A method for predicting oxygen mass transfer effectiveness in a methanol yeast fermentation process

Abstract

The present application relates to a kind of methods for predicting oxygen transfer effect in methanol yeast fermentation process.The method comprises the following steps: step 1, obtaining the methanol feeding amount of fermentation equipment in fermentation process;Step 2: calculate oxygen consumption according to the methanol feeding amount;Step 3: obtain the oxygen consumption of fermentation equipment in stable state;Step 4: obtain oxygen transfer rate according to the oxygen consumption of fermentation equipment in stable state.The method can directly calculate OTR by the methanol feeding amount, and the value of Kla is directly obtained using OTR, which provides data for process optimization and fermentation equipment optimization.

Description

Technical Field

[0001] This invention relates to the field of bioreactor technology, and more specifically to a method for predicting the oxygen mass transfer effect during methanol yeast fermentation. Background Technology

[0002] In yeast fermentation, Kla represents the rate at which oxygen enters the liquid phase from the gas phase. Many factors influence Kla, such as liquid phase properties, bubble size, system geometry, and operating parameters. Due to the low solubility of oxygen in water, the oxygen mass transfer rate (OTR) has always been an important parameter for aerobic microbial culture. During cell culture, oxygen enters the cell from bubbles in two processes: 1) from the gas phase to the liquid phase; 2) from the liquid phase to the cell, which can be expressed by the oxygen mass transfer rate (OTR) and the oxygen uptake rate (OUR). Different types of fermenters exhibit varying Kla values; therefore, understanding the Kla requirements of yeast fermentation is necessary before designing a fermenter. Kla can be determined using both chemical and physical methods.

[0003] Chemical methods, such as the sulfite oxidation method, are used to determine Kla. Physical methods commonly employ dynamic measurement models, which can be further divided into two scenarios: one considering OUR (oral oxygen intake), where oxygen pumping is stopped for a certain period while living cells are present, and OUR and OTR (or active oxygen intake) are measured separately. The first stage determines OUR, and the second stage measures Kla based on OUR and DO (displacement). These testing methods are relatively complex and can cause some damage to cells.

[0004] No effective solution has yet been proposed to address the above problems. Summary of the Invention

[0005] Therefore, it is necessary to provide a method for predicting the oxygen mass transfer effect during methanol yeast fermentation, addressing the aforementioned technical problems. Yeast fermentation mainly consists of three stages: glycerol culture, conversion, and methanol induction. This method is used to predict the oxygen mass transfer effect during the third stage of fermentation, the methanol induction stage.

[0006] During the methanol-induced phase, the initial metabolic stage of methanol in cells occurs in peroxisomes, which proliferate rapidly under methanol-induced conditions, occupying up to 80% of the cell's internal space. In the first step of the methanol utilization pathway, alcohol oxidase (Aox) catalyzes the oxidation of methanol to formaldehyde and hydrogen peroxide (H₂O₂). H₂O₂ is then decomposed into H₂O and O₂ by catalase (CAT). Formaldehyde has two fates: one is the catabolism pathway, where it is catalyzed by formaldehyde dehydrogenase (Fld) and formate dehydrogenase (Fdh) to produce CO₂, providing energy for the cell; the other is the assimilation pathway, where, under the action of dihydroxyacetone synthase (Das), it reacts with xylulose-5-phosphate to produce dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (GAP), subsequently used in the central carbon metabolism pathway to provide precursors and energy for cell growth. Overall, the metabolic and protein synthesis pathways of methanol in cells are complex; therefore, the oxygen consumed by methanol combustion is used to equivalently replace the oxygen consumed during methanol yeast fermentation. Methanol (CH3OH) is the simplest saturated monohydric alcohol. Composed of a methyl group and a hydroxyl group, it exhibits the chemical properties of alcohols. It burns violently in pure oxygen, producing water vapor and carbon dioxide. Its combustion equation is: 2CH3OH + 3O2 = 2CO2 + 4H2O.

[0007] In one aspect, the present invention provides a method for predicting the oxygen mass transfer effect during methanol yeast fermentation, comprising the following steps:

[0008] Step 1: Obtain the amount of methanol fed into the fermentation equipment during the fermentation process;

[0009] Step 2: Calculate oxygen consumption based on methanol feed rate;

[0010] Step 3: Obtain the oxygen consumption of the fermentation equipment when it is in a stable state;

[0011] Step 4: Obtain the oxygen mass transfer rate based on the oxygen consumption when the fermentation equipment is in a stable state.

