Headspace / gas chromatography for reaction monitoring of acrylamide synthesis

Abstract

To provide an improved method for producing an acrylamide aqueous solution.SOLUTION: A method for producing acrylamide aqueous solution includes the steps of: (a) combining water and at least one biocatalyst having nitrile hydratase activity to provide slurry; (b) feeding acrylonitrile into a reactor including the slurry to provide reaction mixture; (c) monitoring the reaction mixture by online GC to measure a concentration of acrylonitrile in a headspace of the reactor by using several detection techniques selected from the group consisting of a hydrogen flame ionization detector, mass spectrometry, a thermal conductivity detector, an electron capture detector, a nitrogen-phosphorus detector and vacuum ultraviolet detector.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure generally relates to the field of acrylamide synthesis, and more particularly to the reaction monitoring of acrylamide synthesis by gas chromatography (GC) in the reactor headspace, called on-line GC. Thus, the present disclosure relates to a method for producing an aqueous acrylamide solution by hydrolyzing acrylonitrile in an aqueous solution in the presence of a biocatalyst, the method including in-line monitoring of the acrylamide synthesis reaction by on-line GC. The present disclosure also generally relates to an aqueous acrylamide solution obtainable by said method and its use for the synthesis of polyacrylamide.

Background Art

[0002] Acrylamide (AM) has been commercially available since the mid-1950s, and the acrylamide market has been growing gradually since then. Acrylamide is mainly used in the production of polyacrylamide, which is used in many applications including water treatment, oil and gas recovery, the paper industry, and mining processes. Acrylamide is produced from acrylonitrile (ACN) by a hydrolysis reaction in the presence of a catalyst.

[0003] Conventionally, the production of acrylamide has been based on chemical catalyst processes, namely sulfuric acid-catalyzed hydration reactions, or copper-catalyzed hydration, which has gradually replaced the sulfuric acid process. In the 1980s, an enzyme-catalyzed acrylamide production process was developed. Advantages over conventional processes are low reaction temperature, operating pressure at atmospheric pressure, complete conversion, low by-product selectivity, and easy downstream processing.

[0004] Microorganisms have two types of nitrile catabolic pathways, the nitrilase and nitrile hydratase (NHase) pathways. Three different enzymes, nitrilase, (NHase), and amidase, are involved in these reactions.

[0005] Nitrilase catalyzes the hydrolysis reaction of a nitrile directly to the corresponding carboxylic acid and ammonia product. In the NHase pathway, NHase first hydrolyzes the nitrile to the corresponding amide product. In the presence of amidase, the amide can be further converted to the corresponding acid and ammonia product. Methods for producing acrylamide from acrylonitrile in the presence of a biocatalyst, such as nitrile hydratase, are described in many patent publications. Monitoring of such reactions, i.e., measuring the concentrations of reaction components including acrylonitrile and acrylamide, and by-products (e.g., acrylic acid) in such processes, using, for example, HPLC-based detection methods, is also known.

[0006] The residual acrylonitrile content should be as low as possible after the bioconversion of acrylonitrile to acrylamide. The residual acrylonitrile depends on the acrylonitrile quality (impurity profile) and the enzyme concentration for the bioconversion. Offline monitoring of acrylonitrile can cause over-investment in enzymes. In fact, the operator prioritizes the compatibility, safety, and environmental specifications of the production unit. An overly low enzyme concentration can lead to higher acrylonitrile concentrations in the production reactor or in the final product, which causes a flammability risk, as well as environmental problems. As a result, aqueous acrylamide solutions are generally produced by over-investment in enzymes, which affects the long-term acrylamide stability (self-polymerization risk) and overall quality, and is accompanied by problems such as turbidity, color correction, and generation of by-products, resulting in low-quality grade acrylamide with respect to polymerization. The enzyme dosage must be continuously adjusted for the quality of optimal and less expensive acrylamide without the need for additional post-treatment.

[0007] The object of the present disclosure is to provide an improved method for producing an aqueous acrylamide solution, in which the reaction state is monitored using gas chromatography (GC). Online gas chromatography (online GC) is an analytical technique that does not require specific sample preparation in the case of gas headspace reactor analysis. This technique also reduces the use of consumables and spare parts such as organic solvents, chromatography columns, or reagents. GC also provides real-time analysis at a higher frequency compared to conventional techniques. The online GC system is also more sensitive, accurate, and reproducible compared to other in-line or online techniques such as Fourier transform near-infrared spectroscopy (FTNIR), mid-infrared spectroscopy (MIR), high-performance liquid chromatography (HPLC), or Raman spectroscopy. The analysis period by GC is longer compared to the above techniques, but it is directly related to the acrylamide synthesis reaction kinetics. Therefore, online GC monitoring provides an increase in sensitivity and stability, as well as production capacity, compared to conventional offline experimental techniques such as HPLC or GC, and also compared to in-line techniques such as MIR, Raman, or FTNIR.

[0008] The GC technique is a chromatography technique that separates chemical substances according to their volatility and chemical properties. The GC detector measures the concentration of the analyte. As an example, the FID detector is based on the principle of ions released during the combustion of chemical substances, while MS is an analytical technique that separates ionized particles according to their charge and respective mass (m / z). Therefore, GC is more suitable for measuring analytes in both aqueous or organic solutions and gases compared to FTNIR, MIR, or Raman techniques. This is because in GC, the separation power is higher with lower sensitivity compared to the above spectroscopic techniques. Furthermore, GC can identify unknown compounds when mass spectrometry is used and limits interference phenomena compared to spectroscopic techniques.

[0009] Online GC technology enables the installation of an analyzer more than about 20 m from a reactor without any hypothetical loss of information, compared to other techniques such as HPLC that use a non-representative sample or a spectroscopic technique (e.g., FTNIR) that uses a long optical fiber cable that can generate more information, for example, due to a blocking phenomenon. By using online GC technology in such an environment, there is no need to provide electrical components or explosion-proof equipment. Further, this installation enables the interconnection of reactors and can generate data across the entire synthesis process.

[0010] Monitoring the headspace of the reactor is easier and more efficient compared to the analysis of the reactor liquid by headspace GC / FID technology due to the limitations from the sample matrix. These limitations are linked to the physical properties of the sample (the presence of insoluble materials such as catalysts), presenting a non-negligible polymerization risk. Such phenomena are difficult to limit by classical headspace GC (offline measurement) or by using spectroscopic techniques such as FTNIR, Raman or MIR. Therefore, this analysis can induce bias during analysis and troubleshooting can also potentially reduce the capabilities (frequency, accuracy, availability) of these techniques, which implicitly means a low sampling frequency and an increase in potential enzyme consumption. Therefore, online GC in the reactor headspace increases sensitivity compared to the aforementioned techniques, allowing the process to be operated with higher efficiency. The use of GC technology also implicitly means the separation of compounds before detection, thus reducing the risk of interference that cannot be completely eliminated by the use of spectroscopic techniques. Despite the monitoring of acrylonitrile, other volatile compounds such as benzene, acrolein, methyl vinyl ketone, oxazole, propionitrile, and any other volatile molecules found in acrylonitrile raw materials can also be analyzed. To enable the accurate quantification of acrylonitrile in the gas, a permeation tube of acrylonitrile is installed in the system, allowing the accuracy of the mathematical model to be verified daily. The combination of this verification with GC technology ensures accurate quantification with low uncertainty compared to other techniques. Spectroscopic techniques always use a mathematical model (advanced chemometric algorithms) based on a reference technique (either liquid or gas conventional chromatography). Therefore, the reference to this different technique implicitly means having higher uncertainty. This does not apply when using online GC which ensures better accuracy.

[0011] Thus, the complete principle of the GC technique by implicitly meaning the analysis of the reactor headspace, by performing chemical separation before detection, and by using an internal calibration curve, can reduce the risk of generating biased results compared to other techniques such as FTNIR, Raman, or MIR as spectral interferences that can generate significant and unpredictable biases.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Non-Patent Documents

[0013]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0014] The present disclosure generally relates to an improved method for producing an aqueous acrylamide solution. The method includes: a) combining water and at least one biocatalyst having nitrile hydratase activity to provide a slurry; b) supplying acrylonitrile into a reactor containing the slurry to provide a reaction mixture; and c) monitoring the reaction mixture by on-line GC to measure the concentration of acrylonitrile in the headspace of the reactor.

[0015] In some embodiments, based on the detected concentration of acrylonitrile in the headspace of the reactor, one or more of (i) the acrylonitrile feed rate, and / or (ii) the amount of water, and / or (iii) at least one biocatalyst and / or its amount, and / or (iv) the temperature and / or pH can be adjusted during the reaction process.

