Method and system for detecting microbial carbon utilization rate based on microwell plate respiratory system

Abstract

The invention provides a method and system for detecting the carbon utilization rate of microorganisms based on a microwell plate respiratory system, and the method comprises the following steps: pre-culturing target microorganisms, and confirming the logarithmic growth phase of the target microorganisms; the method comprises the following steps: inoculating target microorganisms into a plurality of deep holes of a microporous plate respiratory system for culture, wherein each deep hole in the microporous plate respiratory system comprises a bacterial liquid containing a specified carbon source; detecting the initial biomass in the bacterial liquid and the initial carbon dioxide value in the microplate respiratory system at the beginning of culture, and detecting the final biomass in the bacterial liquid and the final carbon dioxide value in the microplate respiratory system at the end of culture; calculating the growth rate of the target microorganisms according to the initial biomass and the end biomass; calculating the respiration rate of the target microorganism according to the initial carbon dioxide value, the end carbon dioxide value and the growth rate; and calculating the carbon utilization rate of the target microorganism according to the growth rate and the respiration rate. The problem that the carbon utilization rate of species level microorganisms is difficult to obtain is solved.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a method and system for detecting microbial carbon utilization based on a microplate respiratory system. Background Technology

[0002] Microbial carbon utilization rate reflects the proportion of substrates converted into biomass carbon by microorganisms, and is a key indicator that determines the rate of organic matter turnover and carbon dioxide emission in an ecosystem.

[0003] Current methods for detecting microbial carbon use efficiency primarily focus on the community level, particularly environmental samples. These methods include isotope tracing, chemometrics, and metabolic flux analysis. While these methods provide important information at the community level, they also have significant limitations. For example, isotope tracing relies on high-end mass spectrometry, which is costly; metabolic flux analysis depends on complex modeling and numerous prior parameters, making it unsuitable for large-scale parallel processing; and chemometrics and some biomass assays are destructive. Furthermore, current methods cannot quantify microbial carbon use efficiency at the species level. Summary of the Invention

[0004] In view of this, the purpose of this application is to propose a method and system for detecting microbial carbon utilization based on a microplate respiration system. By using a microplate respiration system to detect carbon dioxide produced by target microorganisms and combining it with a biomass measurement method, the problem of difficulty in obtaining the carbon utilization of microorganisms at the species level is solved.

[0005] To achieve one of the aforementioned objectives, this application provides a method for detecting microbial carbon utilization based on a microplate respiratory system, the method comprising: The target microorganism was pre-cultured, and its growth curve was measured to confirm the logarithmic growth phase of the target microorganism. The target microorganism is inoculated into multiple deep wells of the microplate respiratory system and cultured until the logarithmic growth phase. Each deep well of the microplate respiratory system contains a bacterial solution containing a specified carbon source. At the start of the culture, the initial biomass in the bacterial culture and the initial carbon dioxide value in the microplate respiration system are measured, and at the end of the culture, the final biomass in the bacterial culture and the final carbon dioxide value in the microplate respiration system are measured. The growth rate of the target microorganism is calculated based on the initial biomass and the final biomass. The respiration rate of the target microorganism is calculated based on the initial carbon dioxide value, the final carbon dioxide value, and the growth rate. The carbon utilization rate of the target microorganism is calculated based on the growth rate and the respiration rate.

[0006] As a further improvement to one embodiment of this application, the step of detecting the initial biomass in the bacterial culture at the start of cultivation and detecting the final biomass in the bacterial culture at the end of cultivation includes: The bacterial solution is irradiated with light of a specific wavelength to obtain the initial and final optical density values ​​of the bacterial solution. The initial and final optical density values ​​are combined with the optical density value and the biomass conversion coefficient to calculate the increased biomass.

[0007] As a further improvement to one embodiment of this application, the step of calculating the growth rate of the target microorganism based on the initial biomass and the final biomass includes: The growth rate of the target microorganism is calculated according to formula (1): (1); in, The target microorganism's growth rate is represented by K, which is the preset optical density value and biomass conversion coefficient. To conclude the optical density value, ν is the initial optical density value, vol is the volume of the bacterial solution, and t is the incubation time.

[0008] As a further improvement to one embodiment of this application, the step of detecting the initial biomass in the bacterial culture at the start of cultivation and detecting the final biomass in the bacterial culture at the end of cultivation includes: The initial cell count, the final cell count, and the cell volume in the bacterial culture were detected. The initial biomass is calculated based on the initial cell number and the cell volume; The final biomass is calculated based on the number of terminated cells and the cell volume.

[0009] As a further improvement to one embodiment of this application, the step of generating the time series to be detected based on the features includes: calculating the growth rate of the target microorganism based on the initial biomass and the final biomass, including: The growth rate of the target microorganism was calculated according to formula (2): (2); (3); C1=n (4); C0=m (5); Where μ is the growth rate of the target microorganism, C1 is the final carbon content of the microorganism, and C0 is the initial carbon content of the microorganism. V is the carbon content of a single cell, n is the cell volume, m is the final cell number, and t is the culture time.

[0010] As a further improvement to one embodiment of this application, the step of detecting the initial carbon dioxide value in the microplate respiration system at the start of the culture and detecting the final carbon dioxide value in the microplate respiration system at the end of the culture includes: The microplate breathing system is placed in an environment of light of a specific wavelength, and the microplate breathing system is equipped with a carbon dioxide sensing gel whose color changes with the carbon dioxide value. The initial carbon dioxide value is determined based on the color of the carbon dioxide sensing gel at the start of the culture, and the final carbon dioxide value is determined based on the color of the gel at the end of the culture.

