Method for detecting cyanide in bacteria-containing fermentation clear liquid

Abstract

The invention relates to a method for detecting cyanide in bacteria-containing fermentation clear liquid. The method comprises the following steps: obtaining a standard curve of absorbance of a series of gradient cyanide standard solutions and concentration of the series of gradient cyanide standard solutions; adding an auxiliary agent into the bacterium-containing fermentation clear liquid sample, and then distilling until cyanide in the bacterium-containing fermentation clear liquid sample containing the auxiliary agent is completely volatilized to obtain a first bacterium-containing fermentation clear liquid; wherein the auxiliary agent comprises a defoaming agent; measuring the absorbance of the first bacterium-containing fermentation clear liquid sample; according to the standard curve and the absorbance of the first bacterium-containing fermentation clear liquid sample, the content of cyanide in the first bacterium-containing fermentation clear liquid sample is obtained. According to the method, the detection accuracy of the cyanide in the bacterium-containing fermentation clear liquid is improved.

Description

Technical Field

[0001] The present application relates to the technical field of cyanide detection, and in particular to a method for detecting cyanide in bacterial fermentation clear liquid. Background Art

[0002] Cyanide is a highly toxic substance that has a significant adverse effect on the fermentation process. Cyanide can inhibit the growth of microorganisms, interfere with the metabolic pathways of microorganisms, and lead to a decrease in the quality and yield of fermentation products. Even at very low concentrations, it can inhibit or completely stop the growth and metabolic activities of microorganisms, thus affecting the normal progress of the fermentation process.

[0003] At present, the detection of cyanide in fermentation broth is difficult because some mycelium remains in the fermentation liquid. When the fermentation liquid sample is boiled, a large number of bubbles will be generated, causing the bubbles to overflow with the steam. The distillation process cannot proceed normally, which becomes a "bottleneck" of detection. Summary of the invention

[0004] The present application provides a method for detecting cyanide in bacterial fermentation supernatant to solve the following technical problem: how to improve the accuracy of cyanide detection in bacterial fermentation supernatant.

[0005] In a first aspect, the present application provides a method for detecting cyanide in a bacterial fermentation supernatant, the method comprising:

[0006] Obtaining a standard curve of the absorbance of a series of gradient cyanide standard solutions and the concentration of the series of gradient cyanide standard solutions;

[0007] Adding an auxiliary agent to the bacterial fermentation clear liquid sample, and then performing distillation until the cyanide in the bacterial fermentation clear liquid sample containing the auxiliary agent is completely volatilized to obtain a first bacterial fermentation clear liquid; wherein the auxiliary agent includes a defoaming agent;

[0008] Measuring the absorbance of the first bacterial fermentation clear liquid sample;

[0009] The content of cyanide in the first bacteria-containing fermentation supernatant sample is obtained according to the standard curve and the absorbance of the first bacteria-containing fermentation supernatant sample.

[0010] Optionally, the defoaming agent includes one of the following: defoaming agent DF104, defoaming agent NV-3, and defoaming agent 31R1.

[0011] Optionally, the defoaming agent is 4 mL to 6 mL relative to 1 L of the bacterial fermentation clear liquid sample.

[0012] Optionally, the auxiliary agent also includes: an anti-boiling agent, a zinc salt, an acid indicator, an organic acid and an alkali solution.

[0013] Optionally, measuring the absorbance of the first bacteria-containing fermentation clear liquid sample comprises:

[0014] Performing a first volume determination on the first bacteria-containing fermentation clear liquid sample;

[0015] performing a color development reaction on the first bacteria-containing fermentation clear liquid sample after the first volume fixation to form a stable colored compound in the first bacteria-containing fermentation clear liquid sample after the first volume fixation to obtain a second bacteria-containing fermentation clear liquid sample;

[0016] Performing a second volume determination on the second bacteria-containing fermentation clear liquid sample;

[0017] The absorbance of the second bacteria-containing fermentation clear liquid sample after the second volume fixation at 613 nm is measured using an ultraviolet-visible spectrophotometer to obtain the absorbance of the first bacteria-containing fermentation clear liquid sample.