[0016] In some embodiments, the sampling loop compressor is not a temperature control loop connected to the GC online system.

[0017] A "sampling loop compressor" is a component used in a gas chromatography (GC) system to improve the accuracy and efficiency of sampling. It is often integrated into a gas monitoring system such as one used to detect specific compounds in an air or gas sample. For example, in a chromatography system for greenhouse gas monitoring, a "sampling loop compressor" can be used to compress a gas sample and inject it into the chromatography system, enabling accurate analysis of different gas levels.

[0018] In some embodiments, the acrylamide concentration is determined by measuring the online refractive index and optimizing the mathematical model used by the online GC.

[0019] In some embodiments, the monitoring of step c) by online GC to measure the concentration of acrylonitrile in the headspace of the reactor can be performed by some detection techniques selected from the group consisting of FID / MS / TCD / NPD / ECD / VUV.

[0020] In some embodiments, an online GC system having a suitable detector (FID or MS or ECD or NPD or TCD or VUV) can be positioned close to the reactor with a short gas sampling line. In some embodiments, the online GC system can be installed outside the reactor and connected to the headspace reactor with a gas sampling line. In some specific embodiments, the reactor may comprise a thermostatically controlled sampling line connected to the online GC. In some embodiments, the online GC system can be positioned within the process and connected to the headspaces of several reactors.

[0021] In some embodiments, the acrylonitrile concentration may be in the range of 0 to 15,000 ppmv (parts per million by volume) and can be measured by on-line GC in the reactor headspace with an accuracy of up to ±10% of the nominal value, more specifically up to ±5% of the nominal value.

[0022] In some embodiments, the acrylonitrile concentration may be in the range of 0 to 15,000 ppmv and can be measured by on-line GC in the reactor headspace with an accuracy of at least ±100 ppmv, more specifically at least ±50 ppmv.

[0023] In some embodiments, the acrylonitrile concentration may be in the range of 0 to 5,000 ppmv and can be measured by on-line GC in the reactor headspace with an accuracy of up to ±5% of the nominal value, more specifically up to ±2.5% of the nominal value.

[0024] In some embodiments, the acrylonitrile concentration may be in the range of 0 to 5,000 ppmv and can be measured by on-line GC in the reactor headspace with an accuracy of at least ±50 ppmv, more specifically at least ±10 ppmv.

[0025] In some embodiments, the acrylonitrile concentration may be in the range of 0 to 300 ppmv and can be measured by on-line GC with an accuracy of up to ±2.5 ppmv of the nominal value.

[0026] In some embodiments, the acrylonitrile concentration may be in the range of 0 to 300 ppmv and can be measured by on-line GC in the reactor headspace with an accuracy of at least ±10 ppmv.

[0027] In some embodiments, the acrylonitrile feed rate is adjusted during the process, whereby the acrylonitrile accumulation in the reactor can be controlled.

[0028] In some embodiments, the reactor may be a semi-batch reactor, a microreactor, or a continuous reactor, a series of continuous reactors, or a series of stirred tank reactors.

[0029] In some embodiments, the reactor may be configured as one or several microreactors operating in a fed-batch or continuous manner.

[0030] In some embodiments, the reactor may be configured as one or several microreactors operating in a fed-batch or continuous manner, and a classical stirred tank reactor.

[0031] In some embodiments, the biocatalyst may contain 0.0001 to 2 kg of dry cells per 3 reaction mixture m.

[0032] In some embodiments, the biocatalyst may be a bacterium selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus, or may include a combination of at least two of any of the above bacteria. More specifically, the biocatalyst may be selected from the group consisting of Rhodococcus, Pseudomonas, Escherichia, and Geobacillus.Or it may be at least two combinations of any of the above.

[0033] In some embodiments, the biocatalyst is the reaction mixture m 3It may contain 0.0001 to 2 kg of dry cells per hit, and may also be a bacterium selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus, or may contain a combination of at least two of any of the above bacteria. More specifically, the biocatalyst may be selected from the group consisting of Rhodococcus, Pseudomonas, Escherichia, and Geobacillus, or alternatively,Alternatively or additionally, it may contain nitrile hydratase derived from any of the above bacteria or any combination of at least two of the above.

[0034] In some embodiments, the biocatalyst may be Rhodococcus rhodochrous or Rhodococcus aetherivorans or nitrile hydratase derived therefrom.

[0035] In some embodiments, the method may further include a step of measuring and adjusting the temperature of the reaction mixture.

[0036] In some embodiments, the method may further include a step of maintaining the temperature of the reaction mixture within the range of 10°C to 35°C, preferably between 15 and 30°C, more preferably between 20 and 25°C, and optionally, the final concentration of acrylonitrile is at most 1000 ppmv.

[0037] In some embodiments, the final concentration of acrylonitrile measured by GC in the headspace of the reactor is at most 1000 ppmv, at most 500 ppmv, at most 150 ppmv, or more specifically at most 50 ppmv.

[0038] In some embodiments, the method may further include a step of cooling the reaction mixture.

[0039] In some embodiments, the method may further include a step of cooling the reaction mixture when the acrylamide concentration reaches 28 wt% to 30 wt%.

[0040] In some embodiments, the method may further include a step of cooling the reaction mixture until the acrylamide concentration reaches 40 wt% to 50 wt%.

[0041] In some embodiments, the method may further include maintaining the temperature of the reaction mixture for 10 seconds to 29 minutes.

[0042] In some embodiments, the method may further include maintaining the temperature of the reaction mixture for 30 minutes to 120 minutes.

[0043] In some embodiments, the method may further include maintaining the temperature of the reaction mixture for 121 minutes to 48 hours.

[0044] In some embodiments, the method may further include cooling the reaction mixture such that the temperature of the reaction mixture is in the range of 5°C to 20°C, more specifically 10°C to 15°C. The present disclosure also generally relates to an aqueous acrylamide solution obtainable by the methods disclosed herein.

[0045] In some embodiments, the aqueous acrylamide solution may have a concentration of the acrylamide solution of 15 wt% to 55 wt%, the concentration of residual acrylonitrile in the acrylamide solution may be measured by on-line GC and, after correlation between the acrylonitrile concentration (ppmv) in the reactor headspace and the concentration (ppm) in the liquid, may be 1000 ppm or less, and the turbidity of the acrylamide solution may be 20 NTU or less as measured from a 0.45 μm filtered acrylamide sample.

[0046] In some embodiments, the concentration of acrylonitrile in the acrylamide headspace reactor measured by on-line GC may be in the range of 0 to 7500 ppmv and may be measured with an accuracy of up to ±5% of the nominal value, more specifically up to ±2.5% of the nominal value.

[0047] In some embodiments, the color of the acrylamide solution may be 20 Hazen or less as measured by a spectrophotometer PtCo (455 nm, cell path length: 10 mm, 25°C) from an acrylamide sample filtered through a 0.45 μm filter.

[0048] In some embodiments, the concentration of the acrylamide solution may be 34 wt% to 55 wt%, more specifically 38 wt% to 40 wt%.

[0049] In some embodiments, the concentration of the acrylamide solution may be 35 wt% to 55 wt%, more specifically 38 wt% to 40 wt%.

[0050] In some embodiments, the concentration of the acrylamide solution may be 38 wt% to 55 wt%.

[0051] In some embodiments, the concentration of residual acrylonitrile in the acrylamide solution measured by on-line GC may be 150 ppmv or less, more specifically 90 ppmv or less, more specifically 50 ppmv or less, and even more specifically 10 ppmv or less.

[0052] In some embodiments, the turbidity of the acrylamide solution may be 15 NTU or less by turbidimeter technology.

[0053] The present disclosure also generally relates to the use of the aqueous acrylamide solution obtainable by the methods disclosed herein in the production of polyacrylamide.

[0054] The present disclosure also generally relates to an aqueous acrylamide solution, - The color of the solution is measured by a spectrophotometer PtCo (455 nm, cell path length: 10 mm, 25 °C) from an acrylamide sample filtered through a 0.45 μm filter and is 20 Hazen or less, - The concentration of the acrylamide solution is 35 to 55 mass%, more specifically 38 to 40 mass%, - The turbidity of the solution is 15 NTU or less.