[0011] As a further improvement to one embodiment of this application, the step of calculating the respiration rate of the target microorganism based on the initial carbon dioxide value, the final carbon dioxide value, and the growth rate includes: The respiration rate of the target microorganism is calculated according to formula (6): (6); (7); (8); (9); Where R is the respiration rate of the target microorganism, R tot The total amount of carbon dioxide accumulated from the initial time to the end time, V is the headspace gas, T is the culture temperature, t is the culture time, μ is the growth rate of the target microorganism, and C0 is the initial carbon content of the microorganism. To conclude the carbon dioxide reading, The initial carbon dioxide value, A0 OD570mean This represents the average initial carbon dioxide value in all deep wells of the microplate breathing system. A, B, and D are relative values ​​representing the amount of carbon dioxide. With A i The parameters in the function that fits the curve between them.

[0012] As a further improvement to one embodiment of this application, the step of calculating the carbon utilization rate of the target microorganism based on the growth rate and the respiration rate includes: The carbon utilization rate of the target microorganism is calculated according to formula (11): CUE = μ / (R+μ) (11); Wherein, CUE is carbon utilization rate, μ is the growth rate of the target microorganism, and R is the respiration rate of the target microorganism.

[0013] Based on the same inventive concept, this application also provides a system for detecting microbial carbon utilization based on a microplate respiration system, comprising: Pre-culture medium, used for pre-culturing target microorganisms; A microplate breathing system includes a deep-well plate and a detection plate. The deep wells of the deep-well plate are used to contain bacterial suspension, and a target microorganism is inoculated into the bacterial suspension. The detection plate is loaded with a carbon dioxide sensing gel, and the detection plate is coupled and sealed to the deep-well plate. A constant-temperature oscillation device, the interior of which is used to contain the pre-culture medium or the microplate respiration system; The detection unit is used to detect the initial biomass in the bacterial solution and the initial carbon dioxide value in the microplate respiration system, and to detect the final biomass in the bacterial solution and the final carbon dioxide value in the microplate respiration system. A data processing unit is configured to calculate the growth rate of the target microorganism based on the initial biomass and the final biomass, calculate the respiration rate of the target microorganism based on the initial carbon dioxide value, the final carbon dioxide value and the growth rate, and calculate the carbon utilization rate of the target microorganism based on the growth rate and the respiration rate.

[0014] As a further improvement to one embodiment of this application, the detection unit includes: An ELISA reader is used to emit light that illuminates the carbon dioxide sensing gel and to detect the initial and final carbon dioxide values ​​in the microplate breathing system. The ELISA reader is used to emit light to illuminate the bacterial solution and to detect the initial and final optical density values ​​of the bacterial solution. And / or, a flow cytometer, said flow cytometer being used to detect the initial number of cells, the final number of cells, and the cell volume in the bacterial culture.

[0015] Compared with existing technologies, the technical advantages of this invention are as follows: It utilizes a microplate respiration system to detect the carbon dioxide value produced by target microorganisms during cultivation, which is suitable for large-scale pure cultures under laboratory conditions. Then, a reliable biomass measurement method is used to detect and calculate the growth efficiency of the target microorganisms, ultimately obtaining the carbon utilization rate of the target microorganisms. This method for obtaining carbon utilization rate can be repeatedly implemented and is reproducible. It can obtain carbon utilization rate at the species level at a low cost, solving the problems of high cost, low throughput, and inability to achieve the same level at the species level in existing detection methods. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in this application or related technologies, the drawings used in the description of the implementation methods or related technologies will be briefly introduced below. Obviously, the drawings described below are only the implementation methods of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A flowchart of a method for detecting microbial carbon utilization based on a microplate respiratory system, provided in one embodiment of this application; Figure 2 A comparison chart of carbon utilization rates of microorganisms in different carbon sources, provided as another embodiment of this application; Figure 3 A comparison graph of two carbon utilization measurements provided for another embodiment of this application. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the specific embodiments shown in the accompanying drawings. However, these embodiments do not limit the present invention, and any structural, methodological, or functional modifications made by those skilled in the art based on these embodiments are included within the scope of protection of the present invention.

[0019] It should be noted that, unless otherwise defined, the technical or scientific terms used in the embodiments of this application should have the ordinary meaning understood by those skilled in the art to which this application pertains. The terms "first," "second," and similar terms used in the embodiments of this application do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects.

[0020] This application provides a method for detecting microbial carbon utilization based on a microplate respiratory system, such as... Figure 1 As shown, it includes the following steps: Step S100: Pre-culture the target microorganism, measure the growth curve of the target microorganism, and confirm the logarithmic growth phase of the target microorganism.

[0021] Specifically, the logarithmic growth phase is a crucial stage in the microbial growth curve. During this phase, cell division reaches its maximum rate, growth rate stabilizes, cell number increases rapidly in an exponential manner, and metabolic activity is vigorous. The logarithmic growth phase is an ideal period for microbial physiological research. Firstly, pre-culturing is used to confirm the logarithmic growth phase of the target microorganism, providing accurate data reference for the detection steps and determining the culture time for the detection.

[0022] In one possible implementation of this application, step S100 includes: Step S110: Place the target microorganism in the pre-cultured bacterial solution.

[0023] Step S120: Place the pre-cultured bacterial solution in an enzyme-linked immunosorbent assay (ELISA) reader with shaking function and incubate it at a specific temperature.

[0024] Step S130: Measure the biomass at regular intervals and plot the growth curve based on the measured values.

[0025] Specifically, the target microorganism is cultured at a specific temperature, and its biomass is monitored at regular intervals. Data is recorded using biomass and time as the x-axis and y-axis, respectively. Connecting the data points forms a growth curve, and the time range of the exponential growth phase can be determined based on the shape of the growth curve.

[0026] It should also be noted that the principle of the ELISA reader is to measure the optical density value of the pre-cultured bacterial solution at a wavelength of 600 nanometers. This optical density value can represent the biomass in the pre-cultured bacterial solution and is recorded as the OD600 value.

[0027] Step S200: The target microorganism is inoculated into multiple wells of the microplate respiratory system and cultured to the logarithmic growth phase. Each well of the microplate respiratory system contains a bacterial solution containing a specified carbon source.

