Method for detecting new carbon source n-propanol in anaerobic fermentation liquor
The detection of n-propanol in anaerobic fermentation broth by gas chromatography solved the problem of lack of detection methods in anaerobic fermentation broth in kitchen waste, confirmed the value of n-propanol as a new carbon source, and formulated standardized processes to achieve Accurate detection of n-propanol in anaerobic fermentation broth.
Patent Information
- Application Number
- CN202411299765.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-18
- Publication Date
- 2024-12-27
AI Technical Summary
At this stage, there is a lack of methods and standardized testing procedures for detecting n-propanol in the anaerobic fermentation broth of kitchen waste, resulting in the failure to discover its potential value as a new carbon source.
Gas chromatography was used to detect n-propanol in anaerobic fermentation broth, and the dilution curve was drawn and compared with standard curves, combined with instrument maintenance and calibration, to ensure the accuracy and reliability of the test results.
It was successfully detected that n-propanol was present in the anaerobic fermentation broth of kitchen waste, and confirmed its possibility as a new carbon source, and standardized testing procedures were formulated, which were suitable for the detection of volatile fatty acids in the anaerobic fermentation broth.
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Figure CN119198944A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biochemistry and environmental science, and in particular to a method for detecting n-propanol, a new carbon source, in anaerobic fermentation liquid. Background Art
[0002] At present, the biological process of sewage needs to add carbon source because the carbon source in the raw water cannot meet the requirements of denitrification and denitrification. The sources of carbon source can be mainly divided into endogenous carbon source and exogenous carbon source. The endogenous carbon source comes from the biodegradable soluble substances produced by microorganisms in their own metabolism. The traditional external carbon source is mainly simple organic matter such as methanol, sodium acetate and glucose. Since simple organic matter has low molecular weight, its metabolic pathway is relatively simple when used as a denitrification carbon source, and it is easier to be used by denitrification-related microorganisms, thus having a relatively high denitrification rate.
[0003] According to the existing carbon source research results, new carbon sources mainly include solid organic carbon sources, high-concentration organic wastewater and anaerobic fermentation products. The cost of preparing carbon sources by anaerobic fermentation of food waste is low. Up to now, no n-propanol has been found in the anaerobic fermentation broth of food waste, and there is no fixed standardized detection method and detection standard method as a guide. In this regard, we proposed a method for detecting n-propanol, a new carbon source, in anaerobic fermentation broth. Summary of the invention
[0004] In order to solve the above technical problems, a method for detecting a new carbon source, n-propanol, in an anaerobic fermentation broth is provided. This technical solution solves the above problems.
[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is: A method for detecting n-propanol, a new carbon source, in an anaerobic fermentation broth. The method for detecting n-propanol, a new carbon source, in the fermentation broth is: Check and ensure that the testing equipment has been maintained and is in optimal condition; Prepare n-propanol standard solution according to the standard points of 50mg / l, 100mg / l, 200mg / l, 400mg / l, 800mg / l and 1000mg / l; Use gas chromatograph to perform sampling and detection on the standard solution of each concentration, record the peak area of the standard solution of each concentration, and draw a standard curve; The fermentation liquid from the anaerobic fermentation reactor was centrifuged at 10000r / min for 10min, and the filtrate was obtained by passing through a 0.22µm membrane. The filtrate was diluted 20 times and put into a sample bottle; Use gas chromatograph to sample and test the diluent, record the peak area of the diluent, and draw a diluent curve; The standard curve was compared with the dilution curve to detect the presence of n-propanol, a new carbon source, in the fermentation broth.
[0006] Preferably, the detection instrument comprises: a gas chromatograph, a hydrogen flame ionization detector, a DB-FATWAX UI chromatographic column, an air generator and a hydrogen generator.
[0007] Preferably, the detection instrument maintenance method is: Evaluate instrument maintenance content based on target peak shape, resolution, peak baseline and the number of times the liner is used as a quantitative indicator; If the liner has been used more than 220 times, replace the liner and septum, and clean the injection needle, ultrasonic needle base, cleaning bottle, and waste bottle with methanol and water, respectively.
