GB2076821A - Process for producing acrylamide or methacrylamide utilizing microorganisms - Google Patents

Abstract

A process for continuously producing acrylamide or methacrylamide in aqueous solution by passing an aqueous solution of acrylonitrile or methacrylonitrile through one or more columns filled with immobilised bacterial cells having a nitrilase activity at a temperature of from the freezing point of the medium, up to 30 DEG C at pH 6 to 10 while feeding acrylonitrile or methacrylonitrile via one or more inlets intermediate between the column inlet and outlet in an amount soluble in the reaction mixture.

Description

SPECIFICATION Process for producing acrylamide or methacrylamide utilizing microorganisms The present invention relates to an improved process for producing acrylamide or methacrylamide utilizing micro-organisms.

As a process for producing acrylamide or methacrylamide, a process of reacting acrylonitril (AN) or methacrylonitrile (MAN) with water has been proposed using reduced copper as a catalyst. However, it has been desired to develop a novel and industrially more advantageous process since catalyst preparation and regeneration is difficult in such a process and the isolation and purification of the amide produced is onerous.

On the other hand, as a process for producing acrylamide or methacrylamide from acrylonitrile or methacrylonitrile utilizing an enzymatic reaction, an interesting process using bacteria belonging to the genus Bacillus, the genus Bacteridium in the sense of Prevot, the genus Micrococcus, the genus Brevibacterium in the sense of Bergy, has been proposed in U.S. Patent 4,001,081. This process is simply based on the discovery that the above-described bacteria hydrolyze various organic nitriles to produce the corresponding organic acid amides. In the case where acrylonitrile or methacrylonitrile (Examples 6-8 in the above-mentioned U.S. Patent) for example are used, this U.S.Patent describes that acrylamide or methacrylamide was obtained almost quantitatively when the acrylonitrile or methacrylonitrile concentration was 8 to 12% by wt, the bacterial cell concentration was 2 to 4% by wt., the pH was 7 tq 9, the temperature was 250C and the reaction time was 20 to 30 minutes. It is true that acrylamide or methacrylamide can be produced at a concentration as high as 10 to 20 wt %, but the bacterial cells so rapidly lose their enzymatic activity under such conditions that it is almost impossible to use them repeatedly. In addition, the solution from which the bacterial cells are separated is very dark yellow and contains various impurities originating from the cells, and hence an onerous purifying step is necessary. Thus, the above-described process is not economically advantageous in industrial applications.

A catalytic process for producing acrylamide or methacrylamide utilizing micro-organism has been investigated and bacteria having an extremely high activity for hydrolyzing acrylonitrile and methacrylonitrile to produce acrylamide or methacrylamide have been discovered. Namely, the strain No77 1 and the strain N-774 belonging to the genus Corynebacterium, and the strain N-775 belonging to the genus Nocardia have been found in the soils around a factory producing acrylonitrile and in the waste water discharged from the factory. (Hereafter the aforementioned bacteria will be referred to as No77 1, N-774 and N-775, respectively). The enzymatic nitrilase activity of these micro-organisms is surprisingly high at low temperatures.As a result of intensive investigations, a process for the hydrolysis of acrylonitrile and methacrylonitrile has been developed wherein the enzymatic activity of the bacterial cells is stably maintained at a high level for a long time, with the accumulation of produced acrylamide or methacrylamide reaching concentrations as high at 10 wt % or more, which process does not require a difficult purifying step.

In a conventional continuous column process, it has been difficult to bring the bacterial cells into uniform contact with a material having a low solubility in water, such as acrylonitrile of methacrylonitrile, at a high concentration to react and, therefore, the conventional process suffers from the defects that a highly concentrated acrylamide or methacrylamide aqueous solution cannot be obtained effectively and smoothly, and that the enzymatic activity of the bacterial cells is sharply reduced.

The inventor attempted the continuous column reaction utilizing immobilized cells to carry out the process economically and effectively.