[0028] Specifically, the target microorganisms are cultured in the deep wells of the microplate respiratory system, consuming carbon from the carbon source during their growth. Furthermore, the carbon concentration is identical in each well, ensuring the accuracy of subsequent calculations.

[0029] It should be noted that the microplate breathing system is a MicroResp measuring device. The MicroResp measuring device has multiple deep wells, which can measure the carbon dioxide value in each deep well.

[0030] Step S300: At the start of cultivation, the initial biomass in the bacterial solution and the initial carbon dioxide value in the microplate respiration system are detected, and at the end of cultivation, the final biomass in the bacterial solution and the final carbon dioxide value in the microplate respiration system are detected.

[0031] Specifically, at the start of cultivation, the initial biomass and initial carbon dioxide levels are simultaneously detected. At the end of cultivation, the final biomass and final carbon dioxide levels are also simultaneously detected. This allows for simultaneous monitoring of the respiration and growth of the target microorganisms, eliminating time errors in traditional measurements. It can accurately capture the metabolic state of microorganisms in the logarithmic growth phase, resulting in more accurate and reliable data on the carbon utilization rate of the target microorganisms.

[0032] In one possible implementation of this application, in order to achieve synchronous detection, step S300 includes detecting the initial biomass in the bacterial solution at the start of cultivation and detecting the final biomass in the bacterial solution at the end of cultivation, including: Step S311: Irradiate the bacterial solution with light of a specific wavelength to obtain the initial and final optical density values ​​of the bacterial solution.

[0033] Step S312: Combine the initial and final optical density values ​​with the conversion coefficient of optical density and biomass to calculate the increased biomass.

[0034] Specifically, the initial optical density value of the bacterial solution is obtained using the OD value method. The OD value method refers to the optical density value of the bacterial solution under light of a specific wavelength. Its optical density value changes with the biomass in the bacterial solution. Therefore, after measuring the OD value at the beginning and end of the culture, the initial and final biomass can be obtained by using the conversion coefficient between the optical density value and the biomass.

[0035] In a specific implementation, the OD600 value is used to represent the optical density of the bacterial solution, that is, the optical density of the bacterial solution under 600 nm light conditions. The OD value method is obtained by utilizing light scattering on cells. The absorbance varies with different cell numbers. At a wavelength of 600 nm, common components in microbial culture media (such as peptone and yeast extract) and most of the pigments of the microorganisms themselves have very weak absorption and will not cause significant interference. This results in higher detection accuracy.

[0036] In another possible implementation of this application, step S300, which involves detecting the initial biomass in the bacterial culture at the start of cultivation and detecting the final biomass in the bacterial culture at the end of cultivation, further includes: Step S321: Detect the initial cell count, final cell count, and cell volume in the bacterial culture; Step S322: Calculate the initial biomass based on the initial cell number and cell volume; Step S323: Calculate the final biomass based on the number of cells at termination and the cell volume.

[0037] Specifically, flow cytometry can also be used to detect biomass. Flow cytometry is a device that automatically analyzes cells and can quickly measure the characteristics of cells in liquids.

[0038] In one possible implementation of this application, step S300 involves detecting the initial carbon dioxide value in the microplate respiration system at the start of the culture and detecting the final carbon dioxide value in the microplate respiration system at the end of the culture, including: Step S331: Place the microplate breathing system in an environment of light of a specific wavelength. The microplate breathing system is equipped with a carbon dioxide sensing gel that changes color with the carbon dioxide value.

[0039] Step S332: Determine the initial carbon dioxide value based on the color of the carbon dioxide sensing gel at the start of incubation, and determine the final carbon dioxide value based on the color of the gel at the end of incubation.

[0040] Specifically, the microplate breathing system uses a colorimetric method to detect carbon dioxide. Each well corresponds to a carbon dioxide sensing gel. The reason why the carbon dioxide sensing gel changes color is that after absorbing carbon dioxide, it dissociates hydrogen ions, causing the pH value to decrease, thereby causing a color change. Within a specific pH range, the indicator in the detection gel has a significant difference in absorbance at a specific wavelength, thus obtaining the carbon dioxide value.

[0041] In a specific implementation, the microplate breathing system is placed in a light environment with a wavelength of 570 nm. The absorbance of the carbon dioxide sensing gel shows the most significant difference at this wavelength. Typically, at low carbon dioxide concentrations, the carbon dioxide sensing gel is relatively alkaline, appearing purple / purple-red, and exhibits a higher absorbance value at 570 nm. At high carbon dioxide concentrations, the carbon dioxide sensing gel becomes acidic, turning yellow, and its absorbance value at 570 nm is lower. The carbon dioxide value is represented by the OD570 value.

[0042] It should also be noted that the carbon dioxide value is not the actual concentration or amount of carbon dioxide, but rather a degree of carbon dioxide content, recorded as the OD570 value. After obtaining the OD570 value, it is necessary to convert the carbon dioxide value into the actual amount of carbon dioxide and the respiration rate of the microorganisms in subsequent calculations, taking into account relevant parameters obtained from prior experiments and the volume of air inside the microplate respiration system.

[0043] In summary, biomass and carbon dioxide levels can be detected using the OD method. At the start of the detection, the bacterial culture is simultaneously monitored at a wavelength of 600 nm, and the carbon dioxide-sensing gel at 570 nm. After the culture ends, the bacterial culture is again monitored at 600 nm, and the carbon dioxide-sensing gel at 570 nm, achieving simultaneous detection of biomass and carbon dioxide levels. Alternatively, flow cytometry can be used to detect biomass, and the OD method to detect carbon dioxide levels. At the start of the detection, flow cytometry is used to detect the initial cell count and cell volume, while the carbon dioxide-sensing gel is simultaneously monitored at 570 nm. After the culture ends, flow cytometry is used again to detect the final cell count and cell volume, while the carbon dioxide-sensing gel is simultaneously monitored at 570 nm.