[0008] If the liner is used less than 220 times, clean the injection needle, ultrasonic needle base, cleaning bottle and waste bottle with methanol and water respectively after each injection.
[0009] If intermittent injection occurs, clean the injection needle or replace the injection needle; If ghost peaks, poor separation and large baseline noise appear in the peak shape, clean the split plate and cut the chromatographic column or increase the dilution factor to reduce matrix interference and increase data accuracy; If the ignition signal reaches several hundred thousand pA and purge is ineffective, clean the collector, nozzle and ceramic piece of the detector.
[0010] Preferably, the method for preparing the n-propanol standard solution is: Prepare pure n-propanol standard, prepare deionized water as diluent, and prepare clean volumetric flasks and pipettes; Use a pipette to take the n-propanol standard and add it into 6 volumetric flasks respectively. Use the dilution method to dilute it with the diluent. The dilution concentrations are 50 mg / l, 100 mg / l, 200 mg / l, 400 mg / l, 800 mg / l and 1000 mg / l standard solutions respectively.
[0011] Preferably, the gas chromatograph detection conditions are as follows: the injection port temperature is set to 280°C, the detector temperature is set to 300°C, the split ratio of the splitter is set to 10:1, the injection volume of the component is set to 0.5µL, and the column box heating program in the temperature control system is set to an initial temperature of 100°C for 2 minutes, and then increased to 200°C at a rate of 10°C for 10 minutes.
[0012] Preferably, the steps for drawing the standard curve are: Draw a scatter plot with the concentration of n-propanol as the X-axis and the corresponding peak area as the Y-axis; Use linear regression to fit the scattered points and get the standard curve equation. The equation calculation formula is: Wherein, Y represents the peak area, a represents the slope, X represents the concentration of n-propanol, and b represents the intercept.
[0013] Preferably, a standard curve of the n-propanol standard solution is drawn, the curve correlation coefficient is greater than 0.999, the precision of the standard solution is within 2%, and the accurate recovery rate of the standard solution is about 100%; Preferably, the sample precision control range is required to be: The liner is used less than 100 times and the sample error range is within 5%; The liner was used 100-150 times, and the sample error range was within 5%-10%; The liner was used 150-220 times, and the sample error range was within 10%-20%.
[0014] Preferably, when the fermentation broth samples are sampled and tested, a blank test is inserted between samples and three parallel data are obtained.
[0015] Preferably, an internal standard is introduced when drawing a standard curve, and the internal standard is mixed with a standard solution of n-propanol and then detected. The peak area of n-propanol is corrected using the peak area of the internal standard as a reference.
[0016] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a method for detecting n-propanol as a new carbon source in anaerobic fermentation broth. The present invention detects the presence of n-propanol in the anaerobic fermentation broth of food waste by capillary gas chromatography. No relevant research content on n-propanol as a carbon source in anaerobic fermentation broth is found in existing literature. The detection method proves that n-propanol can be used as a new carbon source for anaerobic fermentation of food waste and kitchen waste, and a set of standardized detection processes is formulated, which can also be used for the detection of volatile fatty acids in anaerobic fermentation broth. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a flow chart of the method for detecting a new carbon source, n-propanol, in anaerobic fermentation broth. DETAILED DESCRIPTION
[0018] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.
