FTNIR spectroscopy for reaction monitoring of acrylamide synthesis

Abstract

A method for producing an aqueous acrylamide solution by hydrating acrylonitrile in an aqueous solution in the presence of a biocatalyst is provided, which method includes in-line monitoring of the acrylamide synthesis reaction by FTNIR spectroscopy. Also provided are aqueous acrylamide solutions obtainable by the method and their use for the synthesis of polyacrylamide.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 955,309, filed December 30, 2019, and Finnish Application No. 20205067, filed January 23, 2020. The contents of both applications are incorporated by reference in their entirety.

[0002] The present disclosure generally relates to the field of acrylamide synthesis, and more specifically to reaction monitoring of acrylamide synthesis by FTNIR spectroscopy (Fourier transform near-infrared spectroscopy).Accordingly, the present disclosure relates to a method for producing an aqueous acrylamide solution by hydrating acrylonitrile in an aqueous solution in the presence of a biocatalyst, which method includes in-line monitoring of the acrylamide synthesis reaction by FTNIR spectroscopy.The present disclosure also generally relates to an aqueous acrylamide solution obtainable by the method, and its use for synthesizing polyacrylamide. [Background technology]

[0003] Acrylamide (AMD) has been on the market since the mid-1950s, and the acrylamide market has grown steadily since then. Acrylamide is primarily used to manufacture polyacrylamide, which is used in many applications, including water treatment, crude oil recovery, the paper industry, and mining processes. Acrylamide is produced from acrylonitrile (AN) by hydrolysis in the presence of a catalyst.

[0004] Traditionally, the production of acrylamide was based on chemically catalyzed processes, i.e., sulfuric acid-catalyzed hydration or copper-catalyzed hydration, but the sulfuric acid process was gradually replaced by copper-catalyzed hydration. In the 1980s, enzyme-catalyzed processes for the production of acrylamide were developed. The advantages over traditional processes are low reaction temperatures, the ability to be carried out at atmospheric pressure, complete conversion, low by-product selectivity, and ease of downstream processing.

[0005] Microorganisms have two metabolic pathways for nitrile degradation: the nitrilase pathway and the nitrile hydratase (NHase) pathway. These reactions involve three different enzymes: nitrilase, NHase, and amidase.

[0006] Nitrilases catalyze the hydrolysis of nitriles, directly converting them into the corresponding carboxylic acid and ammonia products. In the NHase pathway, NHase and amidase react sequentially. NHase first hydrolyzes the nitrile to the corresponding amide product. In the presence of amidase, the amide can be further converted into the corresponding acid and ammonia products. Numerous patent publications have described methods for producing acrylamide from acrylonitrile in the presence of a biocatalyst (e.g., nitrile hydratase). Monitoring of such reactions, i.e., monitoring the concentrations of reaction components (including acrylonitrile and acrylamide) and by-products (e.g., acrylic acid) in such methods (e.g., using HPLC-based detection methods), is also known.

[0007] One objective of the present disclosure is to provide an improved method for producing aqueous acrylamide solutions, in which the reaction status is monitored using FTNIR spectroscopy (Fourier transform near infrared spectroscopy). Summary of the Invention

[0008] The present disclosure generally relates to an improved method for producing aqueous acrylamide, which may include: a) combining a biocatalyst having nitrile hydratase activity with water to obtain a slurry; b) feeding acrylonitrile to a reactor containing the slurry to obtain a reaction mixture; and c) monitoring the reaction mixture by in-line FTNIR spectroscopy to measure the concentration of acrylonitrile.

[0009] In some embodiments, the acrylonitrile feed rate, and / or the amount of water, and / or the at least one biocatalyst, and / or the temperature may be adjusted during the reaction process based on the detected concentration of acrylonitrile.

[0010] In some embodiments, an FTNIR spectrometer probe (Fourier transform near-infrared spectrometer probe) may be located within the reactor. In some embodiments, an FTNIR spectrometer may be located outside the reactor. In some particular embodiments, the reactor may include a cooling loop connected thereto, and an FTNIR spectrometer probe may be located within the cooling loop. In some embodiments, an FTNIR spectrometer probe may be located within the reactor and another FTNIR spectrometer probe may be located outside the reactor (such as in a cooling loop connected to the reactor). In some embodiments, the FTNIR spectrometer probe located inside or outside the reactor may be a transflection probe.

[0011] In some embodiments, the concentration of acrylonitrile can be in the range of 0-10 wt % and can be measured by FTNIR spectroscopy with an accuracy of at least ±1 wt %, more specifically with an accuracy of at least ±0.5 wt %, and even more specifically with an accuracy of at least ±0.3 wt %.

[0012] In some embodiments, the concentration of acrylonitrile can be in the range of 0-1 wt % and can be measured by FTNIR spectroscopy with an accuracy of at least ±400 ppm, more specifically with an accuracy of at least ±200 ppm, and even more specifically with an accuracy of at least ±180 ppm.

[0013] In some embodiments, the concentration of acrylonitrile may be in the range of 0 to 1000 ppm and may be measured by FTNIR spectroscopy with an accuracy of at least ±100 ppm, more specifically, with an accuracy of at least ±80 ppm.

[0014] In some embodiments, monitoring the reaction mixture may further include measuring the concentration of acrylamide by FTNIR spectroscopy, wherein the concentration of acrylamide may be in the range of 0-50% by weight and may be measured with an accuracy of at least ±5% by weight, more particularly at least ±3.8% by weight, and even more particularly at least ±1.3% by weight.

[0015] In some embodiments, the final concentration of acrylonitrile as measured by FTNIR spectroscopy can be at most 1000 ppm, at most 500 ppm, at most 250 ppm, or more specifically at most 100 ppm.

[0016] In some embodiments, the acrylonitrile feed rate may be adjusted during the process to thereby control acrylonitrile accumulation within the reactor.

[0017] In some embodiments, 38% to 48% of the total amount of acrylonitrile fed to the reactor may be fed between 0 and 60 minutes from the start of feeding acrylonitrile to the reactor.

[0018] In some embodiments, the reactor can be a semi-batch reactor, a continuous reactor, a series of continuous reactors, or a series of stirred tank reactors.

[0019] In some embodiments, the biocatalyst is present at a concentration of 0.1-5 kg ​​of dry cells / ml of reaction mixture. 3 may include:

[0020] In some embodiments, 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, The biocatalyst may be a microorganism selected from the group consisting of Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus, or may comprise a combination of at least two of any of the foregoing microorganisms, more particularly, the biocatalyst may be selected from the group consisting of Rhodococcus, Pseudomonas, Escherichia, and Geobacillus, or may alternatively or additionally comprise a nitrile hydratase derived from any of the foregoing microorganisms or combinations thereof.

