CN115678930B - Preparation method of acrylamide - Google Patents
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- CN115678930B CN115678930B CN202310001221.XA CN202310001221A CN115678930B CN 115678930 B CN115678930 B CN 115678930B CN 202310001221 A CN202310001221 A CN 202310001221A CN 115678930 B CN115678930 B CN 115678930B
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Abstract
The invention provides a preparation method of acrylamide, relating to the technical field of microorganisms, and comprising the following steps: (1) Respectively culturing nocardia and rhodococcus rhodochrous in a seed culture medium, mixing to obtain a composite bacterium A, inoculating the composite bacterium A into a fermentation culture medium for culturing to obtain a fermentation liquid, centrifuging to remove supernatant, and embedding to obtain an immobilized cell A; (2) Culturing pseudomonas aeruginosa in a seed culture medium, inoculating the pseudomonas aeruginosa into a fermentation culture medium for culturing to obtain a fermentation liquid, centrifuging to remove supernatant, and embedding to obtain an immobilized cell B; (3) Mixing the immobilized cell A obtained in the step (1) with deionized water, stirring, adding acrylonitrile in a flowing manner for the first time for reaction, adding the immobilized cell B obtained in the step (2), continuously adding acrylonitrile in a flowing manner, continuously reacting, and filtering to obtain acrylamide. The preparation method is environment-friendly, stable and convenient, and can obtain acrylamide with higher concentration.
Description
Technical Field
The invention relates to the technical field of microorganisms, and in particular relates to a preparation method of acrylamide.
Background
Acrylamide (C) 3 H 5 NO) is the most important and simplest of acrylamide systems, and it has a wide range of applications, and can be used for organic synthesis, paper making, textile sizing, soil improvement, and the like. The traditional preparation method of acrylamide is an acrylonitrile hydration method, but acrylonitrile is a chemical raw material with strong toxicity, so that the preparation process has large pollution and high energy consumption, and the cost is expensive.
The literature: plum blossom, zhang Qing Jue, liu Ming Zi, acrylamide production technology by microbiological method [ J ]. Chemical design communication, 2018, 44 (9): 1, important introduction is made for acrylamide production by microbiological method, and the article refers to the advantages of the microbiological method, namely: (1) the production flow is simple and safe; (2) the conversion rate is high, and acrylonitrile does not need to be recovered and separated; (3) the production efficiency is high, and no by-product is generated; (4) the product has high concentration and no need of extraction. However, the key role of microbial enzymes in the microbial process, i.e., the susceptibility of acrylamide production to enzyme activity, is also emphasized in this article. The literature: influence factors of the microbial method for preparing the acrylamide monomer are analyzed, wherein the influence factors are J, reported by university of oil field workers, 2005 (3): 2, the influence of the catalytic hydration reaction temperature, the pH of reaction liquid, the concentration of acrylonitrile in the reaction liquid, the concentration of acrylamide and the like on the catalytic activity of acrylonitrile hydratase is discussed, and a theoretical basis is provided for the industrial production of the microbial method for preparing the acrylamide.
Currently, the concentration of acrylamide produced by microbiological methods is around 25-35%, for example: patent CN03115536.7 discloses a process for producing acrylamide by microbial catalysis, specifically, a corynebacterium propionate containing nitrile hydratase or a mutant strain cell thereof is produced by fermentation, then acrylamide is synthesized by catalyzing acrylonitrile hydration by a free cell method or an immobilized cell method, and a high-purity acrylamide product is obtained by post-treatment. The invention optimizes the bacterial strain in the process, specifically adopts corynebacterium proprionate and the mutagenized bacterial strain thereof, and has higher biomass and enzyme activity compared with bacteria of the rhodococcus. Although the conversion of the invention can reach 99.99%, the concentration of the acrylamide solution obtained as a whole is only 32%, and the kettle outlet concentration is significant compared with the conventional microbiological method, but the current requirement of high concentration of acrylamide is difficult to meet.