[0044] Step S400: Calculate the growth rate of the target microorganism based on the initial biomass and the final biomass.

[0045] Specifically, the difference between the final biomass and the initial biomass is the biomass growth, and the ratio of the biomass growth to the culture time is the growth rate.

[0046] In one possible implementation of this application, biomass is detected using the OD value method, and step S400 includes: The growth rate of the target microorganism is calculated according to formula (1): (1); Where μ is the growth rate of the target microorganism, K is the preset optical density value and biomass conversion coefficient, Ax_OD600 is the final optical density value, A0_OD600 is the initial optical density value, vol is the volume of the bacterial solution, and t is the culture time.

[0047] The difference between the final optical density value and the initial optical density, combined with the conversion coefficient and the bacterial culture volume, yields the actual biological growth. The ratio of biological growth to culture time is the growth rate.

[0048] In specific implementations, the units of optical density value and biomass conversion coefficient K are gC·OD-1·mL-1, and the unit of cell volume vol is ml.

[0049] In another possible implementation of this application, biomass is detected using flow cytometry, and step S400 includes: calculating the growth rate of the target microorganism according to formula (2): (2); (3); C1=n (4); C0=m (5); Where μ is the growth rate of the target microorganism, C1 is the final carbon content of the microorganism, and C0 is the initial carbon content of the microorganism. V is the carbon content of a single cell, n is the cell volume, m is the final cell number, and t is the culture time.

[0050] After the number and volume of cells are detected by the flow cytometer, the initial microbial carbon content and the final microbial carbon content are obtained according to formulas (3), (4) and (5). The growth rate of the target microorganism is obtained according to the ratio of the initial microbial carbon content, the final microbial carbon content and the culture time.

[0051] Step S500: Calculate the respiration rate of the target microorganism based on the initial carbon dioxide value, the final carbon dioxide value, and the growth rate.

[0052] Specifically, the difference between the final carbon dioxide value and the initial carbon dioxide value is the carbon dioxide value produced by the target microorganism during its growth process. The respiration rate is calculated by combining the carbon dioxide value during the growth process with the growth rate.

[0053] It should be noted that, in the detection method based on the OD value, the initial carbon dioxide value and the final carbon dioxide value here are not specific true values, but a relative degree representation. It is also necessary to conduct experiments in advance to obtain the parameter relationship between the carbon dioxide value and the true quantitative value, and convert the carbon dioxide value into the true carbon dioxide quantity based on the parameter relationship.

[0054] In one possible implementation of this application, step 500 includes: The respiration rate of the target microorganism is calculated according to formula (6): (6); (7); (8); (9); Where R is the respiration rate of the target microorganism, R tot V represents the total carbon dioxide accumulated from the initial time to the end time, T represents the headspace gas, t represents the culture temperature, μ represents the culture time, and C0 represents the initial carbon content of the microorganism. To conclude the carbon dioxide reading, The initial carbon dioxide value, A0 OD570mean This represents the average initial carbon dioxide level in all deep wells of the microplate breathing system. A, B, and D are relative values ​​representing the amount of carbon dioxide. The parameters in the fitted curve function between Ai and Ai.

[0055] It should be noted that Ai specifically represents the normalized data of the change between the initial carbon dioxide value and the contact carbon dioxide value. Therefore, Ai is a specific value in an OD570 value. The relationship between %CO2 and the OD570 value was verified and fitted through a large number of experiments. The relationship obtained is a non-linear relationship. After the fitted curve is expressed by the fitted equation, the form of formula (8) is obtained. Thus, the change of %CO2 can be reflected by the change of absorbance.

[0056] In the specific implementation, A = -0.2265, B = -1.606, and D = -6.771.

[0057] Step S600: Calculate the carbon utilization rate of the target microorganism based on its growth rate and respiration rate.

[0058] In one possible implementation of this application, step S600 includes: The carbon utilization rate of the target microorganism is calculated according to formula (11): CUE = μ / (R+μ) (11); Where CUE is carbon utilization rate, μ is the growth rate of the target microorganism, and R is the respiration rate of the target microorganism.

[0059] In one possible implementation of this application, the method for detecting microbial carbon utilization based on a microplate respiration system further includes: In step S700, a bacterial culture containing a specified carbon source but no microorganisms is placed in the deep wells of the microplate respiration system as a blank group; during the culture time of the target microorganism, the carbon dioxide value and biomass of the blank group are detected.

[0060] Step S700 is executed synchronously with steps S100-S600.

[0061] Specifically, during the cultivation of the target microorganism, a bacterial solution excluding the microorganism is set up as a control group to correct for changes in carbon dioxide caused by non-biological factors and prevent contamination from leading to erroneous results.

[0062] The advantages of using a microplate respiration system to detect microbial carbon use efficiency are as follows: Simultaneous monitoring of microbial respiration and growth on the microplate respiration system completely eliminates the time lag error of traditional stepwise measurements, accurately capturing the metabolic state of microorganisms during the logarithmic growth phase, resulting in more reliable carbon use efficiency. Furthermore, the microplate respiration system design allows for parallel measurements of multiple samples simultaneously, greatly improving research screening and experimental efficiency. It also provides two biomass calculation pathways: OD value method and flow cytometry method, allowing users to choose the appropriate method based on their experimental needs, offering high flexibility and adaptability. This method does not rely on expensive isotope labeling or complex instruments and can be implemented in ordinary microbiology laboratories, significantly reducing the technical threshold and cost of carbon use efficiency determination.

[0063] In one possible implementation of this application, a system for detecting microbial carbon utilization based on a microplate respiration system is also proposed, comprising: a pre-culture medium, a microplate respiration system, a constant temperature oscillation device, a detection unit, and a data processing unit, wherein the pre-culture medium is used to pre-culture the target microorganism.

[0064] The microplate breathing system includes a deep-well plate and a detection plate. The deep wells of the deep-well plate are used to contain bacterial solutions, inoculate the target microorganisms in the bacterial solutions, and load the carbon dioxide sensing gel onto the detection plate. The detection plate is coupled and sealed to the deep-well plate.