[0019] Reference Figure 1 As shown, a method for detecting a new carbon source, n-propanol, in an anaerobic fermentation broth, the method for detecting a new carbon source, n-propanol, in a fermentation broth is: Check and ensure that all instruments used for testing have been maintained and calibrated to achieve optimal working conditions. This is a key step to ensure the accuracy of the test results, as any deviation in the instrument may lead to distortion of the results; The n-propanol standard solution is prepared according to the standard points of 50mg / l, 100mg / l, 200mg / l, 400mg / l, 800mg / l, and 1000mg / l. The concentration points cover the n-propanol concentration range encountered, making the subsequent standard curve more comprehensive and accurate; Use a gas chromatograph to perform sampling and detection on the standard solution of each concentration, record the peak area of the standard solution of each concentration, and draw a standard curve. The peak area reflects the concentration of the target compound in the sample. The standard curve converts the peak area into the corresponding n-propanol concentration, thereby achieving quantitative detection of unknown samples; Obtain the fermentation broth from the anaerobic fermentation reactor. In order to remove solid impurities and microorganisms in the fermentation broth, the fermentation broth is subjected to high-speed centrifugation at a speed of 10,000 r / min for 10 minutes. After centrifugation, it is filtered through a 0.22µm membrane to obtain a clear filtrate. The filtrate is diluted 20 times and placed in a sample bottle. The purpose of the dilution is to adjust the n-propanol concentration in the filtrate to within the detection range of the gas chromatograph and reduce the interference of other impurities. Use gas chromatograph to sample and test the diluent, record the peak area of the diluent, and draw a diluent curve; The standard curve is compared with the dilution curve, and the concentration of the new carbon source n-propanol in the fermentation broth is detected by comparing the peak areas and corresponding concentrations of the two curves. The peak area on the dilution curve matches the concentration point on the standard curve, thereby determining that the fermentation broth contains the corresponding concentration of n-propanol; This detection method is based on gas chromatography. The characteristic of gas chromatography is that gas is used as the mobile phase. Since it requires the components to be in gas state during analysis, it is only suitable for the analysis and separation of low-boiling point and volatile organic compounds.
[0020] The testing instruments include: gas chromatograph, hydrogen flame ionization detector, DB-FATWAX UI chromatographic column, air generator and hydrogen generator; The gas chromatograph consists of a carrier gas system, a sample injection system, a separation system, and a temperature control system; The main function of the carrier gas system is to provide stable and clean carrier gas as the mobile phase. Currently, the main carrier gases used are nitrogen and hydrogen, so air generators and hydrogen generators are used to provide mobile phase for gas chromatographs. The injection system is mainly composed of an injection device and a vaporization chamber. The main function of the injection device is to quantitatively introduce samples. For liquid samples, micro-syringes or automatic injection devices equipped with the instrument are mainly used for injection. The DB-FATWAX UI chromatographic column in the separation system is a type of capillary column. Since the volume of the capillary column is very small and the column capacity is very small, the amount of sample that can be carried is relatively small, so a splitter device is provided in front of the column. At the same time, since the flow rate of the gas in the capillary column is relatively low, a tail blowing device is also provided after the column. Since the gas chromatograph's vaporization chamber, column box, and detector all need to be heated and the temperature controlled, it is equipped with a temperature control system; The working principle of hydrogen flame ionization detector is that the component to be measured burns and ionizes in a hydrogen flame, and the generated positive ions and electrons move in a direction under the action of the electric field to form a current signal. It is characterized by high sensitivity, good stability and wide linear range. Since the hydrogen flame ionization detector requires the component to be measured to be able to burn in a hydrogen flame, it is mainly suitable for organic compounds.
[0021] The maintenance method of the testing instrument is: The instrument maintenance content is evaluated based on the peak shape, separation and peak baseline of the target substance n-propanol. These indicators can directly reflect the performance of the instrument and the accuracy of the test results. At the same time, the number of times the liner is used is used as a quantitative indicator to further determine the specific content of maintenance; When the liner is used more than 220 times, it means that the liner and septum have been greatly worn and contaminated. Therefore, it is necessary to replace the liner and septum in time to ensure the sealing of the instrument and the accuracy of injection. At the same time, in order to thoroughly clean the injection system, methanol and water are used to clean the injection needle, ultrasonic needle base, cleaning bottle and waste liquid bottle respectively to effectively remove residual samples and impurities to avoid interference with subsequent detection; If the liner is used less than 220 times, methanol and water are still needed to clean the injection needle, ultrasonic needle base, cleaning bottle and waste bottle after each injection. Even if the liner has not reached the replacement standard, there may be sample residue or contamination during the injection process. Regular cleaning can keep the injection system clean and accurate. During the injection process, if there is intermittent injection, it may be caused by the injection needle being blocked or damaged. At this time, the injection needle needs to be cleaned or replaced with a new one in time to ensure the continuity and accuracy of the injection; When the peak shape of the instrument has ghost peaks, poor separation or high baseline noise, it is caused by contamination or damage of the split plate or the cut chromatographic column. It is necessary to clean the split plate and the cut chromatographic column in time, or increase the dilution multiple to reduce matrix interference and increase data accuracy, so as to restore the normal performance of the instrument and the accuracy of the test results; If the ignition signal is abnormally high, reaching hundreds of thousands of pA, and purge is ineffective, the detector's collector, nozzle or ceramic piece is contaminated or damaged. Clean the detector's collector, nozzle and ceramic piece to ensure normal operation of the detector and the accuracy of the test results.