Embodiments for Carrying Out the Invention

[0055] 1. Overview According to a first aspect of the present disclosure, a method for producing an aqueous acrylamide solution is provided. More specifically, a method for producing an aqueous acrylamide solution, comprising the steps of combining water and a biocatalyst having nitrile hydratase activity to provide a slurry, feeding acrylonitrile into a reactor containing the slurry to provide a reaction mixture, and monitoring the reaction mixture headspace by on-line GC to measure the concentration of acrylonitrile in the gas. In some embodiments, the on-line GC may be positioned on the reactor. In some embodiments, the reactor may comprise a cooling loop connected thereto and an on-line GC connected within the cooling loop. In some embodiments, the concentration of acrylonitrile can be measured with an accuracy of at least ±100 ppmv, more specifically at least ±30 ppmv.

[0056] According to a second aspect of the present disclosure, an aqueous acrylamide solution obtainable by the method is provided. An aqueous acrylamide solution is provided, characterized in that the concentration of acrylonitrile in a defined headspace can be measured by on-line GC and an association with acrylonitrile in aqueous acrylamide is made based on a mathematical model including all variables from the process. The concentration of acrylonitrile in the headspace can be measured with an accuracy of at least ±50 ppmv, more specifically at least ±10 ppmv.

[0057] In a third aspect of the present disclosure, the use of an aqueous acrylamide solution produced by the method of the present disclosure in the production of polyacrylamide is provided.

[0058] As used herein, "GC" refers to gas chromatography, "FID" refers to a flame ionization detector, "MS" refers to "mass spectrometry", "TCD" refers to a "thermal conductivity detector", "ECD" refers to an "electron capture detector", "NPD" refers to a "nitrogen-phosphorus detector", and "VUV" refers to a vacuum ultraviolet detector.

[0059] As used herein, "biocatalyst" refers to any biocatalyst having at least nitrilase (NHase) activity. A biocatalyst capable of converting acrylonitrile to acrylamide may be a bacterium encoding an enzyme having nitrilase activity (e.g., NHase), or any part of said microorganism having nitrilase activity. In this regard, it is not important whether the microorganism or bacterium naturally encodes nitrilase, or is genetically engineered to encode said enzyme, or whether the microorganism or bacterium that naturally encodes nitrilase is modified to produce more and / or improved nitrilase. Further, it is not important whether the enzyme having nitrilase activity is a naturally occurring enzyme or a modified enzyme. The biocatalyst may be selected from said microorganism or bacterium, the lysed cells of said microorganism, the cell lysate of said microorganism or bacterium, or any combination thereof. In highly specific embodiments, the biocatalyst is nitrilase (NHase).

[0060] As used herein, "platinum-cobalt", "PtCo", or Pt / Co refers to a color scale first introduced in 1892 by chemist Allen Hazen (1869 - 1930) to evaluate the level of contamination in wastewater. Since then, it has been extended to a general method for comparing the lightness of yellowish samples. This is specific to yellow and is based on the dilution of a 500 ppm platinum cobalt solution. The color produced by 1 milligram of platinum cobalt dissolved in 1 liter of water is defined as 1 unit of color on the platinum-cobalt scale. ASTM details the description and procedure in ASTM Designation D1209, "Standard Test Method for Color of Clear Liquids (Platinum-Cobalt Scale)". The color is measured by visual comparison of the sample with the platinum-cobalt standard. 1 unit of color is the color produced by 1 mg / L of platinum in the form of chloroplatinic acid ions. Since even a very small amount of turbidity can interfere with the determination, samples showing visible turbidity are generally clarified by centrifugation. Also, this method is pH-dependent.

[0061] 2. Monitoring of the reaction by on-line-GC The inventors have surprisingly found that the acrylamide synthesis reaction can be monitored by on-line gas chromatography (GC) coupled to at least specific detectors such as FID, MS, NPD, TCD, VUV, ECD, etc., achieving a measurement accuracy of at least ±5 ppmv of acrylonitrile concentration in the reaction mixture headspace. This accuracy is one order of magnitude less than that possible with conventional off-line GC. The preferred detector is FID.

[0062] The reactor may be any suitable reactor, such as a semi-batch reactor, a continuous reactor, a series of continuous reactors, or a series of stirred tank reactors, and in some exemplary embodiments may be a semi-batch reactor. In some embodiments, the on-line GC system may be positioned on each reactor with a short sampling line. In some embodiments, the reactor comprises a cooling loop attached thereto and an on-line GC positioned remotely from the reactor and connected via a gas sampling line. As used herein, positioning the on-line GC remotely from the reactor with a particular gas sampling line is contemplated to enable better monitoring of each reactor as compared to the use of conventional off-line GC systems involving liquid sampling. Further, this is contemplated to result in further improved accuracy in measuring reaction component concentrations, such as in determining acrylonitrile concentration on-line.

[0063] In some embodiments, the on-line GC system is positioned in proximity to the reactor.

[0064] 3. Biocatalyst The biocatalyst may be fresh (i.e., directly from fermentation), may be stored, e.g., cryopreserved (frozen in the wet state), or may be dried before the formation of the slurry.

[0065] After fermentation, the biocatalyst slurry is often typically washed or otherwise suitably treated, optionally or additionally, and then introduced into the slurry or may be stored, e.g., by freezing.

[0066] The biocatalyst can be any biocatalyst having nitrile hydratase (NHase) activity known in the art.

[0067] According to any one of the embodiments of the present disclosure, a biocatalyst capable of converting acrylonitrile into acrylamide may be a microorganism or bacterium encoding an enzyme having nitrile hydratase activity (e.g., NHase), or any part of the microorganism or bacterium having nitrile hydratase activity. In this regard, it is not important whether the microorganism or bacterium naturally encodes nitrile hydratase, or is genetically recombined to encode the enzyme, or whether the microorganism or bacterium naturally encoding nitrile hydratase is modified to produce more and / or improved nitrile hydratase. Further, it is not important whether the enzyme having nitrile hydratase activity is a naturally occurring enzyme or a modified enzyme. The biocatalyst may be selected from the microorganism or bacterium, the lysed cells of the microorganism or bacterium, the cell lysate of the microorganism or bacterium, or any combination thereof. In a very specific embodiment, the biocatalyst is nitrile hydratase (NHase).

[0068] In any one of the embodiments described in this specification, a bacterium encoding nitrile hydratase (for example, encoding nitrile hydratase naturally or genetically recombined to encode it) or any part of the microorganism that can be used as a biocatalyst includes species belonging to a genus selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus.In an exemplary embodiment, the biocatalyst is selected from bacteria of the genera Rhodococcus, Pseudomonas, Escherichia, and Geobacillus. Typically, the biocatalyst is Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus, or any part of said microorganisms or bacteria having nitrile hydratase activity.

[0069] In some embodiments, the biocatalyst is selected from the group consisting of Rhodococcus, such as Rhodococcus pyridinovorans or Rhodococcus rhodochrous or Rhodococcus aetherivorans, Pseudomonas, Escherichia, and Geobacillus, or any part of said microorganisms or bacteria having nitrile hydratase activity.

[0070] In an exemplary embodiment, the biocatalyst is Rhodococcus aetherivorans or Rhodococcus rhodochrous, or any part of said microorganisms or bacteria having nitrile hydratase activity.

[0071] Preferred wild-type nitrile hydratases are Rhodococcus rhodochrous J1-H, Rhodococcus rhodochrous M8, Rhodococcus ruber TH, Rhodococcus pyridinovorans MW3, and P. thermophila JCM3095.

[0072] Improved (mutant) nitrile hydratases are generally formed by substituting the amino acids of wild-type nitrile hydratase in the alpha and / or beta subunits. The amino acid sequences of the wild-type nitrile hydratase to be substituted are publicly available, for example, in the NCBI database such as GenBank (http: / / www.ncbi.nlm.nih.gov / ).

[0073] The improved nitrile hydratase is characterized by modification of the amino acid sequence of wild-type nitrile hydratase, said modification being at least one amino acid modification, preferably at least two amino acid modifications, more preferably at least three amino acid modifications on the beta subunit selected from the following list: 14S, 14Q, 14D, 17G, 17E, 17A, 17V, 17W, 43H, 43Q, 43N, 46H, 46T, 48D, 48W, 48D, 57V, 57M, 57G, 69F, 69T, 77P, 77E, 95Y, 95M, 95V, 97A, 97W, 107M, 107K, 107H, 114Y, 114F, 114W, 114M, 133R, 133G, 133I, 167S, 167T, 167Y, 179C, 179M, 179T, 202P, 202W, 218T, 218H, 218V, 219A, 219N, 219R; and at least one amino acid modification on the alpha subunit selected from the following list: 49H, 49W, 68G, 68T, 112V, 112P, 174K, 174L, 174C, 187A, 187G, 187V.