[0012] In some embodiments, step 5 is included after step 4: obtaining a predicted value of Kla based on the oxygen mass transfer rate.

[0013] In some embodiments, the fermentation equipment is in a stable state when: dC L / dt=0, where C L This represents the dissolved oxygen concentration in the liquid phase.

[0014] In some embodiments, the formula for calculating the oxygen mass transfer rate based on the oxygen consumption of the fermentation equipment in a steady state is as follows:

[0015] OTR=OUR=u O2

[0016] In the formula, OTR is the oxygen mass transfer rate, OUR is the oxygen absorption rate, and u O2 This refers to the oxygen consumption per unit time and unit volume when the fermentation equipment is in a stable state.

[0017] In some embodiments, the formula for calculating the predicted value of Kla based on the oxygen mass transfer rate is as follows:

[0018]

[0019] In the formula, Kla prediction is the predicted value of Kla, OTR is the oxygen mass transfer rate, T is the fermentation temperature, DO actual is the actual dissolved oxygen saturation, and α is the correction coefficient.

[0020] In some embodiments, the correction factor α ranges from 95% to 97%.

[0021] In some embodiments, the formula for calculating oxygen consumption based on the methanol feed rate is as follows:

[0022] F O2 =F 甲醇 ×1.5

[0023] In the formula, F O2 Oxygen consumption per unit time, F 甲醇 This represents the amount of methanol supplied per unit time.

[0024] In some embodiments, the fermentation equipment is a fermentation tank.

[0025] In another aspect, the present invention also provides the application of any of the above-mentioned methods for predicting the oxygen mass transfer effect during methanol yeast fermentation in the optimization of fermentation equipment or fermentation process.

[0026] By implementing the above-described solution of the present invention, the following beneficial effects can be obtained:

[0027] By utilizing the methanol combustion equation, the OTR (Optical Time Tolerance) can be directly calculated from the methanol feed rate, and the Kla value can be directly derived from the OTR, providing data for process optimization and fermentation equipment optimization. This solves the problem of cell damage caused by chemical and physical methods for measuring Kla values. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] In some embodiments, the present invention provides a method for predicting fermentation heat in a fermentation device, which is obtained by the following steps:

[0030] First, calculate the oxygen consumed during methanol combustion based on the amount of methanol added. M is known. 甲醇 Molecular mass (g / mol), ρ 甲醇 Density (kg / m³) 3 W 甲醇 Feed rate (L / h), V: working volume of fermenter (L). The m of methanol can be calculated. b moles per unit mass (mol / kg), C 甲醇 : moles per unit volume (mol / L), F 甲醇 Feed rate per unit time (mol / h).

[0031] m b =1000 / M 甲醇

[0032] C 甲醇 =(ρ×m b ) / 1000

[0033] F 甲醇 =C 甲醇 ×W 甲醇

[0034] According to the combustion equation of methanol, the combustion of 1 mol of methanol requires the consumption of 1.5 mol of oxygen, and F can be calculated. O2 Oxygen consumption per unit time (mol / h), u O2 Oxygen consumption per unit volume per unit time (mmol / L / h). Equipment.

[0035] F O2 =F 甲醇 ×1.5

[0036] u O2 =(F O2 ×1000) / V

[0037] From the oxygen mass balance in the fermentation system, we know that: dC L / dt = OTR-OUR; C L The dissolved oxygen concentration in the liquid phase is given by OTR. According to the two-mode theory and Henry's Law, OTR = Kla(C* - C), where C* is the saturated dissolved oxygen content of the solution, and C is the dissolved oxygen concentration in the solution.

[0038] When the fermentation equipment is in steady state, dC L / dt=0, OTR=OUR=Kla(C*-C).

[0039] When methanol is burned, the oxygen consumption per unit time and unit volume is equivalent to the oxygen consumption per unit time and unit volume of cells in a fermenter. Therefore, OTR = OUR = u O2 Then Kla can be calculated. The dissolved oxygen saturation (DO) in water at different temperatures... f =486 / (31.6+T).