Patent CN201510504967.8 discloses a method for reducing enzyme usage in a process for producing acrylamide by a microbiological method, and the invention mixes nitrile hydratase cell fermentation liquor with storage time of less than or equal to 24 hours and storage time of more than or equal to 240 hours to improve the activity and quality of nitrile hydratase and reduce the usage of nitrile hydratase. The invention mainly explores the dosage of enzyme, takes the acrylamide content of 32-34% as a reaction end point, and further explores the problems of unit consumption and cost, so that the actual operation is difficult to obtain the high-concentration acrylamide.
Patent CN201510940461.1 discloses a method and a device for preparing acrylamide by a continuous microbiological method, which focuses on changing batch production and sampling into continuous and automatic production. The equipment investment and the occupied area are reduced, the efficiency is high, the production cost is reduced, and the product quality is more stable and easy to control. The invention focuses more on the optimization of the device, takes environmental protection as a reference, and does not explore the actual content of acrylamide and the related conversion rate problem.
However, the current microbiological method usually adopts post-treatment such as heating concentration to obtain acrylamide with higher concentration, which causes great environmental pollution problem and the concentration process is easy to generate self-polymerization. Aiming at the problems in the prior art, it is necessary to find a preparation method which is environment-friendly, stable, convenient and fast and can obtain high-concentration acrylamide.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method of acrylamide, which is environment-friendly, stable, convenient and fast and can obtain acrylamide with higher concentration.
In order to realize the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a preparation method of acrylamide, which comprises the following steps:
(1) Respectively culturing nocardia and rhodococcus rhodochrous in a seed culture medium, mixing to obtain a composite bacterium A, inoculating the composite bacterium A into a fermentation culture medium for culturing to obtain a fermentation liquid, centrifuging to remove supernatant, and embedding to obtain an immobilized cell A;
(2) After culturing the strain B in a seed culture medium, inoculating the strain B into a fermentation culture medium for culturing to obtain fermentation liquor, centrifuging to remove supernatant, and embedding to obtain immobilized cells B;
(3) Mixing the immobilized cell A obtained in the step (1) with deionized water, stirring, adding acrylonitrile in a flowing manner for the first time for reaction, adding the immobilized cell B obtained in the step (2), continuously adding acrylonitrile in a flowing manner, continuously reacting, and filtering to obtain acrylamide;
the strain B comprises pseudomonas chlororaphis.
Further, the seed culture medium in the step (1) and the step (2) comprises: 5-12g/L of glucose, 5-8g/L of yeast extract, 3-4g/L of urea, 0.4-0.5g/L of dipotassium phosphate, 1-1.5g/L of magnesium sulfate heptahydrate and 0.3-0.5g/L of NaCl; preferably 8g/L glucose, 5g/L yeast extract, 4g/L urea, 0.4g/L dipotassium hydrogen phosphate, 1.2g/L magnesium sulfate heptahydrate and 0.4g/L NaCl.
Further, the fermentation medium in step (1) and step (2) comprises: 20-25g/L of glucose, 5-8g/L of yeast extract, 5-6g/L of urea, 0.4-0.5g/L of dipotassium phosphate and 1-1.5g/L of magnesium sulfate heptahydrate. Preferably 22g/L glucose, 6g/L yeast extract, 5g/L urea, 0.5g/L dipotassium hydrogen phosphate and 1.2g/L magnesium sulfate heptahydrate.
Further, the embedding in the step (1) and the step (2) is specifically to add sodium alginate, so that the adding amount of the sodium alginate accounts for 2 percent (w/v), stir into slurry, add into 0.4M CaCl 2 After solidification, the solution was washed with water.
Further, the temperature of the culture in the step (1) is 25-35 ℃, and the stirring speed is 150r/min; the temperature of the culture in the step (2) is 28-30 ℃, and the stirring speed is 150r/min.
Further, the temperature of the reaction in the step (3) is 20-28 ℃.
Further, before the immobilized cells B are added in the step (3), the concentration of acrylamide is detected, and the concentration of acrylamide is accumulated to 25%.
Further, the weight ratio of the nocardia to the rhodococcus rhodochrous in the composite bacteria A is 2-4.