[0074] The numbers are positions counted downstream from the N-terminal amino acid residue in the amino acid sequence of the β subunit or the α subunit < <xxx>corresponds to the amino acid residue in (a number containing 1, 2 or 3 digits).

[0075] The possible combinations are listed below. AA means amino acid, and commonly used abbreviations for amino acids such as A for alanine are used. When the position is not mentioned, the amino acid is the amino acid of the wild-type nitrile hydratase. For example, in the following combinations, position β15 is not mentioned, which means that the amino acid at position β15 is the amino acid of the wild-type amino acid at position β15. The following combinations include the modification α174L, and the description also includes the same combinations in which the modification α174L is replaced by the modification α174K or α174C.

[0076] List of combinations α174L, β17G, β46T, β48N, β57M, β95V, β114Y, β167S, β218H, β219A α174L, β17G, β48W, β57M, β95V, β114Y, β167S, β218H, β219A α174L, β17G, β48N, β57M, β95V, β114Y, β167S, β218H, β219A α174L, β17G, β48N, β57M, β95V, β114F, β167S, β218H, β219A α174L, β17G, β48N, β57M, β95V, β114Y, β167T, β218H, β219A α174L, β17G, β48N, β57M, β95V, β114Y, β167S, β218H, β219A α174L, β17G, β48N, β57M, β95V, β114Y, β167T, β218H, β219A α174L, β57M, β95V, β114Y, β167S, β218H, β219A α174L, β57M, β95V, β114F, β167S, β218H, β219A α174L, β46T, β57M, β95V, β114Y, β167S, β218H, β219A α174L, β46T, β48N, β57M, β95V, β114Y, β167S, β218H, β219A α174L, β46T, β48N, β57M, β95V, β114Y, β167S, β218H, β219R α174L, β46T, β48N, β57M, β95V, β114W, β167S, β218H, β219R α174L, β46T, β48N, β57M, β95V, β114Y, β167S, β218H, β219A α174L, β17G, β46T, β48N, β57K, β95V, β114Y, β167S, β218H, β219A α174L, β17G, β46T, β48N, β57K, β95V, β114Y, β167S, β218H, β219A α174L, β17G, β46T, β48N, β57K, β95V, β114Y, β167S, β218H, β219R α174L, β17G, β46T, β48N, β57K, β95V, β114Y, β167S, β218H, β219R α174L, β17X, β48N, β57M, β95V, β114F, β167S, β218H, β219A Throughout the description, X is A or V. α174L, β17X, β48N, β57M, β95V, β114F, β167S, β218H, β219A α174L, β17X, β48N, β57M, β95V, β114F, β167S, β218H, β219R α174L, β17X, β48N, β57M, β95V, β114F, β167S, β218H, β219R α174L, β17X, β46T, β48N, β57M, β95V, β114F, β167S, β218H, β219R α174L, β17X, β46T, β48N, β57M, β95V, β114F, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57M, β95V, β114F, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57K, β95V, β114W, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57K, β95V, β114F, β167S, β218A, β219A α174L, β17X, β46T, β48N, β57K, β95V, β114F, β167S, β218Q, β219A α174L, β17X, β46T, β48N, β57K, β95V, β114F, β167S, β218H, β219R α174L, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57M, β95V, β114W, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57K, β95V, β114W, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57M, β95V, β114W, β167S, β218H, β219R α174L, β17X, β46T, β48N, β57K, β95V, β114W, β167R, β218H, β219R α174L, β17X, β48N, β57K, β95V, β114W, β167S, β218H, β219R α174L, β17G, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β17G, β57M, β95V, β107L, β114Y, β167S, β218H, β219A α174L, β17G, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β17G, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β17G, β48N, β57M, β95V, β107K, β114Y, β167T, β218H, β219A α174L, β17G, β48N, β57M, β95V, β107L, β114Y, β167S, β218H, β219A α174L, β17G, β48N, β57M, β95V, β107L, β114Y, β167T, β218H, β219A α174L, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β46T, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219R α174L, β46T, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219R α174L, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219R α174L, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219R α174L, β17X, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β17X, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β17X, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β46T, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β46T, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57M, β95V, β107H, β114F, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218A, β219A α174L, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218Q, β219A α174L, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β46T, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219R α174L, β17X, β46T, β48N, β57K, β95V, β107K, β114W, β167R, β218H, β219R α174L, β17X, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219R α174L, β14S, β17G, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β17G, β57M, β95V, β107L, β114Y, β167S, β218H, β219A α174L, β14S, β17G, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β17G, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β14S, β17G, β48N, β57M, β95V, β107K, β114Y, β167T, β218H, β219A α174L, β14S, β17G, β48N, β57M, β95V, β107L, β114Y, β167S, β218H, β219A α174L, β14S, β17G, β48N, β57M, β95V, β107L, β114Y, β167T, β218H, β219A α174L, β14S, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β14S, β46T, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219R α174L, β14S, β46T, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219R α174L, β14S, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219R α174L, β14S, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219R α174L, β14S, β17X, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β14S, β17X, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β14S, β17X, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219R α174L, β14S, β17X, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219R α174L, β14S, β17X, β46T, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219R α174L, β14S, β17X, β46T, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α174L, β14S, β17X, β46T, β48N, β57M, β95V, β107H, β114F, β167S, β218H, β219A α174L, β14S, β17X, β46T, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α174L, β14S, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218A, β219A α174L, β17X, β43Q, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218Q, β219A α174L, β17X, β43Q, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β43Q, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β43Q, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β43Q, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β43Q, β46T, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β43Q, β46T, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β43Q, β46T, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219R α174L, β17X, β43Q, β46T, β48N, β57K, β95V, β107K, β114W, β167R, β218H, β219R α174L, β17X, β43Q, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219R α174L, β14S, β17X, β46T, β48N, β57M, β95V, β107H, β114F, β167S, β218H, β219A α174L, β14S, β17X, β46T, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α174L, β14S, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218A, β219A α174L, β17X, β43Q, β46T, β48N, β57K, β77P, β95V, β107K, β114F, β167S, β218Q, β219A α174L, β17X, β43Q, β46T, β48N, β57K, β77P, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β43Q, β46T, β48N, β57K, β77P, β95V, β107K, β114F, β167S, β218H, β219R α174L, β17X, β43Q, β48N, β57K, β77P, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β43Q, β48N, β57M, β77P, β95V, β107K, β114W, β167S, β218H, β219A α174L, β17X, β43Q, β46T, β48N, β57M, β77P, β95V, β107K, β114W, β167S, β218H, β219A α174L, β14S, β46T, β48N, β57M, β97A, β107K, β114W, β167S, β218H, β219R α174L, β14S, β46T, β48N, β57M, β97A, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β17G, β46T, β48N, β57K, β97A, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β46T, β48N, β57K, β97A, β107K, β114Y, β167S, β218H, β219A α174L, β14S, β17G, β46T, β48N, β57K, β97A, β107K, β114Y, β167S, β218H, β219R α174L, β14S, β17G, β46T, β48N, β57K, β97A, β107K, β114Y, β167S, β218H, β219R α174L, β14S, β17X, β48N, β57M, β97A, β107K, β114F, β167S, β218H, β219A α174L, β14S, β17X, β48N, β57M, β97A, β107K, β114F, β167S, β218H, β219A α174L, β14S, β17X, β48N, β57M, β97A, β107K, β114F, β167S, β218H, β219R α49H, β17G, β48N, β57M, β95V, β114Y, β167T, β218H, β219A α49H, β17G, β48N, β57M, β95V, β114Y, β167S, β218H, β219A α49H, β17G, β48N, β57M, β95V, β114Y, β167T, β218H, β219A α49H, β57M, β95V, β114Y, β167S, β218H, β219A α49H, β57M, β95V, β114F, β167S, β218H, β219A α49H, β46T, β57M, β95V, β114Y, β167S, β218H, β219A α49H, β46T, β48N, β57M, β95V, β114Y, β167S, β218H, β219A α49H, β46T, β48N, β57M, β95V, β114Y, β167S, β218H, β219R α49H, β46T, β48N, β57M, β95V, β114W, β167S, β218H, β219R α49H, β17X, β46T, β48N, β57M, β95V, β114F, β167S, β218H, β219R α49H, β17X, β46T, β48N, β57M, β95V, β114F, β167S, β218H, β219A α68T, β17X, β46T, β48N, β57M, β95V, β114F, β167S, β218H, β219A α68T, β17X, β46T, β48N, β57K, β95V, β114W, β167S, β218H, β219A α68T, β17X, β46T, β48N, β57K, β95V, β114F, β167S, β218A, β219A α68T, β17X, β46T, β48N, β57K, β95V, β114F, β167S, β218Q, β219A α68T, β17X, β46T, β48N, β57K, β95V, β114F, β167S, β218H, β219R α68T, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α68T, β46T, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219R α68T, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α68G, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219A α68G, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219A α68G, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219R α68G, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219R α68G, β17X, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α68G, β17X, β46T, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219R α112V, β17X, β46T, β48N, β57M, β95V, β107K, β114F, β167S, β218H, β219A α112V, β17X, β46T, β48N, β57M, β95V, β107H, β114F, β167S, β218H, β219A α112V, β17X, β46T, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α112V, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218A, β219A α112V, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218Q, β219A α112V, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α187G, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α187G, β17X, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α187G, β17X, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219A α187G, β14S, β46T, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219R α187G, β14S, β46T, β48N, β57M, β95V, β107K, β114Y, β167S, β218H, β219A α187V, β14S, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219A α187V, β14S, β17G, β46T, β48N, β57K, β95V, β107K, β114Y, β167S, β218H, β219A α187V, β14S, β17X, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218A, β219A α187V, β17X, β43Q, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218Q, β219A α187V, β17X, β43Q, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α187V, β17X, β43Q, β46T, β48N, β57K, β95V, β107K, β114F, β167S, β218H, β219R α187V, β17X, β43Q, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A α187V, β17X, β43Q, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219A α187V, β17X, β43Q, β46T, β48N, β57M, β95V, β107K, β114W, β167S, β218H, β219A α187V, β17X, β43Q, β46T, β48N, β57K, β95V, β107K, β114W, β167S, β218H, β219A