[0065] The interior of the constant temperature shaking device is used to contain pre-culture medium or microplate breathing system.

[0066] The detection unit is used to detect the initial biomass in the bacterial culture and the initial carbon dioxide value in the microplate respiration system, as well as the final biomass in the bacterial culture and the final carbon dioxide value in the microplate respiration system.

[0067] The data processing unit is used to calculate the growth rate of the target microorganism based on the initial biomass and the final biomass, to calculate the respiration rate of the target microorganism based on the initial carbon dioxide value, the final carbon dioxide value and the growth rate, and to calculate the carbon utilization rate of the target microorganism based on the growth rate and the respiration rate.

[0068] Specifically, during the detection process, the pre-cultured bacterial solution containing the target microorganism is placed in a pre-culture medium, and the pre-culture medium is placed in a constant temperature shaking device for constant temperature incubation. Biomass is measured at regular intervals, and a growth curve is plotted based on the time and the measured biomass.

[0069] During the cultivation process, the bacterial culture containing the target microorganism is placed in the wells of a deep-well plate. A carbon dioxide-sensing gel on the plate corresponds to each well, and changes in color indicate changes in carbon dioxide levels. The biomass and carbon dioxide levels are detected by a detection unit, and the data processing unit receives and calculates the carbon utilization rate of the target microorganism.

[0070] In one possible implementation of this application, the detection unit includes: An ELISA reader is used to emit light that illuminates a carbon dioxide-sensing gel and to detect the initial and final carbon dioxide values ​​in a microplate breathing system. The ELISA reader is used to emit light to illuminate the bacterial solution and to detect the initial and final optical density values ​​of the bacterial solution. And / or, flow cytometer, a flow cytometer is used to detect the initial cell count, final cell count and cell volume in bacterial culture.

[0071] Specifically, an ELISA reader can emit light of one wavelength to detect the carbon dioxide level produced by the bacterial culture, which, when combined with a flow cytometer, yields the biomass of the bacterial culture, providing data for the data processing unit. Alternatively, a dual-wavelength ELISA reader can emit light of two wavelengths, used to detect the carbon dioxide level and the biomass of the bacterial culture, respectively, providing data for the data processing unit.

[0072] In one possible implementation of this application, the data processing unit calculates the growth rate of the target microorganism according to formula (1): (1); in, The target microorganism's growth rate is represented by K, which is the preset optical density value and biomass conversion coefficient. To conclude the optical density value, ν is the initial optical density value, vol is the volume of the bacterial culture, and t is the incubation time.

[0073] In one possible implementation of this application, the data processing unit calculates the growth rate of the target microorganism according to formula (2): (2); (3); C1=n (4); C0=m (5); Where μ is the growth rate of the target microorganism, C1 is the final carbon content of the microorganism, and C0 is the initial carbon content of the microorganism. V is the carbon content of a single cell, n is the cell volume, m is the final cell number, and t is the culture time.

[0074] In one possible implementation of this application, the data processing unit calculates the respiration rate of the target microorganism according to formula (6): (6); (7); (8); (9); Where R is the respiration rate of the target microorganism, R tot V represents the total carbon dioxide accumulated from the initial time to the end time, T represents the headspace gas, t represents the culture temperature, μ represents the culture time, and C0 represents the initial carbon content of the microorganism. To conclude the carbon dioxide reading, The initial carbon dioxide value, A0 OD570mean This represents the average initial carbon dioxide level in all deep wells of the microplate breathing system. A, B, and D are relative values ​​representing the amount of carbon dioxide. The parameters in the fitted curve function between Ai and Ai.

[0075] In one possible implementation of this application, the data processing unit calculates the carbon utilization rate of the target microorganism according to formula (11): CUE = μ / (R+μ) (11); Where CUE is carbon utilization rate, μ is the growth rate of the target microorganism, and R is the respiration rate of the target microorganism.

[0076] One possible implementation of this application proposes a specific method for detecting microbial carbon utilization based on a microplate respiratory system, comprising: Step 101: Prepare materials and equipment.

[0077] The study included 80 different bacterial strains from various phyla, including 36 strains from Proteobacteria, 18 strains from Firmicutes, 19 strains from Actinobacteria, and 7 strains from Bacteroidetes. All strains were stored at -80°C in 60% glycerol for later use.

[0078] It includes a pre-culture medium and a culture solvent. The pre-culture medium includes TSB medium, in which tryptone soybean broth is placed.

[0079] The culture solvent includes: basic salt solution: 5xM9 basic salt solution (each liter contains 33.9g NaHPO4, 15g KH2PO4, 2.5g NaCl, and 5.0g NH4Cl).

[0080] Trace element solution: Each liter contains 1.5g Nitrilotriacetic acid, 3.0g MgSO4·7H2O, 0.5g MnSO4·xH2O, 1.0g NaCl, 0.1g FeSO4·7H2O, 0.1g CoSO4·7H2O, 0.1g CaCl2·2H2O, 0.1g ZnSO4·7H2O, 0.01g CuSO4·5H2O, 0.01g AlK(SO4)2, 0.01g H3BO3, 0.01g Na2MoO4·2H2O, 0.01g NiCl2·6H2O, pH=7.0.

[0081] Vitamin solution: Each liter contains 2.0 mg biotin, 2.0 mg folic acid, 10.0 mg pyridoxine hydrochloride, 5.0 mg thiamine, 5.0 mg riboflavin, 5.0 mg niacin, 5.0 mg calcium pantothenate, 0.1 mg vitamin B12, 5.0 mg para-aminobenzoic acid, and 5.0 mg lipoic acid.

[0082] Other solutions: 1 mol / L MgSO4 solution (sterile), 1 mol / L CaCl2 solution (sterile).

[0083] This includes the working culture medium used to culture the target microorganism. First, prepare the carbon-free basal culture medium and the experimental carbon source.