[0018] Optionally, subjecting the first bacteria-containing fermentation clear liquid sample after the first volume fixation to a color reaction to form a stable colored compound in the first bacteria-containing fermentation clear liquid sample after the first volume fixation to obtain a second bacteria-containing fermentation clear liquid sample comprises:

[0019] An alkaline indicator, an acid solution, a phosphate buffer solution, a chloramine T solution and a pyridine-pyrazolone solution are added to the first bacteria-containing fermentation clear liquid sample after the first fixed volume, and a color reaction is performed to form a stable colored compound in the first bacteria-containing fermentation clear liquid sample after the first fixed volume, so as to obtain a second bacteria-containing fermentation clear liquid sample.

[0020] Optionally, the cyanide content in the first bacterial fermentation clear liquid sample satisfies the following relationship:

[0021]

[0022] In the formula, ρ(CN-) represents the mass concentration of cyanide in the first bacteria-containing fermentation supernatant sample; ρ1 represents the mass concentration of cyanide in the standard curve corresponding to the absorbance of the first bacteria-containing fermentation supernatant sample; V1 represents the volume of the first bacteria-containing fermentation supernatant sample; V2 represents the volume of the first bacteria-containing fermentation supernatant sample after the first constant volume; V3 represents the volume of the second bacteria-containing fermentation supernatant sample after the second constant volume; V represents the volume of the bacteria-containing fermentation supernatant sample.

[0023] Optionally, obtaining a standard curve of absorbance and concentration of a series of gradient cyanide standard solutions comprises:

[0024] A series of gradient cyanide standard solutions are obtained;

[0025] The absorbance of the series of gradient cyanide standard solutions at 613 nm was measured using an ultraviolet-visible spectrophotometer;

[0026] A standard curve of the absorbance of the series of gradient cyanide standard solutions and the concentration of the series of gradient cyanide standard solutions is drawn to obtain a standard curve of the absorbance of the series of gradient cyanide standard solutions and the concentration.

[0027] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0028] The method for detecting cyanide in the bacteria-containing fermentation supernatant provided in the embodiment of the present application includes: obtaining a standard curve of the absorbance of a series of gradient cyanide standard solutions and the concentration of the series of gradient cyanide standard solutions; adding an auxiliary agent to the bacteria-containing fermentation supernatant sample, and then distilling until the cyanide in the bacteria-containing fermentation supernatant sample containing the auxiliary agent is completely volatilized to obtain a first bacteria-containing fermentation supernatant; wherein the auxiliary agent includes a defoaming agent; measuring the absorbance of the first bacteria-containing fermentation supernatant sample; and obtaining the cyanide content in the first bacteria-containing fermentation supernatant sample based on the standard curve and the absorbance of the first bacteria-containing fermentation supernatant sample. The bacterial fermentation clear liquid sample is distilled to produce a large amount of bubbles and foam. The defoamer in the auxiliary agent can destroy the stability of the bubbles, making it difficult for the bubbles to form or break quickly, thereby maintaining the smooth progress of the distillation process, fully distilling the simple cyanide and the complex cyanide, and increasing the concentration of cyanide in the first bacterial fermentation clear liquid sample, and reducing the detection limit; through the absorbance of the series of gradient cyanide standard solutions and the standard curve of the concentration of the series of gradient cyanide standard solutions, as well as the absorbance of the first bacterial fermentation clear liquid sample, a high-accuracy cyanide detection result can be obtained. In summary, the accuracy of cyanide detection in bacterial fermentation clear liquid is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0031] Figure 1 A schematic diagram of a process for detecting cyanide in a bacterial fermentation supernatant provided in accordance with some embodiments of the present application;

[0032] Figure 2 A diagram of a distillation device for providing a bacterial fermentation clear liquid according to some embodiments of the present application;

[0033] Figure 3According to some embodiments of the present application, a standard curve of the absorbance of a series of gradient cyanide standard solutions and the concentration of the series of gradient cyanide standard solutions is provided. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0035] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0036] In this application, the terms "include", "comprising", etc. mean "including but not limited to". In this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0038] Figure 1 This is a flow chart of a method for detecting cyanide in bacterial fermentation supernatant provided in some embodiments of the present application; see Figure 1 The present application provides a method for detecting cyanide in bacterial fermentation clear liquid, the method comprising:

[0039] S1, obtaining a standard curve of the absorbance of a series of gradient cyanide standard solutions and the concentration of the series of gradient cyanide standard solutions;

[0040] In some embodiments, obtaining a standard curve of absorbance and concentration of a series of gradient cyanide standard solutions comprises:

[0041] A series of gradient cyanide standard solutions are obtained;

[0042] The absorbance of the series of gradient cyanide standard solutions at 613 nm was measured using an ultraviolet-visible spectrophotometer;

[0043] A standard curve of the absorbance of the series of gradient cyanide standard solutions and the concentration of the series of gradient cyanide standard solutions is drawn to obtain a standard curve of the absorbance of the series of gradient cyanide standard solutions and the concentration.