[0077] In one embodiment, the amount of the biocatalyst is from 0.0001 kg of dry cells per reaction mixture m 3 to 2 kg of dry cells per reaction mixture m 3 is.

[0078] In some embodiments, the biocatalyst is the reaction mixture m 3 Containing 0.0001 to 0.2 kg of dry cells per hit, the biocatalyst is selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus, and more preferably, the biocatalyst is selected from the group consisting of Rhodococcus, Pseudomonas, Escherichia, and Geobacillus, or a combination of at least two of any of the above.

[0079] During the process, for example, if acrylonitrile begins to accumulate in the reactor, additional biocatalyst may be added. The biocatalyst may be added, for example, as a homogeneous slurry in water. The biocatalyst can be added at any point in the process.

[0080] 4. Progress of the reaction The reaction is carried out at atmospheric pressure, more specifically at 1 absolute bar.

[0081] The slurry can be produced by any known method in the art, for example, by mixing water and biocatalyst in a container or in a reactor. More specifically, the slurry is homogeneous. A strongly aggregated slurry is less active than a homogeneous slurry. The biocatalyst is more active in a homogeneous slurry.

[0082] The reaction of acrylonitrile to acrylamide in an aqueous solution in the presence of a biocatalyst having NHase activity starts when acrylonitrile is fed into the reactor containing the slurry. Thus, by feeding acrylonitrile into the reactor containing the slurry, a reaction mixture containing water, acrylamide, acrylonitrile, and biocatalyst is provided.

[0083] Acrylamide production can be carried out in a discontinuous or continuous process. The combination of reactors can be 1 to 50 reactors, more specifically 1 to 20 reactors, more specifically 1 to 10 reactors distributed in series and / or partially in parallel, more specifically in series. The reactors can be a combination of stirred tank reactors, non-stirred reactors, and plug flow reactors. The reactor size can be from 0.0001 m 3 to 100 m 3 、0.0001 m 3 to 50 m 3 、more specifically 5 to 40 m 3 and may be.

[0084] In the case of a continuous process, in the first reactor or a combination of different reactors, the reactant addition rate is continuously monitored. The overall flow rate is adjusted to achieve a residence time of 0.1 - 20 hours, 0.5 - 15 hours, 1 - 10 hours, specifically 3 - 8 hours. Reactants are listed as acrylonitrile, biocatalyst, water, any organic or inorganic salt solution, any other additives (such as MEHQ acting as a polymerization inhibitor). The last reactor is generally used only for aging without adding additional reactants. In the case of a discontinuous process, acrylonitrile addition is then carried out using different flow rates, from low to high, vice versa, or a combination thereof. In each reactor, the pH is controlled to a defined value of 6 - 9, and each reactor can be monitored at different values using acid or base addition, and the acid and base can be organic or inorganic. The temperature is controlled in each reactor using an internal or external cooling / heating system. At the end of the process, the biocatalyst (with free cells or carriers) can be separated from the aqueous acrylamide solution using well-known separation techniques such as filtration and centrifugation.

[0085] An aqueous solution of acrylamide at a high concentration (e.g., at least 20 wt%, or at least 30 wt%, or at least 40 wt%, or at least 45 wt%, or at least 50 wt%, or more) can be produced by a controlled acrylonitrile supply and process temperature profile. Typically, cooling of the reactor is required to maintain the reaction mixture at the desired reaction temperature. The temperature and acrylonitrile supply rate are relatively high at the start of the reaction to achieve a fast reaction rate and short synthesis time. The acrylonitrile supply rate is relatively low in the last few hours to avoid acrylonitrile accumulation in the reactor.

[0086] The supply of acrylonitrile may be continuous throughout the process, or more specifically, may be continuous throughout the process up to the aging stage. The supply rate of acrylonitrile may vary during the process. The supply of acrylonitrile may be continuous or intermittent. The supply rate of acrylonitrile depends on the reaction rate from acrylonitrile to acrylamide and the rate of biocatalyst inactivation. In one embodiment, the supply of acrylonitrile is continuous throughout the process up to the aging stage.

[0087] In one embodiment, the acrylonitrile supply rate is adjusted during the process to avoid the accumulation of acrylonitrile in the reaction mixture. Acrylonitrile is supplied during the process at a rate such that acrylonitrile is converted to acrylamide. More specifically, the amount of acrylonitrile in the reaction mixture is maintained at less than 5 wt%, or less than 2.5 wt%, more specifically less than 1 wt%, and even more specifically less than 0.5 wt% based on the total amount of the reaction mixture.

[0088] In some embodiments, the acrylonitrile supply rate is adjusted during the process, thereby controlling the accumulation of acrylonitrile in the reactor headspace; 38% - 48% of the total amount of acrylonitrile supplied to the reactor is supplied over a period from 0 minutes or more to 60 minutes or less from the start of the supply of acrylonitrile into the reactor.

[0089] In another embodiment of the method, 30% - 60% of the total amount of acrylonitrile supplied to the reactor is supplied between 0 minutes and 100 minutes from the start of the process; 10% - 40% of the total amount of acrylonitrile is supplied between 100 minutes and 220 minutes of the process; 5% - 20% of the total amount of acrylonitrile is supplied between 220 minutes and 360 minutes of the process. To achieve the remaining amount of 100% of the acrylonitrile supplied, the remaining acrylonitrile is supplied during the process before the aging stage.

[0090] During the aging stage, acrylonitrile is not substantially supplied to the reactor. During aging, acrylonitrile monomers still present in the reaction mixture react to form acrylamide. The reaction mixture is aged until the desired characteristics are achieved.

[0091] It is known that the biocatalyst begins to inactivate in an acrylamide solution of about 25 wt% to 38 wt%. See, for example, WO2019 / 097123. The inactivation of the biocatalyst caused by acrylamide accumulation is strongly dependent on temperature, and cooling of the reaction mixture particularly reduces biocatalyst inactivation. Therefore, the temperature of the reaction mixture is monitored. The monitoring and measurement of temperature can be carried out by any suitable means and methods in the art.

[0092] First, the temperature of the reaction mixture is maintained at 15 - 35 °C. In one embodiment, the temperature is maintained at 19 - 30 °C, more specifically 20 - 28 °C, and even more specifically 22 - 26 °C. In one embodiment, the temperature of the reaction mixture is measured, and the temperature is maintained within the desired range by cooling the mixture or heating the mixture so that the temperature is kept within the desired range. Cooling and / or heating of the reaction mixture can be carried out by known methods in the art.

[0093] In some embodiments, the method includes the step of further cooling the reaction mixture when the acrylamide concentration reaches at least 10 wt%, more specifically 10 wt% - 38 wt%. In one embodiment, the cooling of the reaction mixture is started when the acrylamide concentration reaches 28 wt% - 30 wt%. Cooling of the reaction mixture can be carried out by any suitable methods and means known in the art, for example, by cooling the reactor.