[0040] but

[0041]

[0042] α is a correction coefficient, which can be determined through small-scale experiments.

[0043] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0044] Example 1

[0045] Given molecular mass M 甲醇 32 g / mol, density ρ 甲醇 792kg / m 3 Feeding quantity W 甲醇 80L / h, fermenter working volume V: 20000L, fermentation temperature T: 25℃, dissolved oxygen DO: 30%.

[0046] The value of methanol, m, can be obtained. b moles per unit mass (mol / kg), C 甲醇 : moles per unit volume (mol / L), F 甲醇 Feed rate per unit time (mol / h).

[0047] m b =1000 / 32g / mol = 31.25mol / kg

[0048] C 甲醇 =(ρ×m b ) / 1000=(792kg / m 3 ×31.25mol / kg) / 1000=24.75mol / L

[0049] F 甲醇 =C 甲醇 ×W 甲醇 =24.75mol / L×80L / h=1980mol / h

[0050] F O2 =F 甲醇 ×1.5=1980mol / h×1.5=2970mol / h

[0051] uO2 =(F O2 ×1000) / V=(2970mol / h×1000) / 20000L=148.5mmol / L / h

[0052] OTR=u O2 =148.5mmol / L / h

[0053] Kla 预测 / α=(148.5mmol / L / h×(31.6+25)×32g / mol) / (468mg / L×(10

[0054] 0% - 30% ≈ 821.01h -1 .

[0055] The actual Kla was determined during fermentation using a dynamic method with a dissolved oxygen electrode. The method involved first stopping the gas supply, and then using dC as the dissolved oxygen electrode reading decreased. L / dt = OUR, measure the actual OUR value, and then constantly introduce a certain flow rate of gas, based on dC L / dt=kla(C*-C)-OUR, substituting the previously obtained OUR value, the actual Kla can be calculated to be 789.22h based on the change in dissolved oxygen. -1 The method's prediction was 4.0% higher than the actual result, meaning α was approximately 96.1%.

[0056] Example 2

[0057] Given molecular mass M 甲醇 32 g / mol, density ρ 甲醇 792kg / m 3 Feeding quantity W 甲醇 6.2L / h, fermenter working volume V: 1000L, fermentation temperature T: 25℃, dissolved oxygen DO: 20%.

[0058] The value of methanol, m, can be obtained. b moles per unit mass (mol / kg), C 甲醇 : moles per unit volume (mol / L), F 甲醇 Feed rate per unit time (mol / h).

[0059] m b =1000 / 32g / mol = 31.25mol / kg

[0060] C 甲醇 =(ρ×m b ) / 1000=(792kg / m 3×31.25mol / kg) / 1000=24.75mol / L

[0061] F 甲醇 =C 甲醇 ×W 甲醇 =24.75mol / L×6.2L / h=153.45mol / h

[0062] F O2 =F 甲醇 ×1.5=153.45mol / h×1.5=230.18mol / h

[0063] u O2 =(F O2 ×1000) / V=(230.18mol / h×1000) / 1000L=230.18mmol / L / h

[0064] Kla 预测 / α=OTR / (C*-C)=(OTR×(31.6+T)×32) / (468×(1-DO 实际 ))=

[0065] (230.18mmol / L / h×(31.6+25)×32g / mol) / (468mg / L×(100%-20%))≈1113.52h -1 .

[0066] The test method is the same as in Example 1. Based on the change in dissolved oxygen, the actual Kla can be calculated to be 1067.21 h. -1 The method predicted a result that was 4.3% higher than the actual result, meaning that α was approximately 95.8%.

[0067] Example 3

[0068] Given molecular mass M 甲醇 32 g / mol, density ρ 甲醇 792kg / m 3 Feeding quantity W 甲醇 0.75L / h, fermenter working volume V: 100L, fermentation temperature T: 25℃, dissolved oxygen DO: 15%.

[0069] The value of methanol, m, can be obtained. b moles per unit mass (mol / kg), C 甲醇 : moles per unit volume (mol / L), F 甲醇 Feed rate per unit time (mol / h).