[0021] In some embodiments, the biocatalyst can be Rhodococcus rhodochrous or Rhodococcus aetherivorans or a nitrile hydratase therefrom.

[0022] In some embodiments, the method may further include measuring and regulating the temperature of the reaction mixture.

[0023] In some embodiments, the method may further include maintaining the temperature of the reaction mixture within a range of 15°C to 25°C, cooling the reaction mixture so that the temperature of the reaction mixture is within a range of 10°C to 21°C or within a range of 10°C to 18°C ​​when the acrylamide concentration reaches at least 27% by weight, more particularly when the acrylamide concentration reaches 27% to 38% by weight, or when the acrylamide concentration reaches 37% to 55% by weight, and optionally maintaining the reaction mixture at a temperature range of 10°C to 21°C or 10°C to 18°C, optionally resulting in a final concentration of acrylonitrile of at most 1000 ppm.

[0024] In some embodiments, the method may further include cooling the reaction mixture when the acrylamide concentration reaches 28% to 30% by weight.

[0025] In some embodiments, the method may further include cooling the reaction mixture until an acrylamide concentration of 40% to 50% by weight is reached.

[0026] In some embodiments, the method may further comprise maintaining the temperature of the reaction mixture between 19°C and 25°C, more specifically between 20°C and 22°C, and even more specifically at 22°C.

[0027] In some embodiments, the method can further include maintaining the temperature of the reaction mixture for 30 minutes to 120 minutes.

[0028] In some embodiments, the method may further include cooling the reaction mixture so that the temperature of the reaction mixture is in the range of 10°C to 16°C, more specifically in the range of 13°C to 16°C, and even more specifically 15°C.

[0029] The present disclosure also generally relates to aqueous acrylamide solutions obtainable by the methods disclosed herein.

[0030] In some embodiments, the aqueous acrylamide solution may be characterized in that the concentration of the acrylamide solution may be 35% to 55% by weight, the concentration of residual acrylonitrile in the acrylamide solution may be 1000 ppm or less as measured by FTNIR, and the turbidity of the acrylamide solution may be 20 or less as measured on a 0.45 μm filtered acrylamide sample.

[0031] In some embodiments, the concentration of residual acrylonitrile in the acrylamide solution as measured by FTNIR spectroscopy can be in the range of 0 to 1000 ppm and can be measured with an accuracy of at least ±100 ppm, more specifically, with an accuracy of at least ±80 ppm.

[0032] In some embodiments, the color of the acrylamide solution can be 20 or less as measured by spectrophotometric PtCo(455 nm) on a 0.45 μm filtered acrylamide sample.

[0033] In some embodiments, the concentration of the acrylamide solution can be 34% to 55% by weight, or more specifically, 38% to 40% by weight.

[0034] In some embodiments, the concentration of the acrylamide solution may be 38% to 55% by weight.

[0035] In some embodiments, the concentration of residual acrylonitrile in the acrylamide solution as measured by FTNIR spectroscopy can be 100 ppm or less, more specifically 90 ppm or less, even more specifically 50 ppm or less, even more specifically 10 ppm or less, and even more specifically 0 ppm.

[0036] In some embodiments, the turbidity of the acrylamide solution may be 15 or less.

[0037] The present disclosure also generally relates to the use of the aqueous acrylamide solutions obtainable by the methods disclosed herein in the production of polyacrylamide. [Brief explanation of the drawings]

[0038] [Figure 1] Schematic diagram of the reactor equipped with the FTNIR spectrometer dip probe, fiber optic cable, and FTNIR interferometer is shown. [Figure 2] A typical FTNIR absorption spectrum recorded during an acrylamide synthesis reaction is shown. The inset shows the spectral region where the greatest changes occur during the reaction. The absorption spectrum is plotted based on wavenumber. Absorbance is defined by A = -log10(IT / I0), where IT and I0 are the intensity of the transmitted light and the intensity of the background spectrum, respectively. In this case, the spectrum of air was used as the background spectrum. This spectrum was recorded in the absence of a medium but in the presence of air in the optical slit of the fiber optic probe. Using the ratio of IT to I0 has the advantage of reducing the influence of both the transmitted light path and the characteristics of the measurement system. [Figure 3] 1 shows cross-validation data for AN and AMD concentrations determined by FTNIR and HPLC for acrylamide synthesis experiment 1 shown in Example 2. Time, plotted on the x-axis, is shown as time of day (HH:MM). [Figure 4] 1 shows data for cross-validation of AN and AMD concentrations determined by FTNIR and HPLC for acrylamide synthesis experiment 2 shown in Example 3. Time, plotted on the x-axis, is shown as time of day (HH:MM). [Figure 5] Figure 1 shows cross-validation data for AN and AMD concentrations determined by FTNIR and HPLC for acrylamide synthesis experiment 3 shown in Example 4. Time, plotted on the x-axis, is shown as time of day (HH:MM). [Figure 6]1 shows cross-validation data for AN and AMD concentrations determined by FTNIR ("PLS Experiment") and HPLC ("Lab") for acrylamide synthesis Experiment 4 shown in Example 5. Time, plotted on the X-axis, is shown as time of day (HH:MM). [Figure 7] Figure 1 shows cross-validation data for AN and AMD concentrations determined by FTNIR ("PLS Experiment") and HPLC ("Lab") for acrylamide synthesis Experiment 4 shown in Example 5. Time plotted on the X-axis is shown as time (HH:MM). Data in these plots are limited to time points where the concentration of AN was determined to be less than 1000 ppm. DETAILED DESCRIPTION OF THE INVENTION

[0039] I. Overview According to a first aspect of the present disclosure, there is provided a method for producing an aqueous acrylamide solution.

[0040] More specifically, a method for producing an aqueous acrylamide solution is provided, the method comprising mixing a biocatalyst having nitrile hydratase activity with water to obtain a slurry, feeding acrylonitrile to a reactor containing the slurry to obtain a reaction mixture, and monitoring the reaction mixture by in-line FTNIR spectroscopy to measure the concentration of acrylonitrile.

[0041] In some embodiments, an FTNIR spectroscopic probe may be placed within the reactor.

[0042] In some embodiments, the reactor may include a cooling loop connected thereto and an FTNIR spectroscopic probe disposed within the cooling loop.

[0043] In some embodiments, the concentration of acrylonitrile may be measured with an accuracy of at least ±100 ppm, and more specifically with an accuracy of at least ±80 ppm.

[0044] According to a second aspect of the present disclosure, there is provided an aqueous acrylamide solution obtainable by the method.