[0094] When the cooling of the reaction mixture is started, the temperature of the reaction mixture may be the same as, higher than, or lower than the temperature of the reaction mixture at the start of the process.

[0095] In some embodiments, the cooling of the reaction mixture is continued such that when the acrylamide concentration reaches 35 wt% to 55 wt%, the temperature of the reaction mixture is in the range of 10 °C to 30 °C, or 15 °C to 27 °C. In other words, the period of cooling the reaction mixture to a temperature of 10 °C to 30 °C, or 15 °C to 27 °C, is the period when the acrylamide concentration of at least 27 wt% (more specifically 27 wt% to 38 wt%) increases to an acrylamide concentration of 35 wt% to 55 wt% (more specifically 40 wt% to 50 wt%).

[0096] In one embodiment, the cooling of the reaction mixture is continued such that when the acrylamide concentration reaches 35 wt% to 55 wt%, the temperature is in the range of 10 °C to 30 °C, more specifically 18 °C to 27 °C, and even more specifically the temperature is in the range of 22 to 26 °C. In one embodiment, the cooling is started after the reaction mixture is maintained at 15 °C to 25 °C.

[0097] In one embodiment, the reaction mixture is maintained at a temperature in the range of 15 °C to 25 °C, or 18 °C to 27 °C when the acrylamide concentration reaches 35 wt% to 55 wt%.

[0098] During the aging stage, acrylonitrile is not substantially fed to the reactor, and more specifically, acrylonitrile is not fed to the reactor. During the aging stage, the unreacted acrylonitrile in the reactor reacts to form acrylamide. Aging starts after the reaction mixture is cooled and the temperature of the reaction mixture is in the range of 15 °C to 25 °C, or 15 °C to 27 °C, and / or after the supply of acrylonitrile to the reactor is terminated. More specifically, aging is continued until the final concentration of acrylonitrile in the reactor headspace reaches a maximum of 10000 ppmv, a maximum of 2500 ppmv, a maximum of 1000 ppmv, a maximum of 250 ppmv, or a maximum of 100 ppmv.

[0099] In one embodiment of the method, the temperature of the reaction mixture is maintained at 15°C to 25°C for 30 minutes to 90 minutes, preferably 45 minutes to 60 minutes, and the cooling of the reaction mixture to a temperature of 10°C to 30°C, or 15°C to 27°C, is carried out for a period of 45 minutes to 120 minutes, preferably 60 minutes to 120 minutes.

[0100] Since the temperature is maintained low at the end of the process, less acrylic acid is formed in the process. The activation energy of the reaction to form acrylic acid is higher than that of the main reaction (formation of acrylamide). The amount of acrylic acid in the aqueous acrylamide solution is at most 300 ppm, more specifically at most 200 ppm, and even more specifically at most 100 ppm. When a cationic or nonionic polymer is prepared from the acrylamide solution, a small amount of acrylic acid in the aqueous acrylamide solution is advantageous.

[0101] The resulting aqueous acrylamide solution may be filtered to separate acrylamide from the biocatalyst. Filtration can be carried out using various filtration processes such as centrifugal filtration, vacuum or pressure filtration. The filtration process may be continuous or discontinuous. Various filtration media such as diatomaceous earth, perlite, cellulose or any micrometer filter cloth are possible.

[0102] 5. Aqueous acrylamide solution In a second aspect of the present disclosure, there is provided an aqueous acrylamide solution obtainable or obtained by the method disclosed herein. More specifically, there is provided an aqueous acrylamide solution obtained by the method disclosed herein, characterized in that the concentration of total residual acrylonitrile in the aqueous acrylamide is obtained by analysis of acrylonitrile in the reactor headspace and measured by on-line GC chromatography to be 300 ppmv or less.

[0103] In some embodiments, the turbidity of the aqueous acrylamide solution may be measured by the absorbance at 450 nm of a mixture containing 0.7 ml of HCl (0.1 N), 7 ml of acetone, and 2.3 ml of filtered (0.45 μm) aqueous acrylamide sample, and may be 20 NTU or less. In one embodiment, the turbidity of the solution is 15 or less.

[0104] In some embodiments, the resulting aqueous acrylamide solution may be substantially free of biocatalyst.

[0105] 6. Use of the aqueous acrylamide solution In a third aspect of the present disclosure, there is provided the use of an aqueous acrylamide solution produced by the method of the present disclosure in the production of polyacrylamide. Polyacrylamide is a water-soluble polymer or a water-swellable polymer containing at least acrylamide monomer.

[0106] A water-soluble polymer means a polymer that forms an aqueous solution without insoluble particles when dissolved by stirring at 25°C and at a concentration of 10 g.L -1 in deionized water.

[0107] A water-swellable polymer is a cross-linked polymer that forms a three-dimensional network. These water-swellable polymers are also known as superabsorbent polymers. Generally, water-swellable polymers have a water absorption capacity of more than 10 times their volume.

[0108] Polyacrylamide is a synthetic polymer and more preferably contains at least one non-ionic different form of acrylamide selected from the following list, and / or an anionic and / or cationic hydrophilic monomer. - Nonionic monomers: acrylonitrile, methacrylamide, N-vinylformamide (NVF), N-vinylacetamide, N-vinylpyrrolidone (NVP), N-vinylimidazole, N-vinylsuccinimide, acryloylmorpholine (ACMO), glycidyl methacrylate, glyceryl methacrylate, diacetone acrylamide, N-methylol acrylamide (NMA), hydroxyalkyl (C1-C3)-(meth)acrylate, thioalkyl (C1-C3)-(meth)acrylate and mixtures thereof, - Anionic monomers: acrylic acid, methacrylic acid, itaconic acid, crotonic acid, maleic acid, fumaric acid, acrylamide undecanoic acid, 3-acrylamide 3-methylbutanoic acid, maleic anhydride, 2-acrylamido-2-methylpropanesulfonic acid (ATBS), vinylsulfonic acid, vinylphosphonic acid, methallylphosphonic acid, 2-sulfoethyl methacrylate, sulfopropyl methacrylate, sulfopropyl acrylate, allylphosphonic acid, styrenesulfonic acid, 2-acrylamido-2-methylpropanedisulfonic acid, their salts and mixtures, - Cationic monomers: diallyldialkylammonium salts, such as diallyl dimethyl ammonium chloride (DADMAC); acidified or quaternized salts of dialkylaminoalkyl acrylamides; acidified or quaternized salts of dialkylaminoalkyl methacrylamides, such as methacrylamide-propyltrimethylammonium chloride (MAPTAC), acrylamidepropyltrimethylammonium chloride (APTAC), acidified or quaternized salts of dialkylaminoalkyl acrylates, such as quaternized or chlorinated dimethylaminoethyl acrylate (ADAME), acidified or quaternized salts of dialkylaminoalkyl methacrylates, such as quaternized or chlorinated dimethylaminoethyl methacrylate (MADAME), and mixtures thereof. The alkyl group is C1-C3.

[0109] Optionally, the polyacrylamide comprises zwitterionic hydrophilic monomers selected from the following list: dimethylaminoethyl acrylate derivatives such as 2-((2-(acryloyloxy)ethyl)dimethylammonio)ethane-1-sulfonate, 3-((2-(acryloyloxy)ethyl)dimethylammonio)propane-1-sulfonate, 4-((2-(acryloyloxy)ethyl)dimethylammonio)butane-1-sulfonate, [2-(acryloyloxy)ethyl](dimethylammonio)acetate, dimethylaminoethyl methacrylate derivatives such as 2-((2-(methacryloyloxy)ethyl)dimethylammonio)ethane-1-sulfonate, 3-((2-(methacryloyloxy)ethyl)dimethylammonio)propane-1-sulfonate, 4-((2-(methacryloyloxy)ethyl)dimethylammonio)butane 1-sulfonate, [2-(methacryloyloxy)ethyl](dimethylammonio)acetate, dimethylaminopropyl acrylamide derivatives such as 2-((3-acrylamidopropyl)dimethylammonio)ethane-1-sulfonate, 3-((3-acrylamidopropyl)dimethylammonio)propane-1-sulfonate, 4-((3-acrylamidopropyl)dimethylammonio)butane-1-sulfonate, [3-(acryloyloxy)propyl](dimethylammonio)acetate, dimethylaminopropyl methylacrylamide derivatives such as 2-((3-methacrylamidopropyl)dimethylammonio)ethane-1-sulfonate, 3-((3-methacrylamidopropyl)dimethylammonio)propane-1-sulfonate, 4-((3-methacrylamidopropyl)dimethylammonio)butane-1-sulfonate and [3-(methacryloyloxy)propyl](dimethylammonio)acetate, and mixtures thereof.