Preferably, the weight ratio of the nocardia to the rhodococcus rhodochrous in the composite bacteria A is 3.
Further, the weight ratio of the deionized water, the immobilized cells A (wet weight) and the acrylonitrile fed for the first time in the step (3) is 100. Preferably 100.
Further, the deionized water in the step (3): immobilized cell B (wet weight): acrylonitrile (amount of acrylonitrile fed after addition of immobilized cell B) = 100.
Furthermore, the invention also provides the acrylamide prepared by the preparation method.
The technical effects obtained by the invention are as follows:
the preparation method adopts a microbiological method, microorganism species and process parameters in the acrylamide preparation process are optimized, multiple strains (Nocardia, rhodococcus rhodochrous and Pseudomonas chlororaphis) are embedded to obtain immobilized cells, and then the immobilized enzyme is added through a two-step method, so that the enzyme activity of each strain for producing nitrile hydratase can be effectively improved, meanwhile, the quality of the finally obtained product is easier to control, the yield of the product is improved, the finally obtained acrylamide has higher concentration and the conversion rate of acrylonitrile is higher; in addition, the invention adopts a continuous reaction mode, so that the method is more stable and simple.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It should be noted that the related strains used in the present invention are specifically nocardia ATCC19296, rhodococcus rhodochrous DSM46022, pseudomonas chlororaphis ATCC13985, and the other raw materials are all common commercial products, and thus the sources thereof are not particularly limited.
Example 1
The preparation method of the acrylamide specifically comprises the following steps:
(1) Respectively culturing Nocardia and Rhodococcus rhodochrous in seed culture medium (seed culture medium contains glucose 5g/L, yeast extract 5g/L, and urea)3g/L, dipotassium phosphate 0.4g/L, magnesium sulfate heptahydrate 1g/L, naCl 0.3 g/L), and then mixed to obtain a complex bacterium A (Nocardia and Rhodococcus rhodochrous in a weight ratio of 2: 20g/L glucose, 5g/L yeast extract, 5g/L urea, 0.4g/L dipotassium hydrogen phosphate and 1g/L magnesium sulfate heptahydrate, wherein the inoculation amount is 6% (w/w), the culture temperature is 25 ℃, the stirring speed is 150r/min, fermentation is carried out for 40h to obtain fermentation liquor, the fermentation liquor is centrifuged, supernatant is removed, sodium alginate is added to enable the addition amount of the sodium alginate to be 2% (w/v), the mixture is stirred into slurry, and the slurry is injected into 0.4M CaCl 2 In the solution, after 24h (4 ℃) solidification, washing with water to obtain immobilized cells A;
(2) Culturing Pseudomonas chlororaphis (strain B) in a seed culture medium (the components of the seed culture medium comprise 5g/L of glucose, 5g/L of yeast extract, 3g/L of urea, 0.4g/L of dipotassium phosphate, 1g/L of magnesium sulfate heptahydrate and 0.3g/L of NaCl), inoculating to a fermentation culture medium for culturing (the components of the fermentation culture medium comprise 20g/L of glucose, 5g/L of yeast extract, 5g/L of urea, 0.4g/L of dipotassium phosphate and 1g/L of magnesium sulfate heptahydrate), inoculating in an amount of 5% (w/w), culturing at 28 ℃, stirring at a speed of 150r/min, fermenting for 40h to obtain a fermentation broth, centrifuging the fermentation broth, removing supernatant, adding sodium alginate to make the addition amount of sodium alginate account for 2% (w/v), stirring to obtain slurry, and injecting 0.4M CaCl 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells B;
(3) And (2) injecting deionized water into the reaction kettle, controlling the temperature of the reaction kettle to be 20 ℃, adding the immobilized cell A obtained in the step (1) into the reaction kettle, stirring, adding acrylonitrile in a flowing manner to perform enzyme catalytic hydration reaction, adding the immobilized cell B obtained in the step (2) into the reaction kettle when the concentration of acrylamide is accumulated to reach about 25% (mass fraction), continuing adding acrylonitrile in a flowing manner, reacting for 8 hours, and filtering to obtain acrylamide.