[0022] The method for preparing the n-propanol standard solution is: Prepare pure n-propanol standard, which is the basis for preparing standard solution, prepare deionized water as diluent, and prepare clean volumetric flask and pipette; Use a pipette to take the n-propanol standard. This step requires extremely high precision because the amount of n-propanol will directly affect the concentration of the final standard solution. Add the taken n-propanol into 6 volumetric flasks respectively. Each volumetric flask will be used to make standard solutions of different concentrations. Use the multiple dilution method to dilute with diluent. The multiple dilution method is a commonly used solution dilution method. By gradually increasing the amount of diluent, a series of solutions with decreasing concentrations in a multiple relationship are made. The dilution concentrations are 50mg / l, 100mg / l, 200mg / l, 400mg / l, 800mg / l and 1000mg / l standard solutions respectively.
[0023] Preferably, the gas chromatograph detection conditions are: the injection port temperature is set to 280°C, the detector temperature is set to 300°C, the split ratio of the splitter is set to 10:1, the injection volume of the component is set to 0.5µL, and the column box heating program in the temperature control system is set to an initial temperature of 100°C for 2 minutes, and then to 200°C at a rate of 10°C for 10 minutes; The smaller the split ratio, the greater the amount of actual sample entering the detector, and the higher the response. However, considering the influence of the dirty and viscous fermentation broth sample on the injection system, chromatographic column and detector, and the injection volume is only 0.5µL, the split ratio should not be too large, so the split ratio of the split device is selected as 10:1; Consider using DB-FATWAX UI capillary column for sample determination. The sample is water sample, which directly enters the chromatographic column, which has a certain influence. Therefore, the injection volume is 0.5μL within the column tolerance. The column box heating program in the temperature control system is set to maintain the initial temperature at 100°C for 2 minutes, then increase the temperature to 200°C at a rate of 10°C and maintain it for 10 minutes. This can ensure good separation, no tailing peaks or ghost peaks in the peak shape, and good reproducibility.
[0024] The steps for drawing the standard curve are: Draw a scatter plot with the concentration of n-propanol as the X-axis and the corresponding peak area as the Y-axis; Use linear regression to fit the scattered points and get the standard curve equation. The equation calculation formula is: Wherein, Y represents the peak area, a represents the slope, X represents the concentration of n-propanol, and b represents the intercept.
[0025] A standard curve of n-propanol standard solution was drawn. A curve correlation coefficient greater than 0.999 means that there is an almost perfect linear relationship between concentration and peak area, which provides a solid foundation for quantitative analysis. The precision of the standard solution reflects the repeatability and stability of the measurement results. The precision of the standard solution is required to be within 2% to ensure the reliability of the measurement results. The accurate recovery rate of the standard solution reflects the accuracy of the measurement method. The accurate recovery rate of the standard solution is required to be around 100%, which means that the measurement method can accurately determine the concentration of n-propanol without significant deviation or loss.
