A method for detecting the concentration of dissolved co in a fermentation broth

Abstract

The application discloses a method for detecting the concentration of dissolved CO in fermentation liquor. The method comprises the following steps: (1) preparing CO saturated aqueous solution, oxygen-free aqueous solution, sodium hydrosulfite solution as a reference solution and deoxygenated myoglobin solution; (2) preparing an absorbance-CO saturation standard curve; (3) sampling and detection: taking a sample from the fermentation liquor as a sample to be detected by a sampling device, injecting the sample to be detected into a cuvette, adding 2.5 mL of deoxygenated myoglobin solution in the cuvette in advance and sealing the cuvette, adjusting zero with the reference solution, detecting the absorbance of the cuvette containing the sample to be detected at a wavelength of 415-425 nm by a spectrophotometer, substituting the obtained absorbance into the absorbance-CO saturation standard curve obtained in step (2) to obtain the corresponding CO saturation, and then converting the CO saturation into the concentration of dissolved CO according to the known CO solubility. The detection method has the characteristics of taking and measuring immediately, rapidness and simplicity.

Description

Technical fields:

[0001] This invention relates to the field of biochemical detection technology, specifically to a method for detecting the concentration of dissolved CO in fermentation broth. Background technology:

[0002] In syngas fermentation, CO serves as both a carbon and energy source for microorganisms, and its solubility in the fermentation broth is directly related to the fermentation products. Previous studies have attempted to increase CO solubility by enhancing gas-liquid mass transfer, but this method does not directly yield the dissolved CO concentration in the solution. Furthermore, air bubbles in the fermentation broth and oxygen in the external environment can affect the measurement results, causing discrepancies between the detected values ​​and the actual concentration. Therefore, sampling must eliminate air bubble interference and ensure the solution is unaffected by the external environment. Summary of the Invention:

[0003] This invention solves the problems existing in the prior art. This invention provides a method for detecting the concentration of dissolved CO in fermentation broth, which has the characteristics of immediate measurement, speed and simplicity.

[0004] The purpose of this invention is to provide a method for detecting the concentration of dissolved CO in fermentation broth, comprising the following steps:

[0005] (1) Preparation of solution:

[0006] a. At room temperature, CO gas (purity greater than 99.9%) is continuously bubbled into ultrapure water to obtain a CO saturated aqueous solution for later use;

[0007] b. At room temperature, nitrogen gas (purity greater than 99.9%) is continuously bubbled into ultrapure water to obtain an oxygen-free aqueous solution for later use.

[0008] c. Prepare a sodium dithionite solution as a reference solution using a phosphate buffer solution with a pH of 7 as the solvent.

[0009] d. Prepare myoglobin solution using pH 7 phosphate buffer as solvent, then add sodium dithionite to obtain deoxymyoglobin solution for later use.

[0010] (2) Preparation of absorbance-CO saturation standard curve: Take two cuvettes, add 2.5 mL of reference solution to one cuvette as a reference, and add deoxymyoglobin solution, anaerobic aqueous solution and / or CO saturated aqueous solution to the other cuvette in sequence according to Table 1 to prepare myoglobin solutions 1-5 with different CO saturations. Seal the top of both cuvettes and zero them with the reference solution. Use a spectrophotometer to detect the absorbance of myoglobin solutions 1-5 at wavelengths of 415-425 nm in sequence. Then, plot the absorbance-CO saturation standard curve equation with CO saturation x as the abscissa and absorbance y as the ordinate. The equation is: y = 0.3629x + 0.793, R2 = 0.9995.

[0011] Table 1

[0012] serial number CO saturation Deoxymyoglobin solution (mL) Anaerobic aqueous solution (μL) Saturated CO2 aqueous solution (μL) Myoglobin solution 1 0％ 2.5 20 0 Myoglobin solution 2 25％ 2.5 15 5 Myoglobin solution 3 50％ 2.5 10 10 Myoglobin solution 4 75％ 2.5 5 15 Myoglobin solution 5 100％ 2.5 0 20

[0013] (3) Sampling and testing:

[0014] A 20 μL sample was taken from the fermentation broth using a sampling device as the test sample. The test sample was injected into a cuvette, which was pre-filled with 2.5 mL of deoxymyoglobin solution and sealed. Referring to step (2), the cuvette was zeroed with a reference solution. The absorbance of the cuvette containing the test sample at wavelengths of 415-425 nm was measured using a spectrophotometer. The obtained absorbance value was substituted into the absorbance-CO saturation standard curve obtained in step (2) to obtain the corresponding CO saturation. Then, the dissolved CO concentration was calculated based on the known CO solubility. The dissolved CO concentration was calculated based on the known CO solubility in the CRC Handbook of Chemistry and Physics.