Deionized water in step (3): immobilized cell a (wet weight): acrylonitrile (amount of acrylonitrile fed before the immobilized cell B was added) = 100; deionized water: immobilized cell B (wet weight): acrylonitrile (amount of acrylonitrile fed after addition of immobilized cell B) = 100.
After the reaction, the concentration of acrylamide at the outlet of the reaction vessel was determined to be 56.8%, and the conversion rate of acrylonitrile into acrylamide was determined to be 99.99%.
Example 2
The preparation method of the acrylamide specifically comprises the following steps:
(1) Respectively culturing Nocardia and rhodococcus rhodochrous in a seed culture medium (the components of the seed culture medium are 12g/L of glucose, 8g/L of yeast extract, 4g/L of urea, 0.5g/L of dipotassium phosphate, 1.5g/L of magnesium sulfate heptahydrate and 0.5g/L of NaCl), mixing to obtain a composite bacterium A (the weight ratio of the Nocardia and the rhodococcus rhodochrous is 4) 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells A;
(2) Culturing Pseudomonas chlororaphis (i.e. strain B) in a seed culture medium (the components of the seed culture medium comprise 12g/L of glucose, 8g/L of yeast extract, 4g/L of urea, 0.5g/L of dipotassium phosphate, 1.5g/L of magnesium sulfate heptahydrate and 0.5g/L of NaCl), inoculating to a fermentation culture medium for culture (the components of the fermentation culture medium comprise 25g/L of glucose, 8g/L of yeast extract, 6g/L of urea, 0.5g/L of dipotassium phosphate and 1.5g/L of magnesium sulfate heptahydrate), inoculating 5% (w/w), culturing at 30 ℃, stirring at 150r/min, fermenting for 40h to obtain a fermentation broth, centrifuging the fermentation broth, removing supernatant, adding sodium alginate to make the addition of sodium alginate account for 2% (w/v), stirring to form slurry, and injecting 0.4M CaCl 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells B;
(3) And (2) injecting deionized water into the reaction kettle, controlling the temperature of the reaction kettle to be 28 ℃, adding the immobilized cell A obtained in the step (1) into the reaction kettle, stirring, adding acrylonitrile in a flowing manner to perform enzyme catalytic hydration reaction, adding the immobilized cell B obtained in the step (2) into the reaction kettle when the concentration of acrylamide is accumulated to reach about 25% (mass fraction), continuing adding acrylonitrile in a flowing manner, reacting for 8 hours, and filtering to obtain acrylamide.
Deionized water in the step (3): immobilized cell a (wet weight): acrylonitrile (amount of acrylonitrile fed before feeding into the immobilized cell B) = 100; deionized water: immobilized cell B (wet weight): acrylonitrile (amount of acrylonitrile fed after addition of immobilized cell B) = 100.
After the reaction, the concentration of acrylamide at the outlet of the reaction vessel was determined to be 59.0%, and the conversion rate of acrylonitrile into acrylamide was determined to be 99.99%.
Example 3
The preparation method of the acrylamide specifically comprises the following steps:
(1) Respectively culturing Nocardia and rhodococcus rhodochrous in a seed culture medium (the components of the seed culture medium are 8g/L of glucose, 5g/L of yeast extract, 4g/L of urea, 0.4g/L of dipotassium phosphate, 1.2g/L of magnesium sulfate heptahydrate and 0.4g/L of NaCl), mixing to obtain a composite bacterium A (the weight ratio of the Nocardia and the rhodococcus rhodochrous is 3) 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells A;
(2) Culturing Pseudomonas chlororaphis (strain B) in seed culture medium (the components of the seed culture medium: glucose 8g/L, yeast extract 5g/L, urea 4g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate heptahydrate 1.2g/L, and NaCl 0.4 g/L), inoculating to fermentation culture medium (the components of the fermentation culture medium: glucose 22g/L, yeast extract 6g/L, urea 5g/L, phosphorus 5 g/L), culturing0.5g/L of dipotassium hydrogen phosphate and 1.2g/L of magnesium sulfate heptahydrate), wherein the inoculation amount is 5% (w/w), the culture temperature is 30 ℃, the stirring speed is 150r/min, fermentation is carried out for 40 hours to obtain fermentation liquor, the fermentation liquor is centrifuged, supernatant is removed, sodium alginate is added to enable the addition amount of the sodium alginate to be 2% (w/v), the mixture is stirred into slurry, and 0.4M CaCl is injected 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells B;
(3) And (2) injecting deionized water into the reaction kettle, controlling the temperature of the reaction kettle to be 25 ℃, adding the immobilized cell A obtained in the step (1) into the reaction kettle, stirring, adding acrylonitrile in a flowing manner to perform enzyme catalytic hydration reaction, adding the immobilized cell B obtained in the step (2) into the reaction kettle when the concentration of acrylamide is accumulated to reach about 25% (mass fraction), continuing adding acrylonitrile in a flowing manner, reacting for 8 hours, and filtering to obtain acrylamide.