[0044] In the embodiment of the present application, 0 mL, 0.50 mL, 2.50 mL, 5.00 mL of cyanide standard working solution II and 1.00 mL, 1.50 mL, 2.00 mL of cyanide standard working solution I were taken into a series of 25 mL colorimetric tubes and diluted to about 10 mL. 0.03 mL of phenolphthalein indicator was added and neutralized with acetic acid solution until colorless. 2 mL of phosphate buffer solution and 1.5-2 mL of chloramine T solution were added, and the mixture was shaken and allowed to stand for 1 min. 9 mL of pyridine-pyrazolone solution was added, and the mixture was made up to volume with pure water and shaken and allowed to stand for 30 min. The mass concentrations were 0 mg / L, 0.002 mg / L, 0.01 mg / L, 0.02 mg / L, 0.04 mg / L, 0.06 mg / L and 0.08 mg / L, respectively. The absorbance was measured on a spectrophotometer at a wavelength of 613 nm using a 1 cm colorimetric cup for quantitative analysis of cyanide in the first bacterial fermentation clear liquid.

[0045] S2, adding an auxiliary agent to the bacterial fermentation clear liquid sample, and then distilling until the cyanide in the bacterial fermentation clear liquid sample containing the auxiliary agent is completely volatilized to obtain a first bacterial fermentation clear liquid; wherein the auxiliary agent includes a defoaming agent; Figure 2 A diagram of a distillation apparatus for fermentation supernatant containing bacteria is provided according to some embodiments of the present application; see Figure 2 ;

[0046] In some embodiments, the defoaming agent includes one of the following: defoaming agent DF104, defoaming agent NV-3, and defoaming agent 31R1.

[0047] In the present application embodiment, due to the fermentation clear liquid has a small part of fermentation hyphae, distillation boiling will cause the fermentation clear liquid sample foaming so as to be unfavorable for cyanide distillation, affecting the distillation operation, adding defoamer can reduce the liquid surface tension, eliminate foam, so that the distillation operation is carried out normally. The above-mentioned defoamer DF104, defoamer NV-3, defoamer 31R1 have the characteristics of high molecular weight, high tension and high viscosity, can effectively prevent bubbles from rising in the liquid. And active surfactant is one of the key components in the defoamer, it by adjusting the liquid surface tension, reduces the formation and stability of bubbles, makes the fermentation liquid surface smoother. The above-mentioned bacteria can be to use alcohol clostridium protein as raw material strain to produce fuel ethanol, bacteria is alcohol clostridium, can also be butyric clostridium, acetobutylic clostridium, lactobacillus, bifidobacterium, clostridium botulinum etc.

[0048] In some embodiments, the defoaming agent is 4 mL to 6 mL relative to 1 L of the bacterial fermentation clear liquid sample.

[0049] In the present application embodiment, the role of the defoamer is to destroy the stability of the bubble, so that the foam is not easy to form or quickly break, so as to maintain the smooth progress of the distillation process, and prevent the foam from overflowing, thereby increasing the efficiency of distillation. If the defoamer is added in too much amount, it will remain in the fermentation liquid, affecting the distillation efficiency and reducing the cleaning effect of the distillation apparatus. Too much defoamer will increase unnecessary costs. On the contrary, if the defoamer is added in too little amount, the expected defoaming effect may not be achieved, and foaming will occur during the distillation process, resulting in the distillation process being unable to proceed. Exemplary, the above-mentioned defoamer relative to 1L of the bacterial fermentation clear liquid sample can be 4mL, 4.5mL, 5.0mL, 5.5mL, 6.0mL, etc.

[0050] In some embodiments, the auxiliary agent further comprises: an anti-boiling agent, a zinc salt, an acid indicator, an organic acid and an alkali solution.