However, in the case where a substrate was used at high concentrations, it was found to be difficult to feed a highly concentrated substrate solution in the column and carry out the uniform catalytic reaction with the bacterial cell since the bacterial cell so rapidly lost their enzymatic activity in the conventional continous column process and the solubility of acrylonitrile or methacrylonitrile as a substrate in water was small, and as a result, a highly concentrated acrylamide or methacrylamide aqueous solution was not obtained effectively and smoothly.

According to the present invention, there is provided a process for continuously producing a highly concentrated acrylamide or methacrylamide aqueous solution by passing an aqueous solution of acrylonitrile or methacrylonitrile through a column or columns filled with immobilized bacterial cells having nitrilase activity, at a temperature ranging from the freezing point of the solution to 300C at a pH of 6 to 10, which comprises: (1) using a column having one or more feed inlets provided between the column inlet and the column outlet, continuously feeding an aqueous solution of acrylonitriie or methacrylonitrile via said column inlet and, at the same time, continuously feeding acrylonitrile or methacrylonitrile via said feeding inlet(s) an amount soluble in the reaction medium; or (2) using two or a plurality of columns connected to each other in series, and continuously feeding an aqueous solution of acrylonitrile or methacrylonitrile via the first column inlet and, at the same time, continuously feeding acrylonitrile or methacrylonitrile via the column inlet(s) of the successive column in an amount soluble in the reaction mixture.

As the microorganisms used in the present invention, any one that has the ability to hydrolyze acrylonitrile or methacrylonitrile to produce acrylamide or methacrylamide may be used regardless to the taxonomic position, as well as the aforesaid strains No77 1, N-774 and N-775. For example bacteria from the genus Bacillus, the genus Bacteridium, the genus Micrococcus and the genus Brevibacterium as disclosed in U.S. Patent 4,001,081 may also be used. In addition, it is also possible to use the cellular extract prepared by destroying such bacterial cells, crude enzyme preparations, etc.

To culture the microorganisms used in the present invention, ordinary culture mediums containing a carbon source (e.g., glucose, maltose, etc.), a nitrogen source (e.g., ammonium sulfate, ammonium chloride, etc.), an organic nutrient source (e.g., yeast extract, malt extract, peptone, meat extract, etc.), and an inorganic nutrient source (e.g., phosphate, magnesium, potassium, zinc, iron, manganese, etc.) are used. The culture is aerobically conducted while maintaining the pH of the culture medium at about 6 to 9 at a temperature of about 20 to 350C, preferably about 25 to 300C, for about 1 to 5 days.

The strains N-771, N-774 and N-775 to be used in the present invention were deposited at Fermentation Research Institute, Agency of Industrial Science Er Technology, Ministry of International Trade and Industry, Japan, as FERM-P Nos. 4445, 4446 and 4447, respectively, on March 28th, 1 978. The bacteriological characteristics of each strain are as shown below.

A: Strain No77 1 (a) Morphology (1) Shape and size of cells: (0.5-0.8) y x (2-5) y (2) Pleomorphism of cells: At the initial stage of culture, the bacterial cells are in a long bacillary form of rods without bending, and grow with snapping and, later, break and split into a coccoid or short bacillary form.

(3) Motility: none (4) Spore: none (5) Gram staining: positive (6) Acid fastness: negative (7) Metachromatic granules: positive (b) Growth state in various culture mediums (at 300C) (1) Nutrient agar plate culture: Circular (13 mm in diameter), with solid edges, smooth, hemispherical, opaque with luster, slightly pink.

(2) Nitrient agar slant culture: Middle growth, filament-like, surface-smooth, convex, with luster, slightly pink.

(3) Bouillon liquid culture: Vigorous growth with forming pellicle, middle-degree turbidity with growth, forming a precipitate.

(4) Bouillon gelatin stab culture: Good growth on the surface, funnel-like growth along stab, with almost no growth at the lower portion, no liquefaction of gelatin.

