect of the above strain samples was verified. After 14 days of grouting, the strength was as Figure 2 shown.

[0042] contrast Figure 1 and Figure 2 The data showed that rapid determination of the rate of microbial calcium carbonate production could accurately predict the setting strength of sand columns after 14 days of grouting.

[0043] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for rapidly detecting the mineralization ability of microorganisms, characterized in that: It includes the following steps: Step 1: Place 100 ml of 0.5 M working solution in a 250 ml Erlenmeyer flask and add 1 ml of the test bacterial solution (OD 600 nm=1.00±0.01), and 1 ml of pure water was used as the control; Step 2: Incubate at 30°C with 150 RPM oscillation for 6 hours; Step 3: Centrifuge at 3000 RPM for 10 min to collect the CaCO3 precipitate, and wash it twice with pure water; Step 4: Dry to constant weight and measure the dry weight m1 (g); Step 5: Soak and wash in 0.1M hydrochloric acid solution for several times until no obvious bubbles are generated, then rinse with deionized water, put it back into the oven to dry, and weigh it and record it as m2 (g); Step 6: The difference between the weights of m1 and m2 is the weight of CaCO3 generated by MICP. The rate at which microorganisms produce calcium carbonate is calculated using the formula: The rate at which microorganisms produce calcium carbonate: V (g / h) = (m1-m2) / t.

2. A method for rapidly detecting the mineralization ability of microorganisms according to claim 1, characterized in that: The working solution in step 1 is: a mixed solution containing 0.3M-0.7M urea and 0.3M-0.7M calcium acetate prepared using 0.01M-0.5M PBS buffer (pH 8.0-9.0).

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