[0026] The sample precision control range requirements are: When the liner is used less than 100 times, the error range of the sample is required to be controlled within 5%. Because at this stage, the liner is in relatively good condition, with low wear and contamination, so it should be able to maintain high injection accuracy and sample stability. Through strict instrument maintenance and calibration, it is ensured that the test results of the sample are highly accurate and reliable within this range of use. When the liner is used 100-150 times, the condition of the liner begins to decline, and the error range of the sample is required to be controlled within 5%-10%. At this stage, more attention should be paid to the maintenance and cleaning of the liner, and the liner and septum should be replaced regularly, and the injection needle should be cleaned with methanol and water to reduce the impact of sample residue and contamination on the test results. When the liner has been used more than 150 times but has not reached the 220 times standard for replacement, the error range of the sample is relaxed to within 10%-20%. This is because at this stage, the wear and contamination of the liner are relatively serious, which has a certain impact on the injection accuracy and stability of the sample. However, through regular maintenance and cleaning, the accuracy and reliability of the instrument can still be maintained within an acceptable range, ensuring the availability of the test results.
[0027] When testing the fermentation broth samples, a blank test is performed between samples to ensure the accuracy and reliability of the test results. The purpose of this step is to verify whether there is cross contamination or memory effect in the continuous sampling process of the testing instrument, that is, whether the previous sample will affect the test results of the next sample; The specific operation of interspersed blank test is to insert a needle of blank solvent without any target compound between two fermentation broth samples, and then inject and test the blank solvent according to the normal test process. If the result of the blank test shows the presence of the peak shape or signal of the target compound, it means that there is cross contamination or memory effect in the detection instrument, and the instrument needs to be thoroughly cleaned and calibrated to ensure the accuracy of subsequent sample testing; In addition, in order to ensure the stability and reliability of the test results, three parallel data tests are required for each fermentation broth sample. The three parallel data are the results obtained by three independent injections and tests of the same sample under the same test conditions. The repeatability and stability of the test results are evaluated by comparing indicators such as the peak shape, peak area and retention time of the three parallel data. If the differences between the three parallel data are small and are within an acceptable range, the test results of the sample are considered to be accurate and reliable.
[0028] In the process of drawing the standard curve of n-propanol, in order to further improve the accuracy and reliability of the analysis, an internal standard is introduced. The internal standard is a compound that has similar physical and chemical properties to n-propanol but does not interact with it during the detection process. The internal standard is mixed with the n-propanol standard solution and then detected. The peak area of the internal standard is used as a constant reference point to correct the peak area of n-propanol, thereby eliminating the influence of factors such as instrument fluctuations, injection errors or changes in detection conditions on the results.
[0029] The use process of the present invention is: Check the testing equipment before testing to ensure that it can perform testing work normally and accurately; Prepare n-propanol standard solution and use gas chromatograph for detection to draw a standard curve; Treat and dilute the anaerobic fermentation liquid, detect it using a gas chromatograph, and draw a dilution curve; The standard curve was compared with the dilution curve to verify that the anaerobic fermentation broth contained a new carbon source, n-propanol.
[0030] In summary, the advantages of the present invention are: through the detection method, it is proved that n-propanol can be used as a new carbon source for anaerobic fermentation of food waste and kitchen waste, and a set of standardized detection procedures has been formulated, which can also be used for the detection of volatile fatty acids in anaerobic fermentation broth.
[0031] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.
Claims
1. A method for detecting a new carbon source, n-propanol, in an anaerobic fermentation broth, characterized in that: The method for detecting the new carbon source n-propanol in the fermentation broth is: Check and ensure that the testing equipment has been maintained and is in optimal condition; Prepare n-propanol standard solution according to the standard points of 50mg / l, 100mg / l, 200mg / l, 400mg / l, 800mg / l and 1000mg / l; Use gas chromatograph to perform sampling and detection on the standard solution of each concentration, record the peak area of the standard solution of each concentration, and draw a standard curve; The fermentation liquid from the anaerobic fermentation reactor was centrifuged at 10000r / min for 10min, and the filtrate was obtained by passing through a 0.22µm membrane. The filtrate was diluted 20 times and put into a sample bottle; Use gas chromatograph to sample and test the diluent, record the peak area of the diluent, and draw a diluent curve; The standard curve was compared with the dilution curve to detect the presence of n-propanol, a new carbon source, in the fermentation broth.