[0045] More specifically, an aqueous acrylamide solution is provided, which is characterized in that the concentration of total residual acrylonitrile in the aqueous acrylamide solution is 1000 ppm or less as measured by FTNIR spectroscopy.

[0046] The concentration of total residual acrylonitrile may be measured with an accuracy of at least ±100 ppm, more specifically with an accuracy of at least ±80 ppm.

[0047] 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.

[0048] It is contemplated that any embodiment discussed herein can be implemented in connection with any method, kit, reagent, or composition of the present disclosure, and vice versa. Additionally, the compositions of the present disclosure can be used to achieve the methods of the present disclosure.

[0049] It will be understood that the specific embodiments described herein are shown by way of example, and not limitation. The principal features of this disclosure can be utilized in various embodiments without departing from the scope of the disclosure. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the present disclosure and are covered by the appended claims.

[0050] All publications and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0051] Unless otherwise defined, 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 disclosure belongs. In the event that there are multiple definitions for terms herein, the definitions set forth in this section shall prevail. When reference is made to a URL or other such identifier or address, it is understood that such identifiers may change and particular information on the Internet may come and go, but that equivalent information may be found by searching the Internet. Reference thereto confirms the availability and widespread dissemination of such information.

[0052] As used herein, the singular forms "a," "an," and "the" can mean "one," but can also include plural referents (such as "one or more" and "at least one") unless the context clearly indicates 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 disclosure belongs, unless clearly indicated otherwise.

[0053] As used herein, the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only, or unless the alternatives are mutually exclusive; however, the present disclosure supports definitions that refer to alternatives only, and "and / or."

[0054] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the inherent variation of error for the method employed to determine the value, or that variation exists among test subjects.

[0055] As used herein, approximation terms (such as, but not limited to, "about," "substantial," or "substantially") refer to terms that are understood not to be necessarily absolute or complete when such modifications are made, but would be considered sufficiently close by one of ordinary skill in the art to warrant specifying such terms as existing. The degree to which the description may vary may depend on how great the change that may occur is, and would still allow one of ordinary skill in the art to recognize that the modified feature still possesses the requisite properties and capabilities of the unmodified feature. Generally, within the scope of the foregoing discussion, numerical values ​​set forth herein that are modified by approximation terms (such as "about") may vary from the stated value by at least ±1%, at least ±2%, at least ±3%, at least ±4%, at least ±5%, at least ±6%, at least ±7%, at least ±8%, at least ±9%, at least ±10%, at least ±11%, at least ±12%, at least ±13%, at least ±14%, or at least ±15%.

[0056] As used herein, the word "comprising" (and any form of comprising, such as "comprise" and "comprises"), the word "having" (and any form of having, such as "have" and "has"), the word "including" (and any form of including, such as "includes" and "include")), or the word "containing" (and any form of containing, such as "contains" and "contain")) is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0057] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations that include repeats of one or more items or terms (e.g., BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc.) are expressly included. Those skilled in the art will understand that, unless otherwise clear from the context, there is typically no limit to the number of items or terms in any combination.

[0058] As used herein, "FTNIR" and "FT-NIR" refer to Fourier transform near-infrared spectroscopy.

[0059] As used herein, the term "biocatalyst" refers to any biocatalyst having nitrile hydratase (NHase) activity. A biocatalyst capable of converting acrylonitrile to acrylamide can be a microorganism encoding an enzyme having nitrile hydratase activity (e.g., NHase), or any portion of the microorganism having nitrile hydratase activity. In this regard, it is irrelevant whether the microorganism naturally encodes the nitrile hydratase, whether the microorganism has been genetically modified to encode the enzyme, or whether a microorganism naturally encoding the nitrile hydratase has been modified (such as to produce more nitrile hydratase and / or to produce an enhanced nitrile hydratase). Furthermore, it is irrelevant whether the enzyme having nitrile hydratase activity is a naturally occurring enzyme or a modified enzyme. The biocatalyst can be selected from the microorganism, lysed cells of the microorganism, a cell lysate of the microorganism, or any combination thereof. In a very specific embodiment, the biocatalyst is a nitrile hydratase (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) as a way to assess contaminant levels in wastewater. Since then, this method has expanded into a general method for comparing the intensity of yellowish samples. The method is specific to yellow and is based on dilutions of a 500 ppm platinum-cobalt solution. The color produced by dissolving 1 milligram of platinum-cobalt in 1 liter of water is determined as one unit of color on the platinum-cobalt scale. ASTM provides detailed instructions and procedures in ASTM Designation D1209, "Standard Test Method for Color of Clear Liquids (Platinum-Cobalt Scale)." Color measurements are made by visually comparing the sample to a platinum-cobalt standard. 1 mg / L of platinum produces one unit of color in the form of chloroplatinate ion. Because very small amounts of turbidity can interfere with the determination, samples that exhibit visible turbidity are generally clarified by centrifugation, and this method is pH dependent.

[0061] II. Reaction monitoring with FTNIR The present inventors have surprisingly found that by monitoring an acrylamide synthesis reaction by in-line Fourier transform near-infrared spectroscopy (FTNIR), it is possible to achieve an accuracy of at least ±100 ppm (i.e., at least ±80 ppm) in measuring the acrylonitrile concentration in the reaction mixture. This accuracy is an order of magnitude better than that possible with conventional infrared techniques. Thus, FTNIR can be used to monitor the maturity stage of an acrylamide synthesis reaction when the concentration of acrylonitrile is relatively low (e.g., less than 1000 ppm).

[0062] The reactor can 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 example embodiments is a semi-batch reactor. In some embodiments, an FTNIR spectroscopic probe can be disposed within the reactor. In some embodiments, the reactor includes a cooling loop attached thereto and an FTNIR spectroscopic probe disposed within the cooling loop. It is contemplated herein that disposing the FTNIR spectroscopic probe within the cooling loop will reduce gas bubbles in the reaction mixture compared to using a probe disposed within the reactor. It is further contemplated that this will further improve the accuracy of reactant concentration measurements (e.g., those made in determining the concentration of acrylonitrile).

[0063] FTNIR is a non-destructive technique that requires no sample preparation or consumables (such as solvents, columns, or reagents). FTNIR also provides real-time analysis, which typically requires less than 10 seconds per measurement, e.g., less than 1 second per measurement. When collected at a rate of 10 seconds per measurement, the 3.2 cm -1 Spectral resolution of up to 1000 sq m can be achieved. Therefore, FTNIR monitoring allows for shorter batch cycle times and improved manufacturing capacity compared to traditional wet lab methods (e.g., HPLC).