[0051] In the embodiment of the present application, the anti-boiling agent can be a number of glass beads, ceramic particles, special plastic particles, etc., which can make the bacterial fermentation clear liquid sample heated evenly and prevent the solution from boiling during the distillation process. Zinc salts can be zinc acetate solution (100g / L), zinc oxide, zinc carbonate, zinc sulfate, zinc chloride, zinc phosphate, zinc molybdate, zinc sulfide, zinc chromate, zinc stearate, zinc silicate, etc. On the one hand, it is to eliminate the interference of sulfide (generating zinc sulfide precipitation without being steamed out), and at the same time, it also suppresses the decomposition of each stable complex cyanide. Acidic indicators can be methyl orange indicators, nitrophenol indicators, azo compound indicators, etc., organic acids can be tartaric acid, succinic acid, malic acid, etc., and alkali liquor can be sodium hydroxide, calcium hydroxide, magnesium hydroxide, etc. Exemplary, the color change of methyl orange indicator is as follows: when pH≦3.1, it is red; when the pH value is 3.1-4.4, it is orange, which is a mixed color of red and yellow; when pH≧4.4, it is yellow. Methyl orange is added to determine whether the pH is less than 3.1 after the addition of tartaric acid, in an acidic environment. Tartaric acid acts as a buffer to control the pH value of the reaction system. Because the reaction of cyanide and chloramine T needs to be carried out under certain acidity conditions, and tartaric acid can effectively maintain this suitable acidity environment. Tartaric acid can provide an appropriate amount of hydrogen ions in the reaction system, thereby keeping the pH value of the reaction system within the required range. This acidic environment helps to promote the full reaction of cyanide and chloramine T, as well as the subsequent reaction with pyridine and pyrazolone.

[0052] Cu 2+ , Hg 2+ , Fe 3+ , and S 2- ,I - 、SCN - Plasma has different degrees of interference on the determination of cyanide. After pre-distillation, most of the interference can be eliminated, and the purity of cyanide can be improved. Through pre-distillation under acidic conditions, simple cyanide can be converted into hydrogen cyanide and released from water, and then collected through sodium hydroxide washing liquid, thereby distinguishing simple cyanide from complex cyanide, increasing the concentration of cyanide, and reducing the detection limit.

[0053] S3, measuring the absorbance of the first bacterial fermentation clear liquid sample;

[0054] S4. Obtaining the cyanide content in the first bacteria-containing fermentation supernatant sample according to the standard curve and the absorbance of the first bacteria-containing fermentation supernatant sample.

[0055] In some embodiments, measuring the absorbance of the first bacterial fermentation supernatant sample comprises:

[0056] Performing a first volume determination on the first bacteria-containing fermentation clear liquid sample;

[0057] performing a color development reaction on the first bacteria-containing fermentation clear liquid sample after the first volume fixation to form a stable colored compound in the first bacteria-containing fermentation clear liquid sample after the first volume fixation to obtain a second bacteria-containing fermentation clear liquid sample;

[0058] In the embodiments of the present application, in a neutral or weakly alkaline medium, cyanide ions react with chloramine T to convert into cyanogen chloride, which then reacts with pyridine to generate glutaraldehyde after hydrolysis, and then reacts with pyrazolone to generate a blue polymethylene dye with maximum absorption at a wavelength of 613 nm. Its absorbance is proportional to the mass concentration of cyanide ions within a certain concentration range.

[0059] Performing a second volume determination on the second bacteria-containing fermentation clear liquid sample;

[0060] The absorbance of the second bacteria-containing fermentation clear liquid sample after the second volume fixation at 613 nm is measured using an ultraviolet-visible spectrophotometer to obtain the absorbance of the first bacteria-containing fermentation clear liquid sample.

[0061] In some embodiments, the step of subjecting the first fermentation supernatant containing bacteria to a color reaction to form a stable colored compound in the first fermentation supernatant containing bacteria to obtain a second fermentation supernatant containing bacteria sample comprises:

[0062] An alkaline indicator, an acid solution, a phosphate buffer solution, a chloramine T solution and a pyridine-pyrazolone solution are added to the first bacteria-containing fermentation clear liquid sample after the first fixed volume, and a color reaction is performed to form a stable colored compound in the first bacteria-containing fermentation clear liquid sample after the first fixed volume, so as to obtain a second bacteria-containing fermentation clear liquid sample.