[0070] m b=1000 / 32g / mol = 31.25mol / kg

[0071] C 甲醇 =(ρ×m b ) / 1000=(792kg / m 3 ×31.25mol / kg) / 1000=24.75mol / L

[0072] F 甲醇 =C 甲醇 ×W 甲醇 =24.75mol / L×0.75L / h=18.56mol / h

[0073] F O2 =F 甲醇 ×1.5=18.56mol / h×1.5=27.84mol / h

[0074] u O2 =(F O2 ×1000) / V=(27.84mol / h×1000) / 100L=278.44mmol / L / h

[0075] OTR=u O2 =278.44mmol / L / h

[0076] Kla 预测 / α=OTR / (C*-C)=(OTR×(31.6+T)×32) / (468×(1-DO 实际 ))=

[0077] (278.44mmol / L / h×(31.6+25)×32g / mol) / (468mg / L×(100%-15%))≈1267.75h -1 .

[0078] The test method is the same as in Example 1. Based on the change in dissolved oxygen, the actual Kla can be calculated to be 1213.56 h. -1 The method's prediction was 4.5% higher than the actual result, meaning α was approximately 95.7%.

[0079] The above embodiments demonstrate that the heat of combustion of methanol can be used to equate the heat generation during yeast fermentation and guide the design of the heat exchange capacity of newly built fermenters.

[0080] The metabolic pathways of cells during fermentation are highly complex, and the O2 consumption of yeast and the transport of methanol within cells are difficult to predict. This invention utilizes the methanol combustion equation to directly calculate the OTR (Oxygen Tolerance Rate) based on the methanol feed rate, providing data for process optimization and fermenter optimization. Kla reflects the oxygen supply capacity of the equipment; chemical and physical methods can cause cell damage. This invention can directly calculate Kla from the calculated OTR.

[0081] The embodiments of the present invention have now been described in detail. To avoid obscuring the concept of the invention, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions of this invention based on the above description.

[0082] While specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims

1. A method of predicting the oxygen mass transfer effect in a fermentation process of methylotrophic yeasts, characterized in that, comprising the following steps: Step 1: obtaining the methanol feed amount of the fermentation equipment during the fermentation process; Step 2: calculating the oxygen consumption according to the methanol feed amount; Step 3: obtaining the oxygen consumption when the fermentation equipment is in a stable state; Step 4: obtaining the oxygen mass transfer rate according to the oxygen consumption when the fermentation equipment is in a stable state; After step 4, step 5 is further included: obtaining the predicted value of Kla according to the oxygen mass transfer rate; The calculation formula for obtaining the predicted value of Kla according to the oxygen mass transfer rate is: ； In the formula, Kla 预测 Here are the predicted values ​​for Kla, OTR for oxygen mass transfer rate, T for fermentation temperature, and DO for... 实际 This represents the actual dissolved oxygen saturation. This is a correction factor.

2. The method for predicting the oxygen mass transfer effect in the fermentation process of methanol yeast according to claim 1, characterized in that, The fermentation equipment is in a steady state: dC L / dt = 0, where C L is the liquid-phase dissolved oxygen concentration.

3. The method for predicting the oxygen mass transfer effect in the fermentation process of methanol yeast according to claim 1, characterized in that, The calculation formula for obtaining the oxygen mass transfer rate according to the oxygen consumption when the fermentation equipment is in a stable state is: ； where OTR is the oxygen mass transfer rate, OUR is the oxygen uptake rate, u O2 is the oxygen consumption per unit time and volume when the fermentation equipment is in a steady state.

4. The method for predicting the oxygen mass transfer effect in the fermentation process of methanol yeast according to claim 1, characterized in that, correction factor ranges from 95% to 97%.

5. The method for predicting the oxygen mass transfer effect in the fermentation process of methanol yeast according to claim 1, characterized in that, The calculation formula for calculating the oxygen consumption according to the methanol feed amount is: F O2 = F 甲醇 ×1.5; where F O2 is the oxygen consumption per unit time, F 甲醇 is the methanol feed rate per unit time.

6. The method for predicting the oxygen mass transfer effect in the fermentation process of methanol yeast according to claim 1, characterized in that, The fermentation equipment is a fermentation tank.

7. The application of the method for predicting the oxygen mass transfer effect in the fermentation process of methanol yeast according to any one of claims 1-6 in the optimization of fermentation equipment or fermentation process.

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