[0110] Optionally, the polyacrylamide comprises hydrophobic monomers selected from the following list: C4-C 30 alkyl, arylalkyl (C4-C 30 alkyl, C4-C 30 (meth)acrylate esters having an aryl, propoxylated, ethoxylated or ethoxylated and propoxylated chain; (meth)acrylamide derivatives having a propoxylated, ethoxylated, ethoxylated and propoxylated C1-C3 alkyl, arylalkyl (C4-C 30 alkyl, C4-C 30 aryl), or a dialkyl (C4-C 30 alkyl) chain; alkylaryl sulfonates (C4-C 30 alkyl, C4-C 30 aryl), or C4-C 30 alkyl, arylalkyl (C4-C 30 alkyl, C4-C 30 aryl), propoxylated, ethoxylated, or ethoxylated and propoxylated chain, by a mono- or disubstituted (meth)acrylamide amide; C4-C 30 alkyl, propoxylated arylalkyl (C4-C 30 alkyl, C4-C 30 aryl), ethoxylated, ethoxylated and propoxylated, or C4-C 30 dialkyl chain (meth)acrylamide derivatives; alkylaryl sulfonates (C4-C 30 alkyl, C4-C 30 aryl), and mixtures thereof.

[0111] The water-soluble polyacrylamide preferably contains less than 1 mol% of hydrophobic monomer. This may not contain a hydrophobic monomer.

[0112] The water-soluble polyacrylamide may preferably be linear or structured. A structured polymer refers to a non-linear polymer having side chains that, when the polymer is dissolved in water, result in a strongly entangled state and provide a very high low-shear viscosity.

[0113] The water-soluble polymer according to the invention may be further structured as follows: - At least one structuring agent that can be selected from the group consisting of polyethylene unsaturated monomers (having at least two unsaturated functional groups, such as vinyl functional groups, especially allyl functional groups, acrylic and epoxy functional groups), for example methylene bisacrylamide (MBA), triallylamine, or tetraallylammonium chloride, or 1,2-dihydroxyethylene bis-(N-acrylamide), and / or - Macroinitiators, such as polyperoxides, polyazides and polyagents, such as transfer agents, such as polymer scavenging (co)polymers and polyols, and / or - Functionalized polysaccharides.

[0114] According to the present invention, the water-soluble polyacrylamide may have a linear, branched, star-shaped, comb-shaped, dendritic or block structure. These structures can be obtained by selecting initiators, transfer agents, polymerization techniques, such as controlled radical polymerization known as RAFT (reversible addition-fragmentation-chain transfer), NMP (nitroxide-mediated polymerization) or incorporation of structuring monomers, concentration, etc.

[0115] The water-swellable polyacrylamide is crosslinked with a crosslinking agent that can be selected from polyethylene unsaturated monomers (having at least two unsaturated functional groups, such as vinyl functional groups, especially allyl and acrylic functional groups), or monomers having at least two epoxy functional groups. For example, methylene bisacrylamide (MBA), triallylamine, tetraallylammonium chloride, 1,2-dihydroxyethylene bis-(N-acrylamide) and mixtures thereof can be mentioned. Preferably, the crosslinking agent is methylene bisacrylamide (MBA).

[0116] The amount of the crosslinking agent in the water-swellable polymer is advantageously between 5 and 5000 ppm, more preferably between 100 and 1000 ppm, based on the total mass of the monomers.

[0117] Polyacrylamide does not require the development of any polymerization process. In fact, polyacrylamide can be obtained using any polymerization technique well known to those skilled in the art. Polyacrylamide can be obtained by solution polymerization, gel polymerization, precipitation polymerization, emulsion polymerization (aqueous or inverse), suspension polymerization, polymer reaction extrusion polymerization, polymerization in water-in-water, or micelle polymerization.

[0118] The polymerization is generally free radical polymerization. Free radical polymerization includes free radical polymerization using UV, azo, redox, or thermal initiators, and controlled radical polymerization (CRP) techniques or matrix polymerization techniques.

[0119] The polyacrylamide according to the present invention may be modified after being obtained by polymerization. This is called post-polymerization modification. All known post-polymerization modifications can be applied to the polymers according to the present invention. A preferred modification is post-hydrolysis.

[0120] Post-hydrolysis advantageously consists of the reaction of a hydrolyzable functional group of non-ionic monomer units, more preferably an amide or ester functional group, with a hydrolyzing agent. This hydrolyzing agent may be an enzyme, an ion exchange resin, an alkali metal, or a suitable acidic compound. Preferably, the hydrolyzing agent is a Bronsted base. When the polymer contains monomer amide and / or ester monomer units, the post-hydrolysis reaction produces carboxylate groups.

[0121] According to the present invention, polyacrylamide may be in liquid, gel, or solid form when its preparation involves a drying process, such as spray drying, drum drying, radiation drying, such as microwave drying, or fluidized bed drying.

[0122] The aqueous acrylamide solution obtained by the method of the present invention can be used in the production of polyacrylamide or N-methylenebisacrylamide or N-methylolacrylamide or N,N'-methylenebisacrylamide-2-methylacrylamide.

[0123] Polyacrylamide can be used in fields selected from hydrocarbon recovery, well abandonment and cementing, hydrocarbon well stimulation, water treatment, fermentation process treatment, sludge treatment, manufacturing, construction, wood treatment, hydraulic composition treatment, mining industry, cosmetic formulations, cleaning formulations, fiber manufacturing, battery component manufacturing, geothermal energy, sanitary product manufacturing, or agriculture.

[0124] Polyacrylamide can be used as a flocculant, coagulant, binder, fixing agent, viscosity reducer, thickener, absorbent, anti-friction agent, water remover, water draining agent, charge holding agent, dehydrating agent, conditioning agent, stabilizer, fixing agent, film-forming agent, sizing agent, superplasticizer, clay inhibitor, or dispersant.

Examples

[0125] 7. Examples The following examples are provided for illustrative purposes only and are not limiting.

[0126] Materials and methods for monitoring the acrylamide synthesis reaction used in the examples Without the need to use additional analytical reference techniques compared to NIR with HPLC or GC, a simulation of the acrylamide reaction was carried out in the reactor to build a mathematical model.

[0127] The reaction was carried out as described in Examples 1 to 4. The concentration of acrylonitrile (AN) was determined by on-line GC in the headspace of the reactor. The concentration of the acrylamide (AMD) solution was evaluated by refractive index (RI) measurement. Then, using a calibration curve, the mass percentage concentration was determined by correlation with its refractive index. A few drops of the sample solution were dropped onto the refractometer prism and the corresponding value was read. For example, a Mettler Toledo RM40 refractometer can be used for this process. Additional off-line GC analysis was performed to fully prove that the on-line equipment showed good concentrations.

[0128] On-line GC was performed using on-line equipment from Chromatotec.

[0129]

Table 1

[0130] For offline GC measurement, reaction mixture samples (1.5 mL) were periodically taken out from the reactor, filtered through a 0.2 μm RC syringe filter, and quenched by the addition of sulfuric acid.

[0131] The concentration of AN was determined by offline GC using a Clarus 590 (Perkin Elmer) equipped with a PORAPAK PS packed column, 1 m, 1 / 8" (3.175 mm). Chemical substances were detected by FID. The flow rate was 25 mL / min with nitrogen as the carrier gas. A 0.5 μL sample was injected at 200 °C, and the furnace was isothermal at 170 °C. An approximately 0.5 g of quenched reaction mixture was accurately measured and diluted in 10 ml of Type 1 Milli-Q water to prepare a sample for GC.

[0132] The determined online GC values were correlated with offline GC to generate representative data between acrylonitrile in liquid acrylamide and acrylonitrile in the reactor headspace. Laboratory experiments were conducted to establish the correlation between acrylonitrile concentration in the liquid (offline GC) and acrylonitrile in the reactor headspace (online GC). The acrylamide concentration could be determined by refractive index, and an appropriate and most accurate mathematical model for online GC could be selected.