[0084] Preparation of carbon-free basal medium: Add 200 ml of 5xM9 basal salt solution to an Erlenmeyer flask. Add 2 ml of MgSO4 solution (1 mol / L), 100 μL of CaCl2 solution (1 mol / L), 1 ml of trace elements, and 10 mL of vitamin solution. Add sterile distilled water to a final volume of 1 L. This is the carbon-free M9 basal medium. This medium does not contain any organic carbon source.

[0085] Experimental carbon sources: Fourteen single-carbon-source culture media and one mixed-nutrient medium (TSB medium) were used. The 14 carbon source substrates were glucose, fumarate, acetate, 2-oxoglutarate, ethanol, D-fructose, L-glutamine, L-glutamate, D-lactate, malate, pyruvate, succinate, acetaldehyde, and formate. All carbon source compounds were analytical grade. The stock solutions of each carbon source were prepared with sterile deionized water and sterilized by filtration through a 0.22 μm filter membrane; some were sterilized by autoclaving. Figure 2 As shown, the upper figure is a frequency distribution of carbon utilization in single carbon source culture media and mixed nutrient culture media; the blue in the lower figure represents the carbon utilization of mixed nutrient culture media, and the red represents the carbon utilization of 14 single carbon source culture media.

[0086] Preparation of working medium: Add a specific volume of carbon source stock solution to a carbon-free basal medium (1xM9) to ensure that the final carbon concentration (in terms of carbon atoms) is uniformly 30mM. Taking glucose as an example (molecular weight 180, containing 6 carbon atoms), the concentration of glucose to be added is (30 / 6) = 5mM, that is, 0.90g of glucose to be added per liter of medium.

[0087] Includes instruments and consumables: Microplate breathing system: including a 96-well deep-well plate (each well of the deep-well plate can hold approximately 1.2 mL of liquid, with an actual working volume of 400 μL) and a matching 96-well CO2 detection plate.

[0088] CO2 sensing gel indicator: Prepared according to the manufacturer's instructions, mainly an agar gel containing cresol red (Bromocresol Purple).

[0089] Multifunctional microplate reader: Equipped with temperature control and oscillation functions, it can be used to measure absorbance at 570nm and 600nm.

[0090] Flow cytometer: used for absolute cell counting and size analysis.

[0091] Thermostatic shaking incubator is used for pre-culture and mass culture.

[0092] Sterile 96-well deep well plate (individually packaged, sterilized), sterile 96-well CO2 detection plate.

[0093] Aseptic worktable.

[0094] Other: micropipettes, 0.22μm filter membranes, 96-well plate optical sealing membranes, etc.

[0095] Step S102: Pre-culture the target microorganism, measure the growth curve of the target microorganism, and confirm the logarithmic growth phase of the target microorganism.

[0096] This includes strain resuscitation and pre-culture: each strain was taken out of the -80°C freezer and activated for 3 rounds in TSB liquid medium at 30°C and 120 rpm.

[0097] Growth curve determination includes: taking a small amount of pre-cultured bacterial solution and transferring it to new TSB medium at a ratio of 1:10. Add 20 μL of bacterial solution and 180 μL of TSB medium to a 96-well transparent flat-bottomed culture plate, place it in a microplate reader with shaking function, and continuously incubate at 30°C. The OD600 value is automatically measured every 30 minutes to plot the growth curve and accurately determine the logarithmic growth phase time range for each strain.

[0098] Step S103: Inoculate the target microorganism into multiple wells of the microplate respiratory system and culture it into the logarithmic growth phase. Each well of the microplate respiratory system contains a bacterial solution containing a specified carbon source.

[0099] Includes: bacterial preparation and inoculation.

[0100] In a clean bench, add 540 μL of a specific working medium containing 30 mM carbon to each well of the MicroResp deep well plate.

[0101] Add 60 μL of the above bacterial suspension to each well at a volume fraction of 1 / 10. Three biological replicates are performed for each strain-carbon source combination. Figure 3 The figure shows a comparison of the correlation between carbon utilization rates at two different time points in a mixed nutrient medium. A blank control consisting only of medium without inoculation was also included (to correct for CO2 variations and OD background caused by abiotic factors). The deep-well plates were sealed with an optical membrane to prevent external contamination. Due to the short-term incubation, there was no oxygen restriction during the experiment. The plates were then placed in an incubator and cultured until the logarithmic growth phase.

[0102] Step S104: At the beginning of the culture, the initial biomass in the bacterial solution and the initial carbon dioxide value in the microplate respiration system are detected, and at the end of the culture, the final biomass in the bacterial solution and the final carbon dioxide value in the microplate respiration system are detected.

[0103] Including the determination of the initial value at time T0: Growth monitoring can be performed using one of the following two methods: Method A (OD value method): In a sterile operating table, take 200 μL of bacterial suspension from the deep well plate and transfer it to a 96-well shallow well plate. Use an ELISA reader to directly measure the OD600 value of each well in the shallow well plate and record it as A0-OD600.

[0104] Method B (Flow Cytometry): In a sterile operating room, take 200 μL of bacterial suspension from a deep-well plate. Dilute 100 μL with sterile PBS and perform absolute cell counting using a flow cytometer. Keep the remaining 100 μL as a backup. Record the cell size distribution as A0 - cell count and A0 - mean cell size.

[0105] Respiration (CO2): First, measure the OD570 value of each well on the test plate directly using a microplate reader and record it as A0-OD570. Then, calculate the initial average OD570 value of all wells on the entire test plate and record it as A0. OD570mean Then place it in a sterile operating table, immediately couple and seal the CO2 detection plate with the deep well plate, and then place the entire system in a constant temperature incubator at 30°C and 120 rpm for incubation.

[0106] Determination of the final value at time T6: After 6 hours of incubation, the detection plate was removed, and its absorbance value was measured at 570 nm using an ELISA reader and recorded as A6-OD570.