[0063] In the embodiment of the present application, the alkaline indicator can be a phenolphthalein indicator, a bromophenol blue indicator, a phenol red indicator, a methyl red indicator, etc., and the acid solution can be an acetic acid solution (acetic acid: ultrapure water = 1:3), a formic acid solution, or a butyric acid solution. The preparation method of the above-mentioned phosphate buffer solution (pH 6.8) is as follows: 34.0 g of potassium dihydrogen phosphate (KH2PO4) and 35.5 g of sodium dihydrogen phosphate (Na2HPO4) are weighed and dissolved in pure water, diluted to 1 000 mL, and shaken well. The preparation method of chloramine T solution (10 g / L) is as follows: 0.5 g of chloramine T (C7H7SO2NClNa) is weighed and dissolved in 50 mL of pure water, and prepared immediately before use. Preparation method of pyridine-pyrazolone solution: weigh 0.5g of 1-phenyl-3-methyl-5-pyrazolone (C10H10ON2) and dissolve it in 100mL of anhydrous ethanol, add 0.5mL of pyridine (C5H5N), and shake well. This solution can be stored at room temperature for 7 days. The purpose of the above two volume adjustments is to ensure that the volume of the solution reaches the preset accurate value to meet the needs of the experiment.

[0064] In some embodiments, the cyanide content in the first bacterial fermentation supernatant sample satisfies the following relationship (1):

[0065]

[0066] In the formula, ρ(CN-) represents the mass concentration of cyanide in the first bacteria-containing fermentation supernatant sample; ρ1 represents the mass concentration of cyanide in the standard curve corresponding to the absorbance of the first bacteria-containing fermentation supernatant sample; V1 represents the volume of the first bacteria-containing fermentation supernatant sample; V2 represents the volume of the first bacteria-containing fermentation supernatant sample after the first constant volume; V3 represents the volume of the second bacteria-containing fermentation supernatant sample after the second constant volume; V represents the volume of the bacteria-containing fermentation supernatant sample.

[0067] The present application provides a method for detecting cyanide in bacterial fermentation supernatant, which has the advantages of high sensitivity, simple operation, low cost, etc. It is suitable for the accurate detection of trace cyanide content in ethanol Clostridium protein fermentation supernatant in industrial production, and can better provide reliable analytical data for production fermentation processes.

[0068] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0069] Example 1

[0070] A method for detecting cyanide in bacterial fermentation clear liquid, using a UV-visible spectrophotometer model: SP-752; using a wavelength of 613nm. It includes:

[0071] Step 1 Preparation of standard solution: Pipette 0mL, 0.50mL, 2.50mL, 5.00mL of cyanide standard working solution II and 1.00mL, 1.50mL, 2.00mL of cyanide standard working solution I into a series of 25mL colorimetric tubes, dilute to about 10mL, add 1 drop of phenolphthalein indicator, neutralize with acetic acid solution until colorless, add 2mL of phosphate buffer solution and 6 drops of chloramine T solution, shake well, let stand for 1min, add 9mL of pyridine-pyrazolone solution, make up to volume with pure water, shake well, let stand for 30min;

[0072] Draw a standard curve: the mass concentrations of the prepared cyanide standard solutions are 0 mg / L, 0.002 mg / L, 0.01 mg / L, 0.02 mg / L, 0.04 mg / L, 0.06 mg / L and 0.08 mg / L, respectively. The absorbance is measured on a spectrophotometer at a wavelength of 613 nm using a 1 cm colorimetric cup; the results of the mass concentration and absorbance of the cyanide standard solutions are shown in Table 1.

[0073] Table 1 Cyanide standard solution mass concentration and absorbance

[0074] Sample number Absorbance Concentrationρ(mg / L) Standard solution 0 0.0004 0.0000 Standard solution 1 0.011 0.0020 Standard solution 2 0.0438 0.0100 Standard solution 3 0.0817 0.0200 Standard solution 4 0.1538 0.0400 Standard solution 5 0.2263 0.0600 Standard solution 6 0.2956 0.0800

[0075] According to the mass concentration and absorbance of the cyanide standard solution, a calibration curve was drawn with the mass concentration of cyanide (in terms of CN-) as the abscissa and the absorbance as the ordinate, and the linear regression equation of the mass concentration and absorbance of the cyanide standard solution was obtained: A = 3.6706C + 0.0049, R = 0.99966; Figure 3 According to some embodiments of the present application, a standard curve of absorbance of a series of gradient cyanide standard solutions and concentration of the series of gradient cyanide standard solutions is provided; see Figure 3 .