[0064] FTNIR spectroscopy measures overtone and combination bands of molecular vibrations occurring in the NIR range (approximately 780 nm to 2600 nm). Therefore, FTNIR is suitable for measuring analytes in aqueous solutions, as opposed to FTIR, where the analyte signal can be drowned out by the water signal. Furthermore, while FTNIR spectroscopy is suitable for measuring heterogeneous samples, FTIR cannot probe beneath the surface of a material, resulting in insufficient information when the material is heterogeneous.

[0065] The wavelengths used in the FTNIR method allow for the use of long fiber optic cables, eliminating the need to locate electrical components or ATEX equipment within tens of meters of the reactor.

[0066] FTNIR spectrometers are mechanically simpler than traditional filter-based dispersive NIR instruments because the only continuously moving part in the instrument is the moving mirror in the FTNIR interferometer. Therefore, the possibility of mechanical failure is extremely low. In most dispersive instruments, the diffraction grating and filters must be movable to obtain the spectrum. The advantage of mechanical simplicity is that the scanning mechanism is more reliable and robust, thereby improving the reliability of the analyzer. Furthermore, traditional NIR techniques are prone to sampling challenges due to stray light and have a relatively small resolution (16 cm). -1 or worse), loss of spectral information, wavelength inaccuracies (which are an obstacle in transplantation methods), and low signal-to-noise ratios. In contrast, FTNIR can perform measurements with signal-to-noise ratios of over 10,000.

[0067] Dispersive NIR instruments rely on prisms or diffraction gratings to separate the near-infrared frequencies. The best diffraction gratings offer a frequency resolution of at most 50 cm. -1 On the other hand, most chemical samples are -1These types of samples have spectral information that is resolved at a resolution of 1 / 2 sq. m. Because it is not possible to collect meaningful spectral information for these types of samples using dispersive instruments, dispersive instruments use slit mechanisms to improve resolution. Slits limit the amount of measurement beam, resulting in substantial energy loss, making it difficult or impractical to measure samples with high resolution. Because the resolution in FTNIR systems is determined by the stroke length of the moving mirror, there is no reduction in optical throughput, as occurs with slits in dispersive instruments. Measurements using FTNIR systems can quickly and easily obtain high-resolution spectra without performance degradation. The increased spectral information also reduces the need to rely on sophisticated chemometric algorithms, thereby reducing the criteria required for method development.

[0068] The use of an internal reference laser by FTNIR instruments is called the Cornes advantage. The advantage of internal calibration is that it is 0.1 cm -1 The precision and accuracy surpass those of dispersive NIR instruments. Dispersive NIR instruments utilize mechanically complex prisms or gratings that introduce peak position errors and scan-to-scan inaccuracies. Dispersive instruments have inherent inaccuracies that necessitate the use of reference materials for calibration. Repeated external calibrations are required, making measurements subject to difficulty and operator error. FTNIR instrument artifacts due to wavelength inaccuracies are negligible, requiring fewer standards and providing better results compared to those obtained with dispersive instruments.

[0069] FTNIR offers the advantages of fiber optic probes. FTNIR probes include classic diffuse reflectance probes for solid materials, transmission immersion probes for transparent liquids, and transflection immersion probes for suspensions or emulsions. Examples of transflection immersion probes include those specifically designed for monitoring aqueous acrylamide synthesis reactions. A variety of path lengths are suitable. A variety of probe materials are available, including stainless steel, Hastelloy, or ceramic. Furthermore, probes can be customized to vary length and flange geometry. Thus, FTNIR spectrometer probes can be configured to be placed within a reactor or within a cooling loop connected to a reactor.

[0070] III. Biocatalysis The biocatalyst may be fresh (i.e., as obtained from fermentation), preserved (such as stored as frozen (wet frozen)), or dried prior to preparation of the slurry.

[0071] After fermentation, the biocatalyst slurry is often washed or otherwise appropriately treated or may be further treated, typically before the slurry is put into service or before storage (e.g., before cryopreservation).

[0072] The biocatalyst can be any biocatalyst known in the art that has nitrile hydratase (NHase) activity.

[0073] According to any one of the embodiments of the present disclosure, the biocatalyst capable of converting acrylonitrile to acrylamide can be a microorganism encoding an enzyme having nitrile hydratase activity (e.g., NHase), or any part of the microorganism having nitrile hydratase activity. In this regard, it is irrelevant whether the microorganism naturally encodes the nitrile hydratase, whether the microorganism has been genetically modified to encode the enzyme, or whether the microorganism naturally encoding the nitrile hydratase has been modified (such as to produce more nitrile hydratase and / or to produce an enhanced nitrile hydratase). Furthermore, it is irrelevant whether the enzyme having nitrile hydratase activity is a naturally occurring enzyme or a modified enzyme. The biocatalyst can be selected from the microorganism, lysed cells of the microorganism, a cell lysate of the microorganism, or any combination thereof. In a very specific embodiment, the biocatalyst is a nitrile hydratase (NHase).

[0074] A microorganism encoding a nitrile hydratase (e.g., a microorganism that naturally encodes a nitrile hydratase or a microorganism that has been genetically modified to encode a nitrile hydratase) or any portion of such a microorganism can be used as a biocatalyst in any one of the embodiments described herein, including, but not limited to, Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas s, 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 exemplary embodiments, the biocatalyst is selected from bacteria of the genera Rhodococcus, Pseudomonas, Escherichia, and Geobacillus.Typically, biocatalysts include Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiel la, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Coryne The enzyme is selected from the group consisting of: bacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus, or any part of such a microorganism having nitrile hydratase activity.

[0075] In some embodiments, the biocatalyst is selected from the group consisting of Rhodococcus (e.g., Rhodococcus pyridinovorans or Rhodococcus rhodochrous or Rhodococcus aetherivorans), Pseudomonas, Escherichia, and Geobacillus, or any part of such a microorganism that has nitrile hydratase activity.

[0076] In exemplary embodiments, the biocatalyst is Rhodococcus aetherivorans or Rhodococcus rhodochrous, or any part of such a microorganism that has nitrile hydratase activity.

[0077] In one embodiment, the amount of biocatalyst is 0.1 kg of dry cells / m of reaction mixture. 3~5 kg of dry cells / m of reaction mixture 3 is.

[0078] In another embodiment, the amount of biocatalyst is, based on the final AMD amount, from 0.1 g dry cells / 100% AMD kg to 3 g dry cells / 100% AMD kg, more specifically from 0.2 g dry cells / 100% AMD kg to 3 g dry cells / 100% AMD kg, more specifically from 0.2 g dry cells / 100% AMD kg to 2.5 g dry cells / 100% AMD kg, In another embodiment, the amount of biocatalyst is from 0.5 g dry cells / 100% AMD kg to 2 g dry cells / 100% AMD kg, or more specifically from 1.1 g dry cells / 100% AMD kg to 1.5 g dry cells / 100% AMD kg.