Deionized water in the step (3): immobilized cell a (wet weight): acrylonitrile (amount of acrylonitrile fed before feeding into the immobilized cell B) = 100; deionized water: immobilized cell B (wet weight): acrylonitrile (amount of acrylonitrile fed after addition of immobilized cell B) = 100.
After the reaction was completed, the acrylamide concentration at the outlet of the reaction vessel was measured to be 61.4%, and the conversion rate of acrylonitrile into acrylamide was measured to be 99.99%.
Comparative example 1
The preparation method of the acrylamide specifically comprises the following steps:
(1) Respectively culturing Nocardia and rhodococcus rhodochrous in a seed culture medium (the components of the seed culture medium are 5g/L of glucose, 5g/L of yeast extract, 3g/L of urea, 0.4g/L of dipotassium phosphate, 1g/L of magnesium sulfate heptahydrate and 0.3g/L of NaCl), mixing to obtain a compound bacterium A (the weight ratio of the Nocardia and the rhodococcus rhodochrous is 2Adding sodium alginate in an amount of 2% (w/v), stirring to obtain slurry, and adding 0.4M CaCl 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells A;
(2) Culturing Pseudomonas chlororaphis in a seed culture medium (the components of the seed culture medium comprise 5g/L of glucose, 5g/L of yeast extract, 3g/L of urea, 0.4g/L of dipotassium phosphate, 1g/L of magnesium sulfate heptahydrate and 0.3g/L of NaCl), inoculating the culture medium with the culture medium (the components of the fermentation culture medium comprise 20g/L of glucose, 5g/L of yeast extract, 5g/L of urea, 0.4g/L of dipotassium phosphate and 1g/L of magnesium sulfate heptahydrate), inoculating the culture medium with the inoculation amount of 5% (w/w), culturing at 28 ℃, stirring at 150r/min, fermenting for 40h to obtain a fermentation liquid, centrifuging the fermentation liquid, removing supernatant, adding sodium alginate to enable the addition amount of the sodium alginate to be 2% (w/v), stirring the fermentation liquid into a slurry, and injecting 0.4M CaCl 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells B;
(3) And (2) injecting deionized water into the reaction kettle, controlling the temperature of the reaction kettle to be 20 ℃, adding the immobilized cells A and the immobilized cells B obtained in the step (1) into the reaction kettle, stirring, feeding acrylonitrile for the first time to perform enzyme catalytic hydration reaction, feeding the acrylonitrile continuously when the concentration of the acrylamide is accumulated to reach about 25% (mass fraction), reacting for 8 hours, and filtering to obtain the acrylamide.
Deionized water in the step (3): immobilized cell a (wet weight): acrylonitrile (amount of acrylonitrile fed in first feed) = 100; deionized water: immobilized cell B (wet weight): acrylonitrile (amount of acrylonitrile continuously fed) = 100.
That is, it is different from example 1 mainly only in that the obtained immobilized cells B are directly added together with the obtained immobilized cells A in step (3) without stepwise addition.