[0076] Step 2: Distillation of fermentation liquid: Take 250mL of fermentation liquid in a 500mL all-glass distillation bottle, put a few glass beads, connect the cooling system (the whole system cannot leak), connect a 50mL measuring cylinder containing 5mL of sodium hydroxide solution to the lower end of the condenser, and insert the lower end of the condenser below the liquid surface of the sodium hydroxide solution. Add 1-1.5mL of defoamer, 10mL of zinc acetate solution and 1-1.5mL of methyl orange indicator to the distillation bottle and shake well. Quickly add 2g of tartaric acid, at this time the solution should be red (if it is yellow, tartaric acid should be added until the solution is red), immediately cover the bottle cap, open the condensed water and heat for distillation. The heating temperature should be controlled during distillation so that there are no bubbles on the absorption liquid surface. When the total volume of the solution in the receiving measuring cylinder is close to 50mL, stop distillation and dilute to 50mL with pure water for measurement;

[0077] Step 3: Absorbance measurement of fermentation clear liquid: Take 10.00mL of distillate in a 25mL colorimetric tube, add 1 drop of phenolphthalein indicator, neutralize with acetic acid solution until colorless, add 2mL of phosphate buffer solution and 6 drops of chloramine T solution, shake well, let stand for 1 min, add 9mL of pyridine-pyrazolone solution, make up to volume with pure water and shake well. After standing for 30min, measure its absorbance at a wavelength of 613nm on a spectrophotometer with a 1cm colorimetric cup and a reagent blank sample as a reference; where the reagent blank sample: take 250mL of pure water instead of the sample, repeat steps 2 and 3 to determine the measurement accuracy of the fermentation clear liquid.

[0078] For the fermentation supernatant sample containing 0.0260 mg / L cyanide, steps 2 to 3 were performed to measure cyanide 8 times, and the cyanide content in the fermentation supernatant sample was calculated using formula 1. Please refer to the result table 2.

[0079] For the fermentation supernatant sample containing 0.0423 mg / L cyanide in steps 2 to 3, cyanide was measured 8 times, and the cyanide content in the fermentation supernatant sample was calculated using formula 1. Please refer to the results in Table 3.

[0080] Table 2 Cyanide measurement results of fermentation liquid samples containing 0.0260 mg / L cyanide

[0081] Sample name Cyanide mass concentration ρ(mg / L) T2R2 serum 0.026039 T2R2 serum 0.026002 T2R2 serum 0.025984 T2R2 serum 0.026007 T2R2 serum 0.026041 T2R2 serum 0.025968 T2R2 serum 0.026020 T2R2 serum 0.025995

[0082] Table 3 Cyanide measurement results of fermentation liquid samples containing 0.0423 mg / L cyanide

[0083] Sample name Cyanide mass concentration ρ(mg / L) T2R2 serum 0.042301 T2R2 serum 0.042299 T2R2 serum 0.042301 T2R2 serum 0.042299 T2R2 serum 0.042301 T2R2 serum 0.042301 T2R2 serum 0.042300 T2R2 serum 0.042300

[0084] According to Table 2 and Table 3, the relative standard deviation of the fermentation liquid sample containing 0.0260 mg / L cyanide was 0.25% after 8 determinations, and the relative standard deviation of the fermentation liquid sample containing 0.0423 mg / L cyanide was 0.17%, indicating that the method for detecting cyanide in bacterial fermentation liquid provided by the present application has high precision and high accuracy.

[0085] Comparative Example 1

[0086] Difference from Example 1 is: fermentation clear liquid distillation: take fermentation clear liquid 250mL in 500mL all-glass distilling bottle, put several glass beads, connect cooling system (whole system can not leak), condenser tube lower terminal is connected with a 50mL graduated cylinder filled with 5mL sodium hydroxide solution, and the lower mouth of condenser tube will be inserted under the sodium hydroxide solution liquid level. Add zinc acetate solution 10mL and methyl orange indicator 1-1.5mL in distilling bottle, shake up. Add tartaric acid 2g quickly, now solution should be red (if yellow, tartaric acid should be added until solution is red), cover bottle cap immediately, open condensed water and heat distillation. Because defoamer is not added in distilling bottle, when distilling, a large amount of bubbles are produced after solution boiling, rise to bottle mouth and distilling tube, cause distillation experiment and subsequent steps to be unable to carry out.