[0107] Growth monitoring can be performed using one of the following two methods: Method A (OD method): Take 200 μL of bacterial suspension from the deep well plate and transfer it to a 96-well shallow well plate. Use an ELISA reader to directly measure the OD600 value of each well in the shallow well plate and record it as A6-OD600.

[0108] Method B (flow cytometry): Take 100 μL of bacterial suspension from the deep well plate, dilute it appropriately with sterile PBS, and use a flow cytometer to perform absolute cell counting and record the cell size distribution as A6-cell number and A6-mean cell size.

[0109] Step S105: Calculate the growth rate of the target microorganism based on the initial biomass and the final biomass.

[0110] You can choose one of the following two methods: Method A (OD method): Microbial biomass carbon (MBC) throughout the growth curve was calculated using a conversion factor of 195 g C·OD⁻¹·ml⁻¹ (BioNumber 109836). However, due to technical difficulties in collecting biomass from small cell cultures, the MBC of these groups was underestimated. Therefore, a non-ideal biomass conversion factor was used uniformly. The growth rate calculation formula is as follows: ; ; Where A6-OD600 represents the OD600 value of the bacterial culture at the end of the formal culture, A0-OD600 represents the OD600 value of the bacterial culture at the beginning of the formal culture, and vol represents the culture system in ml. In this experiment, the culture system was 400 μL.

[0111] Method B (flow cytometry): Growth rate needs to be calculated by combining cell number and cell diameter. The carbon content of a single microbial cell is calculated using the following formula: ; By combining the cell number n, the initial carbon content C1 and C0 can be calculated.

[0112] The growth rate is calculated as follows: ; Step S106: Calculate the respiration rate of the target microorganism based on the initial carbon dioxide value, the final carbon dioxide value, and the growth rate.

[0113] The respiration rate was calculated using the formula derived from the microplate respiration system and the bacterial exponential growth model, resulting in the following formula: ; The specific derivation process is as follows: Reasoning: In a bacterial culture system in the exponential growth phase, the total accumulation of carbon dioxide (CO2) depends on the population biomass and its respiration rate per unit biomass at any given time during the culture period. By accurately measuring the total accumulated CO2 over the entire experimental period and combining this with the mathematical relationships of the bacterial exponential growth model, the respiration rate of the population per unit time can be derived. Therefore, the CO2 accumulation rate (dRtot / dt) is directly proportional to the total population biomass C(t) at that time, and the proportionality constant is the biomass-specific respiration rate (R) to be calculated. ; Where Rtot is the total amount of CO2 accumulated over time; t represents time; C(t) represents the population biomass at time point t; R: the respiration rate per unit biomass per unit time, which is a constant that needs to be solved.

[0114] Since the cultured bacteria are in the exponential growth phase, the biomass C(t) at any time point t can be expressed by the following formula: ; C0 represents the initial population biomass; This indicates the growth rate of bacteria.

[0115] We obtain a differential equation describing the relationship between CO2 accumulation rate and time: ; Solve the integral of the above expression: ; C1 represents the integration constant, and the solution is required.

[0116] Solve for C1; let t=0 in the above equation, and simplify to obtain the following equation: ; The formula for calculating the respiratory rate R after algebraic transformation is as follows: ; Since the initial CO2 accumulation is 0, therefore R tot (0) is 0, therefore the final formula for calculating the respiratory rate is: ; R tot The total respiratory volume can be calculated using the MicroResp component: ; Where V is the headspace volume (ul), which was actually measured to be 1032ul in the experiment; T is the incubation temperature (K), which was 30°C in the experiment; and the incubation time t was 6 hours. %CO2 was calculated using the following formula: ; Where A = -0.2265, B = -1.606, D = -6.771; Ai is normalized data, calculated by dividing the final colorimetric value by the initial colorimetric value and then multiplying by the average of the initial colorimetric values, i.e.: ; Step S107: Calculate the carbon utilization rate of the target microorganism based on its growth rate and respiration rate.

[0117] Carbon utilization rate is derived from the following formula: ; Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of this application (including the claims) is limited to these examples; this manner of description is merely for clarity, and those skilled in the art should consider the specification as a whole. Within the framework of this application, the above embodiments or the technical features of different embodiments can also be appropriately combined, the steps can be implemented in any order, and there are many other variations of different aspects of the embodiments of this application as described above, which are not provided in the details for the sake of brevity.

[0118] Additionally, to simplify the description and discussion, and to avoid obscuring the embodiments of this application, the well-known power / ground connections to integrated circuit (IC) chips and other components may or may not be shown in the provided drawings. Furthermore, the apparatus may be shown in block diagram form to avoid obscuring the embodiments of this application, and this also takes into account the fact that the details of the implementation of these block diagram apparatuses are highly dependent on the platform on which the embodiments of this application will be implemented (i.e., these details should be entirely within the understanding of those skilled in the art). While specific details (e.g., circuits) are set forth to describe exemplary embodiments of this application, it will be apparent to those skilled in the art that the embodiments of this application can be implemented without these specific details or with variations thereof. Therefore, these descriptions should be considered illustrative rather than restrictive.

[0119] Although this application has been described in conjunction with specific embodiments thereof, many substitutions, modifications, and variations of these embodiments will be apparent to those skilled in the art from the foregoing description. For example, other memory architectures (e.g., dynamic RAM (DRAM)) may use the embodiments discussed.

[0120] The embodiments described herein are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and principles of the embodiments described herein should be included within the protection scope of this application.

Claims

1. A method for detecting microbial carbon utilization based on a microplate respiratory system, characterized in that, The method includes: The target microorganism was pre-cultured, and its growth curve was measured to confirm the logarithmic growth phase of the target microorganism. The target microorganism is inoculated into multiple deep wells of the microplate respiratory system and cultured until the logarithmic growth phase. Each deep well of the microplate respiratory system contains a bacterial solution containing a specified carbon source. At the start of the culture, the initial biomass in the bacterial culture and the initial carbon dioxide value in the microplate respiration system are measured, and at the end of the culture, the final biomass in the bacterial culture and the final carbon dioxide value in the microplate respiration system are measured. The growth rate of the target microorganism is calculated based on the initial biomass and the final biomass. The respiration rate of the target microorganism is calculated based on the initial carbon dioxide value, the final carbon dioxide value, and the growth rate. The carbon utilization rate of the target microorganism is calculated based on the growth rate and the respiration rate.