After the reaction was completed, the acrylamide concentration at the outlet of the reaction vessel was measured to be 45.5%, and the conversion rate of acrylonitrile into acrylamide was measured to be 98.4%.
Comparative example 2
The preparation method of the acrylamide specifically comprises the following steps:
(1) Nocardia, rhodococcus rhodochrous and pseudomonas chlororaphis are respectively cultured in a seed culture medium (the components of the seed culture medium are 5g/L of glucose, 5g/L of yeast extract, 3g/L of urea, 0.4g/L of dipotassium phosphate, 1g/L of magnesium sulfate heptahydrate and 0.3g/L of NaCl), mixed to obtain a compound bacterium (the weight ratio of the Nocardia, the rhodococcus rhodochrous and the pseudomonas chlororaphis is 2 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells;
(3) And (2) injecting deionized water into the reaction kettle, controlling the temperature of the reaction kettle to be 20 ℃, adding part of the immobilized cells obtained in the step (1) into the reaction kettle, stirring, adding acrylonitrile in a flowing manner to perform enzyme catalytic hydration reaction, adding the rest of the immobilized cells into the reaction kettle when the concentration of the acrylamide is accumulated to reach about 25% (mass fraction), continuing to add the acrylonitrile in a flowing manner, reacting for 8 hours, and filtering to obtain the acrylamide.
Deionized water in the step (3): partially immobilized cell a (wet weight): acrylonitrile (amount of acrylonitrile fed before the addition of the remaining immobilized cell a) = 100; deionized water: remaining immobilized cell a (wet weight): acrylonitrile (amount of acrylonitrile fed after the addition of part of the immobilized cells a) = 100.
That is, the difference from example 1 is mainly that nocardia, rhodococcus rhodochrous, and pseudomonas chlororaphis were subjected to seed culture, respectively, and then the three were mixed to obtain a composite bacterium, and the subsequent operations were continued.
After the reaction, the concentration of acrylamide at the outlet of the reaction vessel was determined to be 40.7%, and the conversion rate of acrylonitrile into acrylamide was determined to be 97.5%.
Comparative example 3
The preparation method of the acrylamide specifically comprises the following steps:
(1) Respectively culturing Nocardia and rhodococcus rhodochrous in a seed culture medium (the components of the seed culture medium are 5g/L of glucose, 5g/L of yeast extract, 3g/L of urea, 0.4g/L of dipotassium phosphate, 1g/L of magnesium sulfate heptahydrate and 0.3g/L of NaCl), mixing to obtain a compound bacterium A (the weight ratio of the Nocardia to the rhodococcus rhodochrous is 2 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells A;
(2) Culturing Rhodococcus rhodochrous in seed culture medium (the components of the seed culture medium comprise glucose 5g/L, yeast extract 5g/L, urea 3g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate heptahydrate 1g/L and NaCl 0.3 g/L), inoculating to fermentation culture medium (the components of the fermentation culture medium comprise glucose 20g/L, yeast extract 5g/L, urea 5g/L, dipotassium hydrogen phosphate 0.4g/L and magnesium sulfate heptahydrate 1 g/L), inoculating 5% (w/w), culturing at 28 deg.C and stirring at 150r/min for 40 hr to obtain fermentation liquid, centrifuging the fermentation liquid, removing supernatant, adding sodium alginate to make the addition amount of sodium alginate 2% (w/v), stirring to obtain slurry, and injecting 0.4M CaCl 2 In the solution, after curing for 24h (4 ℃), washing with water to obtain immobilized cells B;
(3) And (2) injecting deionized water into the reaction kettle, controlling the temperature of the reaction kettle to be 20 ℃, adding the immobilized cell A obtained in the step (1) into the reaction kettle, stirring, feeding acrylonitrile for enzyme-catalyzed hydration reaction, adding the immobilized cell B obtained in the step (2) into the reaction kettle when the concentration of acrylamide is accumulated to reach about 25% (mass fraction), feeding acrylonitrile continuously, reacting for 8 hours, and filtering to obtain acrylamide.