[0015] Preferably, in step (2), the absorbance of myoglobin solutions 1-5 at a wavelength of 421 nm is detected sequentially using a spectrophotometer, and in step (3), the absorbance of the cuvette containing the sample to be tested at a wavelength of 421 nm is detected using a spectrophotometer.

[0016] Preferably, the sampling device includes a T-shaped tube, a filter screen, and a screw cap. The T-shaped tube has a T-shaped opening in the middle, and a filter screen for filtering impurities in the fermentation broth is provided at the T-shaped opening. The bottom of the filter screen extends into the T-shaped tube, and the outer diameter of the filter screen is the same as the inner diameter of the T-shaped opening. The outside of the T-shaped opening is provided with a thread for connecting to the screw cap, and a circular opening for sampling is provided in the center of the screw cap.

[0017] In a further preferred embodiment, the T-shaped tube is provided with an inlet valve for the fermentation liquid to enter and an outlet valve for the fermentation liquid to exit on both sides.

[0018] The detailed steps of step (3) are as follows: Connect the inlet of the sampling device to the sampling port of the fermenter containing the fermentation broth, open the inlet and outlet valves of the sampling device at the same time, release a certain volume of fermentation broth and remove the air in the device, then close the outlet valve. After the device is filled with liquid, close the inlet valve. Use an airtight microsyringe to take 20 μL of sample from the center of the cap as the test sample, and immediately inject the sample into a cuvette (pre-fill the cuvette with 2.5 mL of deoxymyoglobin solution and seal it). Referring to step (2), zero the instrument with the reference solution and use a spectrophotometer to detect the absorbance of the cuvette containing the test sample at a wavelength of 421 nm. Substitute the obtained absorbance value into the absorbance-CO saturation standard curve equation to calculate the corresponding CO saturation, and then convert it into the dissolved CO concentration according to the known CO solubility in the CRC Handbook of Chemistry and Physics.

[0019] More preferably, the T-shaped opening is a circular opening with a diameter of 1.5-1.8 cm.

[0020] In a further preferred embodiment, a butyl rubber stopper is provided between the screw cap and the T-shaped opening to enhance the sealing effect.

[0021] Further preferably, the bottom of the filter screen is conical, and the filter screen aperture is 50 mesh.

[0022] More preferably, the diameter of the circular opening is 4.5-5.5 mm.

[0023] Preferably, in step c, a sodium dithionite solution with a concentration of 2 g / L is prepared as a reference solution.

[0024] Preferably, in step d, a myoglobin solution with a concentration of 0.0119 mM is prepared, and then sodium dithionite is added at a concentration of 2 g / L to obtain a deoxymyoglobin solution.

[0025] Compared with the prior art, the present invention has the following advantages: the sampling device and sampling method proposed in the present invention can eliminate the interference of air bubbles during the sampling process and keep the solution from being affected by the external environment, and has the characteristics of immediate sampling and testing, and is fast and convenient. Attached image description:

[0026] Figure 1 This is a schematic diagram of the sampling device;

[0027] Explanation of reference numerals in the attached diagram: 1. Inlet valve, 2. Outlet valve, 3. Filter screen, 4. Butyl rubber stopper, 5. Cap. Detailed implementation method:

[0028] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0029] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental materials and reagents used herein are commercially available products conventionally available in this technical field.

[0030] like Figure 1 As shown, the sampling device includes a T-tube, a filter screen 3, a butyl rubber stopper 4, and a screw cap 5. The main body of the device is a T-shaped glass tube, with a valve at each end, namely an inlet valve 1 and an outlet valve 2. A T-shaped opening is located in the middle of the T-tube, at which a movable filter screen 3 filters impurities from the fermentation broth. The filter screen 3 is made of corrosion-resistant materials such as stainless steel, has a 50-mesh pore size, and is cylindrical in shape with a hollow top and a conical bottom that extends into the glass tube. The outer diameter of the filter screen 3 matches the inner diameter of the glass tube at the T-shaped opening. The T-shaped opening of the T-tube is relatively short, generally less than 2 cm. In the following embodiments, a circular opening with a diameter of 1.5-1.8 cm is preferred. The T-shaped opening is enlarged to fix the filter screen, and threads are machined on the outside of the T-shaped opening. The screw cap 5 is made of metal and has internal threads that engage with the threads at the T-shaped opening. A butyl rubber stopper 4 is provided between the screw cap 5 and the T-shaped opening for sealing. The center of the screw cap 5 has a circular opening with a diameter of 4.5-5.5 mm, which can be used for needle sampling. The sampling device is characterized by the fact that the filter screen 3 can be removed, cleaned and reused, and the butyl rubber stopper 4 can be replaced periodically.