(5) Litmus milk: no change (c) Physiological characteristics (1) Reduction of nitrate: positive (2) Denitrification: negative (3) MR test: negative (4) VP test: negative (5) Indole production: negative (6) Hydrogen sulfide production: negative (7) Hydrolysis of starch: negative (8) Citric acid use: Koser's culture medium: negative Christiansen's culture medium: positive (9) Use of inorganic nitrogen source: Nitrate: positive Ammonium salt: positive (10) Pigment production: negative (11) Urease: positive (12) Oxidase: negative (13) Catalase: positive (14) Hydrolysis of cellulose: negative (15) Growth range: pH: 5-10; temp.: 5-370C (16) Oxygen relation: aerobic (17)0-Ftest F (18) Heat resistance (in 10% skim milk, at 72 OC for 15 minutes): none (19) Acid and gas production from sugar Acid production Gas production L-Arabinose + D-Xylose D-Glucose + D-Mannose D-Fructose + D-Galactose Maltose Sucrose Lactose Trehaiose D-Sorbitol + D-Mannitol + I nositol Glycerin + Starch Salicin B:Strain N-774 (a) Morphology (1) Shape and size of cells: (0.5-0.8) y x (2-5) y (2) Pleomorhism of cells; At the initial stage of culture, the bacterial cells are in a long bacillary form of rods without bending, and grow with snapping and, later, break and split into a coccoid or short bacillary form.

(3) Motility: none (4) Spore: none (5) Gram staining: positive (6) Acid fastness: negative (7) Metachromatic granules: positive (b) Growth state in various culture mediums (at 300C) (1) Nutrient agar plate culture: Circular (13 mm in diameter), slightly irregular, smooth with surface-drying tendency, flat, opaque, slightly pink.

(2) Nutrient agar slant culture: Middle growth, filament-like, surface-smooth, convex with drying tendency, slightly pink.

(3) Bouillon liquid culture: Vigorous growth with forming pellicle, slight turbidity, forming a precipitate with growth.

(4) Bouillon gelatin stab culture: Good growth on the surface, funnel-like growth along stab, with almost no growth at the lower stab portion, no liquefaction of gelatin.

(5) Litmus milk: no change (c) Physiological characteristics (1) Reduction of nitrate: positive (2) Denitrification: negative (3) MR test: negative (4) VP test: negative (5) Indole production: negative (6) Hydrogen sulfide production: negative (7) Hydrolysis of starch: negative (8) Citric acid use: Koser's culture medium: negative Christiansen's culture medium: positive (9) Use of inorganic nitrogen source: Nitrate: positive Ammonium salt: positive (10) Pigment production: negative (11) Urease: positive (12) Oxidase: negative (13) Catalase: positive (14) Hydrolysis of cellulose: negative (15) Growth of range: pH: 5-10; temp.: 1 01C00 C (1 6) Oxygen relation: aerobic (17)0-Ftest: F (18) Heat resistance (in 10% skim milk, at 720C for 1 5 minutes): none (19) Acid and gas production from sugar Acid production Gas production L-Arabinose + D-Xylose D-Glucose + D-Mannose + D-Fructose + D-Galactose Maltose + Sucrose Lactose Trehalose + D-Sorbitol + D-Mannitol + I nositol Glycerin + Starch Saiicin + C: Strain N-775 (a) Morphology (1) Shape and size of cells: (0.6-1.0) y x (5-15) ju (2) Pleomorphism of cells: At the initial stage of culture, the bacterial cells are in a long bacillary form with hypha-like appearance, and grow with branching and, later, break and split into a coccoid or short bacillary form.

(3) Motility: none (4) Spore: none (5) Gram staining: positive (6) Acid fastness: weakly positive (7) Metachromatic granules: positive (b) Growth state in various culture mediums (at 300C) (1) Nutrient agar plate culture: Circular (13 mm in diameter), irregular, smooth, in relief, opaque, slightly lustrous, slightly red.

(2) Nutrient agar slant culture: Middle growth, filament-like, surface-smooth, flat trapezoid cross section with slight luster, slightly red.