[0133] (Example 1) Acrylamide synthesis at laboratory scale 627 g of deionized water is charged into a 1 L glass reactor equipped with a double jacket thermally controlled by an external circulator and a mechanical stirrer. Stirring is started and set to 180 RPM. The pH of the solution is adjusted to a value of 8 by dropwise addition of a 10% sodium hydroxide solution. The medium temperature is adjusted to 18 °C. 500 μL of a 9% wt solution of a biocatalyst NHase prepared from Rhodococcus rhodochrous J-1 strain having an activity of 880 U (the unit U means generating 1 micromole of amide compound from a nitrile compound in 1 minute) is added. During the addition of acrylonitrile and the aging step, the temperature control is set to 20 °C. Continuous acrylonitrile addition is carried out at a flow rate of 1.77 g / min for 105 minutes, then at 1.03 g / min for 116 minutes and at 0.54 g / min for 126 minutes. The reaction medium is left for 3 hours under continuous stirring. The reactor gas headspace is analyzed using the online method described previously. Liquid samples are taken at the end of each acrylonitrile addition and every hour until the final aging and prepared as described previously for refractive index measurement. The results are reported in Table 2. Offline GC is used to measure the residual acrylonitrile in the liquid phase at the end of the process, and a value of 15 ppm is measured.

[0134]

Table 2

[0135] (Example 2) Acrylamide Experiment in Industrial Process Ten stirred continuous reactors of 10 m3 each are combined in series. Acrylonitrile is continuously fed to the first five reactors, and the nitrile hydratase catalyst and water defined in Example 1 are fed to Reactor 1 (Table 3). In Reactor 1, the pH is continuously adjusted to pH 7.5 using a 10% wt caustic solution. The flow rate of the catalyst is continuously adjusted. The flow rates are reported in Table 4. The temperature is set to 25 °C in each reactor.

[0136]

Table 3

[0137] Using on-line GC, the reactor headspace in reactor 8 is continuously measured. The values are used to continuously adjust the catalyst flow rate. Catalyst adjustment is necessary due to fluctuations in acrylonitrile quality resulting from impurity content. The values are reported in table 4.

[0138]

Table 4

[0139] After reactor 10, the acrylamide solution is centrifuged to remove the catalyst bacterial cells. The residual acrylonitrile amount is measured by an off-line GC method. Values of less than 100 ppm are always measured each day. The average catalyst consumption during the production campaign was 3.06 kg / h.

[0140] (Example 3) Acrylamide experiment in an industrial process, only off-line analysis Acrylamide was synthesized under the same industrial operating conditions as described in Example 2, except that there was no on-line GC analysis in the reactor headspace and only off-line GC analysis was performed once a day in each reactor. The production days, unlike those in Example 2, may result in acrylonitrile quality fluctuations.

[0141] Using the acrylonitrile values, the catalyst flow rate is adjusted daily. The measured values are reported in table 5. After reactor 10, the acrylamide solution is centrifuged to remove the catalyst bacterial cells used in the polymerization.

[0142] Compared with Example 2, the overall catalyst consumption was higher, and the average catalyst consumption during the production campaign was 3.93 kg / h, 28% higher than in the case of Example 2. Furthermore, the residual acrylonitrile amount sometimes exceeded 100 ppm in R10.

[0143]

Table 5

[0144] (Example 4) Polymerization During the 3rd day, the acrylamide solutions prepared in Examples 2 and 3 are evaluated in bulk polymerization. 425 g of the acrylamide solution is diluted with 560 g of water. The solution is cooled to 1 °C. With stirring, 20 mg of sodium persulfate, 10 mg of sodium hypophosphite and 1.2 g of 2,2'-dimethyl-2,2'-azobis(propionitrile) are added. The pH is adjusted to a value of 4 with an acetic acid solution. The solution is poured into a 2 L Dewar flask. Nitrogen degassing is carried out for 30 minutes through a deep nozzle into the solution. Then, 10 mg of a 50% molar salt solution is added to the mixture. Polymerization generally starts after 5 minutes under continuous degassing. The temperature rises naturally to 85 °C. The polymer is aged for 3 hours. Then the internal polymer is cut, crushed and dried. Finally, polymer powder is obtained. UL viscosity measurements are carried out for Examples 2 and 3. The values are reported in Table 6.

[0145] The UL viscosity is measured between 23 and 25 °C using a Brookfield viscometer equipped with a UL adapter, which rotates at 60 revolutions per minute (0.1 mass percent polymer in 1 M sodium chloride physiological saline).

[0146]

Table 6

[0147] In the case of Example 2, the polymer shows a higher UL viscosity compared to Example 3.

[0148] (Example 5) Stability test The acrylamide solutions prepared in Examples 2 and 3 are evaluated for stability with respect to self-polymerization. At each production date, four sealed bottles of 50 mL of acrylamide solution with a 2×5 cm, 1 mm thick carbon steel plate are placed in an oven at 70 °C. The appearance of the solution is checked daily, and if more than 50% of the solution begins to polymerize with an increase in viscosity, the polymerization delay is recorded. The polymerization delay is reported in Table 7 (in days). In the case of the acrylamide solution production described in Example 2, the average stability delay is improved.

[0149]

Table 7

Claims

1. 1. A method for producing an aqueous acrylamide solution, comprising: (a) combining water and at least one biocatalyst having nitrile hydratase activity to provide a slurry; (b) providing acrylonitrile into a reactor containing the slurry to provide a reaction mixture; (c) monitoring the reaction mixture by on-line GC to measure the concentration of acrylonitrile in the reactor headspace using several detection techniques selected from the group consisting of a flame ionization detector, mass spectrometry, a thermal conductivity detector, an electron capture detector, a nitrogen-phosphorus detector, and a vacuum ultraviolet detector; A method comprising:

2. 2. The method of claim 1, wherein, based on the detected concentration of acrylonitrile in the reactor headspace, one or more of: (i) acrylonitrile feed rate; (ii) amount of water; (iii) at least one biocatalyst and / or its amount; or (iv) temperature and / or pH are adjusted during the reaction process.

3. 3. The method of claim 1 or 2, wherein the sampling loop compressor is not a temperature control loop connected to a GC online system.

4. 4. The method according to any one of claims 1 to 3, wherein the concentration of acrylonitrile is in the range of 0 to 15000 ppmv, measured by online GC from the reactor headspace with an accuracy of at least ±100 ppmv, more particularly at least ±50 ppmv.

5. 5. The method according to any one of claims 1 to 4, wherein the concentration of acrylonitrile is in the range of 0 to 5000 ppmv, measured by GC online in the reactor headspace with an accuracy of at least ±50 ppmv, more particularly at least ±10 ppmv.

6. 6. The method according to any one of claims 1 to 5, wherein the concentration of acrylonitrile is in the range of 0 to 300 ppmv, as measured by GC online in the reactor headspace spectroscopy with an accuracy of at least ±10 ppmv.

7. 7. The process of any one of claims 1 to 6, wherein the residual concentration of acrylonitrile as measured by GC in the reactor headspace is at most 1000 ppmv, at most 500 ppmv, at most 150 ppmv, or more particularly at most 50 ppmv.

8. - the acrylonitrile feed rate is adjusted during the process to control acrylonitrile accumulation in the reactor headspace; - 38% to 48% of the total amount of acrylonitrile fed to the reactor is fed over a period ranging from 0 minutes to 60 minutes from the start of feeding acrylonitrile to the reactor.

9. 9. The process of any one of claims 1 to 8, wherein the reactor is a semi-batch reactor, a microreactor, a continuous reactor, a series of continuous reactors, or a series of stirred tank reactors.

10. - the biocatalyst is added to the reaction mixture m 3 Each contains 0.0001-0.2 kg of dry cells. - Biocatalysts include Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus crococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces 10. The method according to any one of claims 1 to 9, wherein the biocatalyst is a bacterium selected from the group consisting of Bacillus subtilis, Pichia, Rhodotorula, Comomonas, and Pyrococcus, more preferably the biocatalyst is selected from the group consisting of Rhodococcus, Pseudomonas, Escherichia, and Geobacillus, or a combination of at least two of any of the above.

11. - measuring the temperature of the reaction mixture; - maintaining the temperature of the reaction mixture within the range of 10°C to 35°C, and optionally providing a final concentration of acrylonitrile in the reactor headspace of up to 1000 ppmv; 11. The method of claim 1, further comprising:

12. An aqueous solution of acrylamide, - the color of the aqueous solution is less than or equal to 20 Hazens, as measured by a spectrophotometer PtCo (455 nm, cell path length: 10 00, 25°C) from a 0.45 μm filtered acrylamide sample; the concentration of the acrylamide solution is between 35 and 55% by weight, more particularly between 38 and 40% by weight, - The turbidity of the solution is less than 15 NTU; Acrylamide aqueous solution.

13. 13. Use of the aqueous acrylamide solution according to claim 12 for the production of polyacrylamide or N-methylenebisacrylamide or N-methylolacrylamide or N,N'-methyleneacrylamide-2-methylacrylamide.

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