Deionized water in the step (3): immobilized cell a (wet weight): acrylonitrile (amount of acrylonitrile fed before the immobilized cell B was added) = 100; deionized water: immobilized cell B (wet weight): acrylonitrile (amount of acrylonitrile fed after addition of immobilized cell B) = 100.
That is, the difference from example 1 is mainly only that Pseudomonas chlororaphis of step (2) is replaced with Rhodococcus rhodochrous.
After the reaction was completed, the acrylamide concentration at the outlet of the reaction vessel was determined to be 50.9%, and the conversion rate of acrylonitrile into acrylamide was determined to be 96.6%.
From the above results, the preparation method of the present invention, as a whole, reduces the concentration step of obtaining high concentration acrylamide, and is more environment-friendly and stable; in addition, because the optimization of the strains and the preparation method adopt continuous two-step reaction, the finally obtained acrylamide has higher concentration and higher conversion rate of acrylonitrile.
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and do not limit the protection scope of the present invention, and those skilled in the art can make simple modifications or equivalent substitutions on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A preparation method of acrylamide is characterized in that: the method comprises the following steps:
(1) Respectively culturing Nocardia ATCC19296 and Rhodococcus rhodochrous DSM46022 in a seed culture medium, mixing to obtain a compound bacterium A, inoculating the compound bacterium A into a fermentation culture medium for culturing to obtain a fermentation liquid, centrifuging to remove a supernatant, and embedding to obtain an immobilized cell A;
(2) After culturing the strain B in a seed culture medium, inoculating the strain B into a fermentation culture medium for culturing to obtain fermentation liquor, centrifuging to remove supernatant, and embedding to obtain immobilized cells B;
(3) Mixing the immobilized cell A obtained in the step (1) with deionized water, stirring, adding acrylonitrile in a flowing manner for the first time for reaction, adding the immobilized cell B obtained in the step (2), continuously adding acrylonitrile in a flowing manner, continuously reacting, and filtering to obtain acrylamide;
the strain B is Pseudomonas chlororaphis ATCC13985;
before the immobilized cell B is added in the step (3), the concentration of acrylamide is detected, and the concentration of the acrylamide is accumulated to 25%;
the weight ratio of the nocardia to the rhodococcus rhodochrous in the compound bacteria A is (2-4) to 1;
in the step (3), the weight ratio of the deionized water to the immobilized cells A to the acrylonitrile fed in the first time is 100 (2-4) to (15-20).
2. The method of claim 1, wherein: the fermentation medium in the step (1) and the step (2) comprises: 20-25g/L of glucose, 5-8g/L of yeast extract, 5-6g/L of urea, 0.4-0.5g/L of dipotassium phosphate and 1-1.5g/L of magnesium sulfate heptahydrate.
3. The production method according to claim 1, characterized in that: the embedding in the step (1) and the step (2) is specifically to add sodium alginate, stir into slurry and add CaCl 2 After solidification in the solution, the solution was washed with water.
4. The method of claim 1, wherein: the temperature of the culture in the step (1) is 25-35 ℃, and the stirring speed is 150r/min; the temperature of the culture in the step (2) is 28-30 ℃, and the stirring speed is 150r/min.
5. The method of claim 1, wherein: the temperature of the reaction in the step (3) is 20-28 ℃.
6. The method of claim 1, wherein: the weight ratio of the nocardia to the rhodococcus rhodochrous in the composite bacteria A is 3.
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| CN101186911B (en) * | 2007-09-20 | 2010-06-02 | 清华大学 | Method for constructing nitrile hydratase gene engineering bacterium, genetic engineering strain and application thereof |
| CN101463358B (en) * | 2009-01-15 | 2011-05-11 | 清华大学 | Nitrile hydratase gene cluster and use thereof |
| WO2016050819A1 (en) * | 2014-09-30 | 2016-04-07 | Basf Se | Method for preparing an acrylamide solution having a low acrylic acid concentration |
| WO2019097123A1 (en) * | 2017-11-20 | 2019-05-23 | Kemira Oyj | A process for producing aqueous acrylamide solution, aqueous acrylamide solution and use thereof |
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