[0079] In yet another embodiment, the amount of biocatalyst is based on the final AMD amount from 0.5g dry cells / 50% AMD kg to 1g dry cells / 50% AMD kg, more specifically from 1.6g dry cells / 50% AMD kg to 1.8g dry cells / 50% AMD kg.

[0080] In another embodiment, the amount of biocatalyst is m of the reaction mixture at the end of maturation of the reaction mixture. 3 In another embodiment, the amount of biocatalyst is between 0.1 kg and 1.5 kg of dry cells per m of reaction mixture. 3 ~1.0 kg of dry cells / m of reaction mixture 3 is.

[0081] During the process, for example, if acrylonitrile begins to accumulate in the reactor, the amount of biocatalyst added is increased. The biocatalyst may be added, for example, as a homogeneous slurry in water.

[0082] IV. Reaction Progress The reaction is carried out at ambient pressure, more particularly at 1 bar.

[0083] The slurry can be prepared by any method known in the art, such as mixing water and biocatalyst in a vessel or reactor. More specifically, the slurry is homogeneous. Strongly flocculated slurries have lower activity compared to homogeneous slurries. Biocatalysts are more active in homogeneous slurries.

[0084] The reaction of acrylonitrile to acrylamide in an aqueous solution in the presence of a biocatalyst having NHase activity begins when acrylonitrile is fed to a reactor containing the slurry. Thus, feeding acrylonitrile to a reactor containing the slurry produces a reaction mixture containing water, acrylamide, acrylonitrile, and the biocatalyst.

[0085] By controlling the acrylonitrile feed profile and the process temperature profile, a highly concentrated aqueous acrylamide solution (e.g., at least 35 wt. %, or at least 40 wt. %, or at least 45 wt. %, or at least 50 wt. %) can be obtained. Cooling the reactor is typically required to maintain the reaction mixture at the desired reaction temperature. To increase the reaction rate and shorten the synthesis time, the temperature and acrylonitrile feed rate are relatively high at the beginning of the reaction. Since biocatalyst deactivation due to acrylamide accumulation is significantly reduced at low temperatures compared with high temperatures, cooling of the reactor is initiated after the start of the reaction (e.g., 60 minutes after the start of the reaction). To avoid acrylonitrile accumulation in the reactor, the acrylonitrile feed rate is relatively slowed during the last few hours.

[0086] The supply of acrylonitrile can be continued throughout the process, more specifically, the supply of acrylonitrile can be continued throughout the process until the maturity stage is reached. The acrylonitrile supply rate can be changed during the process. The supply of acrylonitrile can be continuous or intermittent. The acrylonitrile supply rate depends on the reaction rate of acrylonitrile to acrylamide and the deactivation rate of the biocatalyst. In one embodiment, the supply of acrylonitrile is continued throughout the process until the maturity stage is reached.

[0087] In one embodiment, the acrylonitrile feed rate is adjusted during the process to avoid acrylonitrile accumulation in the reaction mixture. The acrylonitrile is fed during the process at a rate at which the acrylonitrile is converted to acrylamide. More specifically, the amount of acrylonitrile in the reaction mixture is maintained at less than 3 wt.% or less than 2 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 another embodiment of the process, 38% to 48% of the total amount of acrylonitrile fed to the reactor is fed to the reactor between 0 and 60 minutes from the start of the process, 22% to 30% of the total amount of acrylonitrile is fed between 60 and 120 minutes of the process, 12% to 18% of the total amount of acrylonitrile is fed between 120 and 180 minutes of the process, and 8% to 12% of the total amount of acrylonitrile is fed between 180 and 240 minutes of the process. The remaining acrylonitrile is fed during the process prior to the maturation period to balance 100% of the fed acrylonitrile.

[0089] During the maturation period, substantially no, more specifically no, acrylonitrile is fed to the reactor. During maturation, any acrylonitrile monomer still present in the reaction mixture reacts to form acrylamide. The reaction mixture is maturated until the desired characteristics are achieved.

[0090] It is known that biocatalysts begin to deactivate in acrylamide solutions of approximately 25% to 38% by weight. See, for example, WO 2019 / 097123. Because biocatalyst deactivation caused by acrylamide accumulation is strongly temperature-dependent, cooling the reaction mixture significantly reduces biocatalyst deactivation. Therefore, the temperature of the reaction mixture is monitored. This monitoring and measurement can be performed using any suitable means and methods known in the art.

[0091] Initially, the temperature of the reaction mixture is maintained between 15 and 25° C. In one embodiment, the temperature is maintained between 19 and 25° C., more specifically between 20 and 22° C., and even more specifically at 22° C. In one embodiment, the temperature is maintained within the desired range by measuring the temperature of the reaction mixture and cooling the mixture or heating the mixture so that the temperature is within the desired range. Cooling and / or heating the reaction mixture can be accomplished using methods known in the art.

[0092] In some embodiments, the method includes further cooling the reaction mixture when the acrylamide concentration reaches at least 27% by weight, more specifically, 27% to 38% by weight. In one embodiment, cooling of the reaction mixture is initiated when the acrylamide concentration reaches 28% to 30% by weight. Cooling the reaction mixture can be carried out by any suitable method and means known in the art, such as by cooling the reactor.

[0093] When cooling of the reaction mixture is initiated, the temperature of the reaction mixture can be the same as, higher than, or lower than the temperature of the reaction mixture at the start of the process.

[0094] In some embodiments, cooling of the reaction mixture is continued such that when the acrylamide concentration reaches 37% to 55% by weight, the temperature of the reaction mixture is within a range of 10° C. to 18° C. or 10° C. to 21° C. In other words, the time period during which the reaction mixture is cooled to a temperature of 10° C. to 18° C. or 10° C. to 21° C. is the time period during which the acrylamide concentration increases from at least 27% by weight (more specifically, 27% to 38% by weight) to 37% to 55% by weight (more specifically, 40% to 50% by weight).

[0095] In one embodiment, cooling of the reaction mixture is continued such that the temperature is within the range of 10° C. to 16° C., more specifically within the range of 13° C. to 16° C., and even more specifically 15° C., when the acrylamide concentration reaches 37% to 55% by weight. In one embodiment, cooling is initiated, for example, after the reaction mixture has been maintained at 15° C. to 25° C. In one embodiment, the reaction mixture is cooled by at least 10° C., more specifically at least 5° C., and even more specifically at least 4° C. Cooling can be performed linearly or stepwise, and is typically performed linearly.

[0096] In one embodiment, the reaction mixture is matured at a temperature in the range of 10°C to 18°C ​​or 10°C to 21°C when the acrylamide concentration reaches 37% to 55% by weight.