[0031] Example 1

[0032] A method for detecting the concentration of dissolved CO in fermentation broth includes the following steps:

[0033] (1) Take a small glass bottle, add 10 mL of ultrapure water, add a butyl rubber stopper and seal it with a pressure cap. Then insert a long needle into the bottom of the glass bottle and a short needle into the top of the glass bottle for venting. At room temperature, continuously introduce CO gas (purity greater than 99.9%) through the long needle and bubble for 30 min to obtain a CO saturated aqueous solution.

[0034] Similarly, add 10 mL of ultrapure water to a small glass bottle, add a butyl rubber stopper and seal it, insert two needles of different lengths, and continuously bubble nitrogen gas (purity greater than 99.9%) for 30 minutes at room temperature to obtain an oxygen-free aqueous solution.

[0035] Add 50 mL of phosphate buffer (Shanghai Maclean Company) (pH 7, 10 mM) to a 75 mL glass bottle, weigh 0.1 g of sodium dithionite (Shanghai Maclean Company) and add it to the solution. After adding the butyl rubber stopper, cap the bottle and shake well to dissolve the solution completely, thus obtaining the reference solution.

[0036] Add 50 mL of phosphate buffer (pH 7, 10 mM) to a 75 mL glass bottle. Then weigh 0.01 g of myoglobin lyophilized powder (Shanghai Yuanye Company) (purity greater than 95%) and add it to the solution. Shake gently to mix, then add 0.1 g of sodium dithionite to the solution. Immediately add a butyl rubber stopper and seal the bottle. Then shake gently to dissolve the myoglobin completely to obtain a deoxymyoglobin solution.

[0037] (2) Set the wavelength of the spectrophotometer (Biochrom Libra S12, Biochrom, UK) to 421 nm. Add 2.5 mL of reference solution to a cuvette and seal it with a butyl rubber stopper. Use this cuvette to adjust the zero point of the spectrophotometer. Then, add 2.5 mL of deoxymyoglobin solution to another cuvette and seal it with a butyl rubber stopper. Next, use a microsyringe to slowly inject the anoxic aqueous solution and CO-saturated aqueous solution into the cuvette sequentially (the total volume of the anoxic aqueous solution and the CO-saturated aqueous solution is 20 μL, with volume ratios of 20:0, 15:5, 10:10, 5:15, and 0:20, respectively). After gentle shaking, measure the absorbance of each cuvette at 421 nm. Plot the absorbance value y on the ordinate and the corresponding CO saturation x on the abscissa to obtain the absorbance-CO saturation standard curve equation: y = 0.3629x + 0.793(R²). 2 =0.9995).

[0038] (3) Connect the sampling port of the fermenter containing the fermentation broth to the inlet of the sampling device on one side of the inlet valve 1. At the same time, open the inlet valve 1 and the outlet valve 2 of the device to drain the fermentation broth in the sampling tube of the fermenter and remove the air in the device. After draining a certain volume of fermentation broth, close the outlet valve 2 of the device. After the fermentation broth fills the device, close the inlet valve 1 and separate the sampling device from the sampling port of the fermenter. Use an airtight microsyringe to take 20 μL of sample from the center of the cap 5 as the sample to be tested. Immediately and carefully inject the above sample into the cuvette (pre-fill the cuvette with 2.5 mL of deoxymyoglobin solution and seal it with a butyl rubber stopper). Referring to step (2), zero the instrument with the reference solution and use a spectrophotometer to detect the absorbance of the cuvette containing the sample to be tested at a wavelength of 421 nm. Substitute the obtained absorbance value into the absorbance-CO saturation standard curve equation to calculate the corresponding CO saturation. Then, convert the CO solubility known in the CRC Handbook of Chemistry and Physics into the dissolved CO concentration.