[0087] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for detecting cyanide in bacterial fermentation supernatant, characterized in that: The method comprises: Obtaining a standard curve of the absorbance of a series of gradient cyanide standard solutions and the concentration of the series of gradient cyanide standard solutions; Adding an auxiliary agent to the bacterial fermentation clear liquid sample, and then performing distillation until the cyanide in the bacterial fermentation clear liquid sample containing the auxiliary agent is completely volatilized to obtain a first bacterial fermentation clear liquid; wherein the auxiliary agent includes a defoaming agent; Measuring the absorbance of the first bacterial fermentation clear liquid sample; The content of cyanide in the first bacteria-containing fermentation supernatant sample is obtained according to the standard curve and the absorbance of the first bacteria-containing fermentation supernatant sample.

2. The method according to claim 1, characterized in that: The defoamer includes one of the following: defoamer DF104, defoamer NV-3, and defoamer 31R1.

3. The method according to claim 1, characterized in that: Relative to 1L of the bacterial fermentation clear liquid sample, the defoaming agent is 4mL to 6mL.

4. The method according to claim 1, characterized in that: The auxiliary agent also includes: an anti-boiling agent, a zinc salt, an acid indicator, an organic acid and an alkali solution.

5. The method according to claim 1, characterized in that The step of measuring the absorbance of the first bacterial fermentation clear liquid sample comprises: Performing a first volume determination on the first bacteria-containing fermentation clear liquid sample; performing a color development reaction on the first bacteria-containing fermentation clear liquid sample after the first volume fixation to form a stable colored compound in the first bacteria-containing fermentation clear liquid sample after the first volume fixation to obtain a second bacteria-containing fermentation clear liquid sample; Performing a second volume determination on the second bacteria-containing fermentation clear liquid sample; The absorbance of the second bacteria-containing fermentation clear liquid sample after the second volume fixation at 613 nm is measured using an ultraviolet-visible spectrophotometer to obtain the absorbance of the first bacteria-containing fermentation clear liquid sample.

6. The method according to claim 1, characterized in that The step of subjecting the first fermentation clear liquid sample containing bacteria to a color reaction after the first volume fixation to form a stable colored compound in the first fermentation clear liquid sample containing bacteria after the first volume fixation to obtain a second fermentation clear liquid sample containing bacteria comprises: An alkaline indicator, an acid solution, a phosphate buffer solution, a chloramine T solution and a pyridine-pyrazolone solution are added to the first bacteria-containing fermentation clear liquid sample after the first fixed volume, and a color reaction is performed to form a stable colored compound in the first bacteria-containing fermentation clear liquid sample after the first fixed volume, so as to obtain a second bacteria-containing fermentation clear liquid sample.

7. The method according to claim 6, characterized in that The cyanide content in the first bacterial fermentation clear liquid sample satisfies the following relationship: In the formula, ρ(CN-) represents the mass concentration of cyanide in the first bacteria-containing fermentation supernatant sample; ρ1 represents the mass concentration of cyanide in the standard curve corresponding to the absorbance of the first bacteria-containing fermentation supernatant sample; V1 represents the volume of the first bacteria-containing fermentation supernatant sample; V2 represents the volume of the first bacteria-containing fermentation supernatant sample after the first constant volume; V3 represents the volume of the second bacteria-containing fermentation supernatant sample after the second constant volume; V represents the volume of the bacteria-containing fermentation supernatant sample.

8. The method according to claim 1, characterized in that The method of obtaining a standard curve of absorbance and concentration of a series of gradient cyanide standard solutions comprises: A series of gradient cyanide standard solutions are obtained; The absorbance of the series of gradient cyanide standard solutions at 613 nm was measured using an ultraviolet-visible spectrophotometer; A standard curve of the absorbance of the series of gradient cyanide standard solutions and the concentration of the series of gradient cyanide standard solutions is drawn to obtain a standard curve of the absorbance of the series of gradient cyanide standard solutions and the concentration.

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