[0097] During maturation, substantially no acrylonitrile is fed to the reactor, more specifically, none. During maturation, unreacted acrylonitrile in the reactor reacts to form acrylamide. Maturing begins after the reaction mixture is cooled and the temperature of the reaction mixture is within the range of 10°C to 18°C ​​or 10°C to 21°C, and / or after the feed of acrylonitrile to the reactor has been terminated. More specifically, maturation continues until the final concentration of acrylonitrile in the reaction mixture is at most 1000 ppm, at most 500 ppm, at most 250 ppm, at most 100 ppm, at most 50 ppm, at most 10 ppm, or at most 0 ppm.

[0098] 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 (such as 45 minutes to 60 minutes), and cooling of the reaction mixture to a temperature of 10°C to 18°C ​​or 10°C to 21°C is carried out over 45 minutes to 120 minutes (such as 60 minutes to 120 minutes).

[0099] Since the temperature is kept low at the end of the process, less acrylic acid is formed during the process. The activation energy of the reaction in which acrylic acid is formed 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. A low amount of acrylic acid in the aqueous acrylamide solution is advantageous when preparing cationic polymers from the acrylamide solution.

[0100] The resulting aqueous acrylamide solution can be centrifuged to separate the acrylamide from the biocatalyst.

[0101] IV. Acrylamide aqueous solution In a second aspect of the present disclosure, an aqueous acrylamide solution is provided that is obtainable or obtainable by the method disclosed herein. More specifically, an aqueous acrylamide solution is provided that is obtainable by the method disclosed herein, characterized in that the total residual acrylonitrile concentration in the aqueous acrylamide solution is 1000 ppm or less as measured by FTNIR spectroscopy. In one embodiment, the total residual acrylonitrile concentration in the solution as measured by FTNIR spectroscopy is at most 1000 ppm, at most 500 ppm, at most 250 ppm, at most 100 ppm, at most 90 ppm, more specifically at most 75 ppm, even more specifically at most 50 ppm, and most specifically at most 10 ppm. In one embodiment, the residual acrylonitrile concentration is 0 ppm.

[0102] In some embodiments, the concentration of acrylonitrile is measured by FTNIR spectroscopy with an accuracy of at least ±5000 ppm, at least ±3000 ppm, at least ±1000 ppm, at least ±500 ppm, at least ±400 ppm, at least ±300 ppm, at least ±200 ppm, at least ±100 ppm, or at least ±80 ppm.

[0103] In some embodiments, the concentration of total residual acrylonitrile is measured by FTNIR spectroscopy with an accuracy of at least ±100 ppm, more specifically with an accuracy of at least ±80 ppm.

[0104] In some embodiments, the concentration of acrylamide and the concentration of acrylic acid in the aqueous acrylamide solution can also be measured by FTNIR spectroscopy. In some embodiments, the concentration of acrylamide in the aqueous acrylamide solution is 34% to 55% by weight, or 38% to 55% by weight, or 50% to 55% by weight. In some embodiments, 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. A low amount of acrylic acid in the aqueous acrylamide solution is advantageous when preparing a cationic polymer from the acrylamide solution.

[0105] In some embodiments, the turbidity of the aqueous acrylamide solution can be 20 or less when measuring 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 solution sample. In one embodiment, the turbidity of the solution is 15 or less.

[0106] In some embodiments, the resulting aqueous acrylamide solution may be substantially free of biocatalysts.

[0107] V. Use of Aqueous Acrylamide 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. [Example]

[0108] VI. Working Examples The following examples are offered by way of illustration only, and not by way of limitation.

[0109] Materials and methods for monitoring the acrylamide synthesis reaction used in the examples Four acrylamide synthesis reactions were carried out as described in Examples 1 to 4. For comparative validation of the method, the concentrations of acrylamide (AMD), acrylonitrile (AN), and acrylic acid (AA) were determined by in-line FTNIR spectroscopy and HPLC.

[0110] FTNIR spectroscopy was performed with an i-RED FTNIR spectrometer (Infrarot Systeme GmbH, Austria) using a fiber-optic Falcata 12 transflection probe (Hellma, Germany) placed in the semi-batch reactor. Figure 1 shows a schematic diagram of the reactor equipped with FTNIR. Table 1 lists the FTNIR spectrometer parameters. Figure 2 shows a typical FTNIR absorption spectrum. [Table 1]

[0111] For HPLC measurements, reaction mixture samples (1.5 mL) were periodically taken from the reactor by pipette, filtered through a 0.45 μm PVDF syringe filter, and the reaction was stopped by adding 10 μL of 0.7 M CuSO4·5H2O.

[0112] The concentrations of AN, AMD, and AA were determined by HPLC using an 1100 Series HPLC (Agilent Technologies, USA) equipped with a Kintex® 5 μm C18 100Å LC column (250 × 4.6 mm). Detection of these components was performed by UV absorbance at 200 nm, 225 nm, and 260 nm using a 1260 Diode Array Detector (Agilent Technologies, USA), depending on the component and its UV absorbance linearity. The flow rate was 1 mL / min, and 3.8 × 10 M H3PO4 was used as the eluent. Samples for HPLC were prepared by accurately measuring approximately 0.2 g of the quenched reaction mixture and diluting it to 100 mL in Type 1 MilliQ water, which had been pretreated with UV irradiation for 1 hour to destroy impurities. A 1961 ppm acrylamide internal standard was analyzed in triplicate prior to measurement to ensure proper instrument calibration. The 0.1 μL acrylamide program was used in the Agilent software.

[0113] Multivariate Data Analysis The FTNIR data and HPLC-determined reference values ​​for AN and AMD concentrations were subjected to multivariate data analysis. Analysis was not possible for AA, as all concentrations of this by-product were determined to be 0.0% or <0.01%.

[0114] For quantitative determination of the concentrations of reaction mixture components, the recorded FTNIR spectra, along with HPLC-derived reference values, were subjected to multivariate data analysis using the PLS method. Correlations between the reference values ​​and FTNIR spectra for AN and AMD were therefore investigated. Furthermore, evaluation or prediction models were developed to calculate concentrations from the measured FTNIR spectra.

[0115] To more easily assess the quality (predictive power, stability) of such models and to better estimate the resulting mean measurement error, the data were subjected to cross-validation. Specifically, in the modeling method, data samples were split out, a model was created with the remaining data / samples, and then this model was applied to the split-out data / samples. This ensured that the model was not tested on samples or measurements already included in the model.