2. The method for detecting a new carbon source, n-propanol, in an anaerobic fermentation broth according to claim 1, characterized in that: The detection instruments include: gas chromatograph, hydrogen flame ionization detector, DB-FATWAX UI chromatographic column, air generator and hydrogen generator.
3. The method for detecting n-propanol as a new carbon source in anaerobic fermentation broth according to claim 1, characterized in that: The maintenance method of the testing instrument is: Evaluate instrument maintenance content based on target peak shape, resolution, peak baseline and the number of times the liner is used as a quantitative indicator; If the liner is used more than 220 times, replace the liner and septum, and use methanol and water to clean the injection needle, ultrasonic needle base, cleaning bottle and waste bottle respectively; If the liner is used less than 220 times, use methanol and water to clean the injection needle, ultrasonic needle base, cleaning bottle and waste liquid bottle after each injection. If intermittent injection occurs, clean the injection needle or replace the injection needle; If ghost peaks, poor separation and large baseline noise appear in the peak shape, clean the split plate and cut the chromatographic column or increase the dilution factor to reduce matrix interference and increase data accuracy; If the ignition signal reaches several hundred thousand pA and purge is ineffective, clean the collector, nozzle and ceramic piece of the detector.
4. The method for detecting n-propanol as a new carbon source in anaerobic fermentation broth according to claim 1, characterized in that: The method for preparing the n-propanol standard solution is: Prepare pure n-propanol standard, prepare deionized water as diluent, and prepare clean volumetric flasks and pipettes; Use a pipette to take the n-propanol standard and add it into 6 volumetric flasks respectively. Use the dilution method to dilute it with the diluent. The dilution concentrations are 50 mg / l, 100 mg / l, 200 mg / l, 400 mg / l, 800 mg / l and 1000 mg / l standard solutions respectively.
5. The method for detecting n-propanol as a new carbon source in anaerobic fermentation broth according to claim 1, characterized in that: The gas chromatograph detection conditions were as follows: the injection port temperature was set at 280°C, the detector temperature was set at 300°C, the split ratio of the splitter was set at 10:1, the injection volume of the component was set at 0.5µL, and the column box heating program in the temperature control system was set to an initial temperature of 100°C for 2 min, then increased to 200°C at a rate of 10°C and held for 10 min.
6. The method for detecting n-propanol as a new carbon source in anaerobic fermentation broth according to claim 1, characterized in that: The steps for drawing the standard curve are: Draw a scatter plot with the concentration of n-propanol as the X-axis and the corresponding peak area as the Y-axis; Use linear regression to fit the scattered points and get the standard curve equation. The equation calculation formula is: Wherein, Y represents the peak area, a represents the slope, X represents the concentration of n-propanol, and b represents the intercept.
7. The method for detecting n-propanol as a new carbon source in anaerobic fermentation broth according to claim 1, characterized in that: A standard curve of n-propanol standard solution was drawn, and the correlation coefficient of the curve was greater than 0.999, the precision of the standard solution was within 2%, and the accurate recovery rate of the standard solution was about 100%.
8. The method for detecting n-propanol as a new carbon source in anaerobic fermentation broth according to claim 1, characterized in that: The sample precision control range requirements are: The liner is used less than 100 times and the sample error range is within 5%; The liner was used 100-150 times, and the sample error range was within 5%-10%; The liner was used 150-220 times, and the sample error range was within 10%-20%.
9. The method for detecting n-propanol as a new carbon source in anaerobic fermentation broth according to claim 1, characterized in that: When the fermentation broth samples are sampled and tested, a blank test is required between samples, and three parallel data are required.
10. The method for detecting n-propanol as a new carbon source in anaerobic fermentation broth according to claim 1, characterized in that: When drawing the standard curve, an internal standard is introduced, and the internal standard is mixed with the n-propanol standard solution for detection. The peak area of n-propanol is corrected with the peak area of the internal standard as a reference.