[0039] The dissolved CO concentration data of the three fermentation broths measured using this method are shown in Table 2 below:

[0040] Table 2

[0041] Sample Name absorbance CO saturation Dissolved CO concentration Fermentation broth 1 1.120 90.10％ 0.02486g / L Fermentation broth 2 0.847 14.88％ 0.00410g / L Fermentation broth 3 0.828 9.64％ 0.00266g / L

[0042] The method proposed in this invention measures the absorbance of CO-bound myoglobin in the wavelength range of 415-425 nm, with the best results achieved at a wavelength of 421 nm.

[0043] The above description of the embodiments is only for the purpose of helping to understand the technical solution and core idea of ​​the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for detecting the concentration of dissolved CO in fermentation broth, characterized in that, Includes the following steps: (1) Preparation of solution: a. At room temperature, CO gas is continuously bubbled into ultrapure water to obtain a CO saturated aqueous solution; b. At room temperature, nitrogen gas is continuously bubbled into ultrapure water to obtain an oxygen-free aqueous solution; c. Prepare a sodium dithionite solution as a reference solution using a phosphate buffer solution with a pH of 7 as the solvent. d. Prepare myoglobin solution using pH 7 phosphate buffer as solvent, and then add sodium dithionite to obtain deoxymyoglobin solution. (2) Preparation of absorbance-CO saturation standard curve: Take two cuvettes, add 2.5 mL of reference solution to one cuvette as a reference, and add deoxymyoglobin solution, anaerobic aqueous solution and / or CO saturated aqueous solution to the other cuvette in sequence according to Table 1 to prepare myoglobin solutions 1-5 with different CO saturation. Seal the top of both cuvettes and zero them with the reference solution. Use a spectrophotometer to detect the absorbance of myoglobin solutions 1-5 at wavelengths of 415-425 nm in sequence. Then plot the absorbance-CO saturation standard curve equation with CO saturation x as the abscissa and absorbance y as the ordinate: y = 0.3629x +0.793, R² = 0.9995; Table 1 (3) Sampling and testing: Connect the inlet of the sampling device to the sampling port of the fermenter containing the fermentation liquid. Simultaneously open the inlet and outlet valves of the sampling device, release a certain volume of fermentation liquid and remove the air in the device, then close the outlet valve. After the device is filled with liquid, close the inlet valve. Use an airtight microsyringe to take 20 μL of sample from the center of the cap as the test sample. Immediately inject the above sample into the cuvette. Add 2.5 mL of deoxymyoglobin solution to the cuvette beforehand and seal it. Refer to step (2), zero the cuvette with the reference solution, and use a spectrophotometer to detect the absorbance of the cuvette containing the test sample at a wavelength of 415-425 nm. Substitute the obtained absorbance into the absorbance-CO saturation standard curve equation obtained in step (2) to obtain the corresponding CO saturation. Then, convert the known CO solubility into the dissolved CO concentration.

2. The method according to claim 1, characterized in that, The sampling device includes a T-shaped tube, a filter screen, and a screw cap. The T-shaped tube has a T-shaped opening in the middle, and a filter screen is provided at the T-shaped opening to filter impurities in the fermentation broth. The bottom of the filter screen extends into the T-shaped tube, and the outer diameter of the filter screen is the same as the inner diameter of the T-shaped opening. The outside of the T-shaped opening is provided with a thread for connecting to the screw cap, and the center of the screw cap is provided with a circular opening for sampling.

3. The method according to claim 2, characterized in that, The T-shaped pipe is equipped with an inlet valve for the fermentation liquid to enter and an outlet valve for the fermentation liquid to exit on both sides.

4. The method according to claim 2, characterized in that, The T-shaped opening is a circular opening with a diameter of 1.5-1.8 cm.

5. The method according to claim 2, characterized in that, A butyl rubber stopper is provided between the screw cap and the T-shaped opening to enhance the sealing effect.

6. The method according to claim 2, characterized in that, The bottom of the filter screen is conical, and the filter screen has a mesh size of 50 mesh.

7. The method according to claim 2, characterized in that, The diameter of the circular opening is 4.5-5.5 mm.

8. The method according to claim 1 or 2, characterized in that, In step c, a sodium dithionite solution with a concentration of 2 g / L is prepared as a reference solution.

9. The method according to claim 1 or 2, characterized in that, In step d, a myoglobin solution with a concentration of 0.0119 mM was prepared, and then sodium dithionite was added at a concentration of 2 g / L to obtain a deoxymyoglobin solution.

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