[0116] In cross-validation, two parameters (R 2 and RMSECV). These two quantities are explained as follows: -R 2 : Correlation coefficient. R 2 is a dimensionless measure of the correlation between the reference value and the FTNIR spectral data. Its value ranges from 0 to 1, with larger values ​​indicating better correlation. - RMSECV: Root mean square error of cross-validation. This parameter describes the average deviation of the FTNIR values ​​from the reference value in the cross-validation for each measurement unit. This parameter indicates the magnitude of the predicted mean error of the measurement method based on this evaluation model.

[0117] Data analysis was performed for each experiment separately, and one overall model including data from all experiments was examined.

[0118] Example 1: Acrylamide synthesis experiment 1 A homogeneous slurry was prepared by vortexing 9.96 g of thawed biocatalyst in 190 g of TRIS buffer (TRIS-HCl (pH 8.0±0.1) prepared with deionized water) at 1000 rpm for 15 minutes. The dry cell content of the biocatalyst in the slurry was determined to be 2.189 wt%. A semi-batch reactor equipped with a FTNIR spectrometer probe was charged with 570.0 g of TRIS buffer (pH 8.0) and 3.09 g of the biocatalyst slurry and mixed at 400 rpm to prevent settling of the biocatalyst.

[0119] The reactor was cooled to an initial temperature of 21°C before the acrylonitrile feed was started at initial conditions.

[0120] The acrylamide synthesis reaction was initiated by feeding acrylonitrile to the reactor containing the slurry to form a reaction mixture. A total of 226.91 g of acrylonitrile was fed to the reactor over approximately 3 hours. After 5 hours, the reaction mixture was cooled to 20°C.

[0121] Table 2 shows the reaction mixture component concentrations determined by HPLC for this experiment. [Table 2]

[0122] Cross-validation for AN and AMD concentrations is shown in Figure 3. The calculation model for AN concentration showed good correlation (R ) between the reference values ​​and the FTNIR measurement data. 2 = 0.978). The predicted mean error of measurement (RMSECV) of the spectrophotometric method for AN content is less than 0.5% in the concentration range of 0-10%.

[0123] The calculation model for AMD concentrations showed some correlation (R 2 = 0.715). The predicted mean error of measurement (RMSECV) of the spectrophotometric method for AMD content is less than 3.5% in the concentration range of 0–24%.

[0124] Example 2: Acrylamide synthesis experiment 2 To determine the precision and accuracy of FTNIR for acrylamide in the concentration range of 0% to 50%, the following reaction protocol was designed to achieve a final acrylamide concentration of at least 50%. Compared to acrylamide synthesis experiment 1, the amount of acrylonitrile fed to the reaction mixture was increased by 57.3%.

[0125] A homogeneous slurry was prepared by vortexing 10 g of thawed biocatalyst in 190 g of deionized water at 1000 rpm for 15 minutes. The dry cell concentration of the biocatalyst in the slurry was determined to be 0.669 wt%. Deionized water (545.6 g) and the slurry (17.33 g) were added to a semi-batch reactor equipped with an FTNIR spectrometer probe and mixed at 300 rpm to prevent settling of the biocatalyst.

[0126] The reactor was cooled to an initial temperature of 22°C before the acrylonitrile feed was started at initial conditions.

[0127] The acrylamide synthesis reaction was initiated by feeding acrylonitrile to the reactor containing the slurry to form a reaction mixture. A total of 357 g of acrylonitrile was fed to the reactor over a 3-hour period. Specifically, 167.8 g (47.0 wt%) of acrylonitrile was fed to the reactor during the first 60 minutes (i.e., 0 to 60 minutes). 117.8 g (33.0 wt%) of acrylonitrile was fed to the reactor during the next 60 minutes (i.e., 60 to 120 minutes). 71.4 g (20.0 wt%) of acrylonitrile was fed to the reactor during the next 60 minutes (i.e., 120 to 180 minutes). After 3 hours, the acrylonitrile feed was stopped.

[0128] After 5 hours, the reaction mixture was cooled to 20°C. The FTNIR probe was then removed, and air was purged into the reaction mixture for 5 minutes (air supply rate 0.5 nL / min) through a porous glass sinter submerged below the liquid surface. The reaction mixture was then left overnight to continue the AMD maturation phase.

[0129] Table 3 shows the reaction mixture component concentrations as determined by HPLC. [Table 3]

[0130] Cross-validation for AN and AMD concentrations is shown in Figure 4. The calculation model for AN concentration showed good correlation (R ) between the reference values ​​and the FTNIR measurement data.2 = 0.847). The predicted mean square error of measurement (RMSECV) of the spectrophotometric method obtained from this experiment for AN content is less than 0.3%.

[0131] The calculation model for AMD concentrations showed a very good correlation (R 2 = 0.966). The predicted mean error of measurement (RMSECV) of the spectroscopic method obtained from this experiment for AMD content is less than 3.8%.

[0132] Example 3: Acrylamide synthesis experiment 3 To determine the precision and accuracy of FTNIR for acrylonitrile in the concentration range of 0% to 1%, the following reaction protocol was used.

[0133] A homogeneous slurry was prepared by vortexing 10.05 g of thawed biocatalyst in 190 g of deionized water. The dry cell concentration of the biocatalyst in the slurry was determined to be 0.819 wt%. Deionized water (542.5 g) and the slurry (20.47 g) were added to a semi-batch reactor equipped with an FTNIR spectrometer probe and mixed at 300 rpm to prevent settling of the biocatalyst.

[0134] The reactor was cooled to an initial temperature of 22°C before the acrylonitrile feed was started at initial conditions.

[0135] The acrylamide synthesis reaction was initiated by feeding acrylonitrile to the reactor containing the slurry to form a reaction mixture. During the first 60 minutes (i.e., 0 to 60 minutes), 168 g (47.0 wt%) of acrylonitrile was fed to the reactor. During the next 60 minutes (i.e., 60 to 120 minutes), 118 g (33.0 wt%) of acrylonitrile was fed to the reactor. During the next 60 minutes (i.e., 120 to 180 minutes), 71 g (20.0 wt%) of acrylonitrile was fed to the reactor. After 3 hours, the acrylonitrile feed was stopped. After 5 hours, the reaction mixture was cooled to 20°C.

[0136] Table 4 shows the reaction mixture component concentrations as determined by HPLC. [Table 4]

[0137] Cross-validation for AN and AMD concentrations is shown in Figure 5. The calculation model for AN concentration showed good correlation (R ) between the reference values ​​and the FTNIR measurement data. 2 =0.893). The calculation model for AMD concentration also showed good correlation (R ) between the reference values ​​and the FTNIR measured data. 2 =0.898).