2. The method for detecting microbial carbon utilization based on a microplate respiratory system according to claim 1, characterized in that, The step of detecting the initial biomass in the bacterial culture at the start of cultivation and detecting the final biomass in the bacterial culture at the end of cultivation includes: The bacterial solution is irradiated with light of a specific wavelength to obtain the initial and final optical density values ​​of the bacterial solution. The initial and final optical density values ​​are combined with the optical density value and the biomass conversion coefficient to calculate the increased biomass.

3. The method for detecting microbial carbon utilization based on a microplate respiratory system according to claim 2, characterized in that, The step of calculating the growth rate of the target microorganism based on the initial biomass and the final biomass includes: The growth rate of the target microorganism is calculated according to formula (1): （1）； in, The target microorganism's growth rate is represented by K, which is the preset optical density value and biomass conversion coefficient. To conclude the optical density value, ν is the initial optical density value, vol is the volume of the bacterial solution, and t is the incubation time.

4. The method for detecting microbial carbon utilization based on a microplate respiration system according to claim 1, characterized in that, The step of detecting the initial biomass in the bacterial culture at the start of cultivation and detecting the final biomass in the bacterial culture at the end of cultivation includes: The initial cell count, the final cell count, and the cell volume in the bacterial culture were detected. The initial biomass is calculated based on the initial cell number and the cell volume; The final biomass is calculated based on the number of terminated cells and the cell volume.

5. The method for detecting microbial carbon utilization based on a microplate respiration system according to claim 4, characterized in that, The step of calculating the growth rate of the target microorganism based on the initial biomass and the final biomass includes: The growth rate of the target microorganism was calculated according to formula (2): （2）； （3）； C1=n (4); C0=m (5); Where μ is the growth rate of the target microorganism, C1 is the final carbon content of the microorganism, and C0 is the initial carbon content of the microorganism. V is the carbon content of a single cell, n is the cell volume, m is the final cell number, and t is the culture time.

6. The method for detecting microbial carbon utilization based on a microplate respiratory system according to claim 1, characterized in that, The step of detecting the initial carbon dioxide value in the microplate respiration system at the start of the culture and detecting the final carbon dioxide value in the microplate respiration system at the end of the culture includes: The microplate breathing system is placed in an environment of light of a specific wavelength, and the microplate breathing system is equipped with a carbon dioxide sensing gel whose color changes with the carbon dioxide value. The initial carbon dioxide value is determined based on the color of the carbon dioxide sensing gel at the start of the culture, and the final carbon dioxide value is determined based on the color of the gel at the end of the culture.

7. The method for detecting microbial carbon utilization based on a microplate respiratory system according to claim 1, characterized in that, The step of calculating the respiration rate of the target microorganism based on the initial carbon dioxide value, the final carbon dioxide value, and the growth rate includes: The respiration rate of the target microorganism is calculated according to formula (6): （6）； （7）； （8）； （9）； Where R is the respiration rate of the target microorganism, R tot The total amount of carbon dioxide accumulated from the initial time to the end time, V is the headspace gas, T is the culture temperature, t is the culture time, μ is the growth rate of the target microorganism, and C0 is the initial carbon content of the microorganism. To conclude the carbon dioxide reading, The initial carbon dioxide value, A0 OD570mean This represents the average initial carbon dioxide value in all deep wells of the microplate breathing system. A, B, and D are relative values ​​representing the amount of carbon dioxide. The parameters in the fitted curve function between Ai and Ai.

8. The method for detecting microbial carbon utilization based on a microplate respiratory system according to claim 1, characterized in that, The step of calculating the carbon utilization rate of the target microorganism based on the growth rate and the respiration rate includes: The carbon utilization rate of the target microorganism is calculated according to formula (11): CUE = μ / (R+μ) (11); Wherein, CUE is carbon utilization rate, μ is the growth rate of the target microorganism, and R is the respiration rate of the target microorganism.

9. A system for detecting microbial carbon utilization based on a microplate respiration system, characterized in that, The system includes: Pre-culture medium, used for pre-culturing target microorganisms; A microplate breathing system includes a deep-well plate and a detection plate. The deep wells of the deep-well plate are used to contain bacterial suspension, and a target microorganism is inoculated into the bacterial suspension. The detection plate is loaded with a carbon dioxide sensing gel, and the detection plate is coupled and sealed to the deep-well plate. A constant-temperature oscillation device, the interior of which is used to contain the pre-culture medium or the microplate respiration system; The detection unit is used to detect the initial biomass in the bacterial solution and the initial carbon dioxide value in the microplate respiration system, and to detect the final biomass in the bacterial solution and the final carbon dioxide value in the microplate respiration system. A data processing unit is configured to calculate the growth rate of the target microorganism based on the initial biomass and the final biomass, calculate the respiration rate of the target microorganism based on the initial carbon dioxide value, the final carbon dioxide value and the growth rate, and calculate the carbon utilization rate of the target microorganism based on the growth rate and the respiration rate.

10. The system for detecting microbial carbon utilization based on a microplate respiration system according to claim 9, characterized in that, The detection unit includes: An ELISA reader is used to emit light that illuminates the carbon dioxide sensing gel and to detect the initial and final carbon dioxide values ​​in the microplate breathing system. The ELISA reader is used to emit light to illuminate the bacterial solution and to detect the initial and final optical density values ​​of the bacterial solution. And / or, a flow cytometer, said flow cytometer being used to detect the initial number of cells, the final number of cells, and the cell volume in the bacterial culture.

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