[0138] The predicted mean square root mean square error (RMSECV) of the spectroscopic measurements obtained from this experiment was less than 0.04% (less than 400 ppm) for AN content and less than 1.3% for AMD content. While these error values ​​represent an improvement over the error levels achieved in acrylamide synthesis experiments 1 and 2, the presence of air bubbles was observed within the reactor. The presence of air bubbles within the reactor is expected to increase the error in the FTNIR measurements compared to FTNIR measurements performed in the absence of air bubbles. Therefore, it is contemplated that placing the FTNIR probe outside the reactor (i.e., within the cooling loop attached to the reactor) will further reduce the error in the FTNIR measurements of AN and AMD concentrations due to the reduced presence of air bubbles within the cooling loop compared to the reactor.

[0139] Example 4: Acrylamide synthesis experiment 4 The following experiment was designed to further control the acrylonitrile concentration in the very low concentration range of 0–1% (10,000 ppm) and to obtain more data points in this concentration range. Notably, the biocatalyst used in this reaction was denatured. Therefore, it was expected that this biocatalyst would not catalyze acrylamide synthesis. Therefore, this experiment served as a "mock" acrylamide synthesis reaction. In this example experiment, the initial acrylamide content of the reactor was 36.95 wt%. A total of only 9.3 g of AN was fed to the reactor, gradually increasing the AN concentration from 0.0% to 0.99 wt%. As expected, the AMD concentration remained relatively constant. However, a slight decrease in AMD concentration was observed due to dilution caused by the addition of AN, dropping the AMD concentration to 36.58 wt%.

[0140] Table 5 shows the reaction mixture component concentrations as determined by HPLC. [Table 5-1] [Table 5-2]

[0141] Cross-validation for AN and AMD concentrations is shown in Figure 6. The calculation model for AN concentration showed excellent correlation (R ) between the reference values ​​and the FTNIR measured data. 2 The predicted mean error of measurement (RMSECV) of the spectrophotometric method for AN content is less than 0.02% (i.e., less than 0.018% (180 ppm)) in the concentration range of 0-1% (0-10,000 ppm).

[0142] The calculation model for AMD concentration also showed good correlation (R 2 The predicted mean error of measurement (RMSECV) of the spectrophotometric method for AMD content is less than 0.01% in the concentration range of 36.6–37%.

[0143] To determine the predicted mean measurement error for acrylonitrile concentrations in the very low range of 0-0.1% (0-1000 ppm, the expected range of acrylonitrile concentrations in aqueous acrylamide products), the data from this experiment were restricted to values ​​where [AN] < 1000 ppm. A model was developed and tested.

[0144] Figure 7 shows the cross-validation of AN and AMD concentrations for data with [AN] < 1000 ppm. The calculation model for AN concentration showed very good correlation (R 2 The predicted mean error of measurement (RMSECV) of the spectrophotometric method for AN content is less than 80 ppm in the concentration range of 0 to 1000 ppm.

Claims

1. (a) mixing at least one biocatalyst having nitrile hydratase activity with water to obtain a slurry; (b) feeding acrylonitrile into a reactor containing the slurry to obtain a reaction mixture; and (c) monitoring the reaction mixture by in-line FTNIR spectroscopy to measure the concentration of acrylonitrile in the reaction mixture; Including, an FTNIR spectroscopic probe located inside the reactor and / or outside the reactor; the FTNIR spectrometer probe comprises a transflection immersion FTNIR probe; The final concentration of acrylonitrile is in the range of 0-1000 ppm and is measured in real time by FTNIR spectroscopy with an accuracy of at least ±100 ppm at a rate of 10 seconds or less per measurement; A method for producing an aqueous acrylamide solution.

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

3. 3. The method of claim 1 or claim 2, wherein the reactor includes a cooling loop connected thereto, and the FTNIR spectrometer probe is disposed within the cooling loop.

4. 4. The method of claim 1, wherein the concentration of acrylonitrile is in the range of 0 to 10% by weight, as measured by FTNIR spectroscopy with an accuracy of at least ±1% by weight.

5. 5. The method of any one of claims 1 to 4, wherein the concentration of acrylonitrile is in the range of 0 to 1 wt %, as measured by FTNIR spectroscopy with an accuracy of at least ±400 ppm.

6. 6. The method according to any one of claims 1 to 5, wherein the final concentration of acrylonitrile is measured by FTNIR spectroscopy with an accuracy of at least ±80 ppm.

7. 7. The method of any one of claims 1 to 6, wherein monitoring the reaction mixture further comprises measuring the concentration of acrylamide by FTNIR spectroscopy, wherein the concentration of acrylamide is in the range of 0 to 50 wt% and is measured with an accuracy of at least ±5 wt%.

8. 8. The method of any one of claims 1 to 7, wherein the final concentration of acrylonitrile as measured by FTNIR spectroscopy is at most 500 ppm.

9. i. adjusting the acrylonitrile feed rate during the process to thereby control acrylonitrile accumulation in the reactor; ii. The method of any one of claims 1 to 8, wherein 38% to 48% of the total amount of acrylonitrile fed to the reactor is fed during a time period spanning from 0 minutes to 60 minutes, inclusive, from the start of feeding acrylonitrile to the reactor.

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

11. i. The biocatalyst is prepared at a concentration of 0.1-5 kg ​​of dry cells / ml of reaction mixture. ３ Including, ii. If the biocatalyst is Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Br. adyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesor hizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium , Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hansoniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus, or a combination of at least two of any of said microorganisms; and / or iii. the biocatalyst is Rhodococcus rhodochrous or Rhodococcus aetherivorans, and / or iv. The method of any one of claims 1 to 10, wherein the biocatalyst comprises or additionally comprises a composition comprising a nitrile hydratase from any of the microorganisms.

12. i. Measuring and regulating the temperature of the reaction mixture; ii. Maintaining the temperature of the reaction mixture within the range of 15°C to 25°C; cooling the reaction mixture when the acrylamide concentration reaches 27% to 38% by weight so that the temperature of the reaction mixture is within the range of 10° C. to 21° C. when the acrylamide concentration reaches 37% to 55% by weight; maintaining the reaction mixture at a temperature range of 10°C to 21°C to achieve a final acrylonitrile concentration of at most 1000 ppm; iii. Cooling the reaction mixture when the acrylamide concentration reaches 28% to 30% by weight. iv. Cooling the reaction mixture until an acrylamide concentration of 40% to 50% by weight is reached; v. Maintaining the temperature of the reaction mixture between 19°C and 25°C; vi. maintaining the temperature of the reaction mixture for 30 minutes to 120 minutes; and / or vii. Cooling the reaction mixture so that the temperature of the reaction mixture is within the range of 10°C to 16°C; The method of any one of claims 1 to 11, further comprising:

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