(3) Bouillon liquid culture: Vigorous growth with forming pellicle, transparent solution, slightly forming a precipitate with growth.

(4) Bouillon gelatin stab culture: Good growth on the surface, funnel-like growth along stab, with almost no growth at the lower stab portion, no liquefaction of gelatin.

(5) Litmus milk: no change (c) Physiological characteristics (1) Reduction of nitrate: positive (2) Denitrification: negative (3) MR test: negative (4) VP test: negative (5) Indole production: negative (6) Production of hydrogen sulfide: negative (7) Hydrolysis of starch: negative (8) Citric acid use: Koser's culture medium: positive Christiansen's culture medium: positive (9) Use of inorganic nitrogen source: Ammonium salt: positive Nitrate: positive (10) Pigment production: negative (11) Urease: positive (12) Oxidase: negative (13) Catalase: positive (14) Hydrolysis of cellulose: negative (15) Growth range: pH: 6-10;temp.: 10-400C (16) Oxygen relation: aerobic (17) 0-F test: 0 (18) Heat resistance (in 10% skim milk, at 72 OC for 15 minutes): none (19) Acid and gas production from sugar: Acid production Gas production D-Arabinose + D-Xylose + D-Glucose + D-Mannose D-Fructose + D-Galactose + Maltose Sucrose + Lactose Trehalose + Acidproduction Gas production D-Sorbitol + D-Mannitol + Inositol - - Glycerin + Starch - Salicin - To determine taxonomic positions of the bacteria based on the above-described bacteriological characteristics accordingto Bergy's Manual of Determinative Bacteriology, 7th ed. (1957) and 8th ed.

(1974), the strains N-771 and N--774 fall under the aerobic, Gram-positive, non-acid fastness and catalase-positive bacillary category forming no endo-spores and no flagella. From the fact that the bacteria are in a long bacillary form at the initial stage of growth, not showing filament-like appearance but showing snapping growth without branching and that the bacteria break and split into a coccoid or short bacillary form, it is clear that they fall under the category of Coryneform bacteria.In addition, comparison with the Coryneform bacteria described in the Bergy's Manual precludes the bacteria of the present invention from belonging to: (1) the genus Cellulomonas, because they do not have cellulosedecomposing ability, (2) the genusArthrobacter, because Gram-staining is not variable, (3) the genus Microbacterium, because they do not have heat resistance in 10% skim milk at 720C for 1 5 minutes, and (4) the genus Kurthia because they do not have flagella. Accordingly, it is concluded the bacteria of the present invention belong to the genus Corynebacterium.

The strain N-775 falls under the aerobic, Gram-positive, weakly acid fastness and catalasepositive bacillary category forming no endospores and no flagella. From the fact that the bacteria are in a long bacillary form at the initial stage of growth, showing hypha-like appearance, and grow with branching to break and split later in to a coccoid or short bacillary form, they are considered to beiong to the genus Nocardia.

In practicing the process of the present invention, all that is required is to select a microorganism having the abiity to hydrolyze acrylonitrile or methacrylonitrile or one of the above-described microorganisms, culture it for 2 to 3 days in the aforesaid manner, collect the bacterial cells from the culture solution by centrifugation, suspend the cells in water or physiological saline and subject acrylonitrile or methacrylonitrile to the action of the cells.

That is, the reaction is usually conducted with about 1 to 10 dry wt % of bacterial cells and 0.5 to 10 wt % of acrylonitrile or methacrylonitrile at a temperature ranging from about the freezing point of the medium to 300C, preferably from about the freezing point to 1 50C, at a pH of about 6 to 10, preferably about 7 to 9.

Additionally, upon reaction, it is preferable to subsequently add acrylonitrile or methacrylonitrile as their concentration in the system falls while limiting the concentration of acrylonitrile or methacrylonitrile in the system to a level of not higher than 2 wt %, since they possess a strong toxicity and would inhibit the enzymatic reaction.

During the reaction, the pH is preferably controlied to be in the range of about 7 to 9 by consecutively adding a caustic alkali, ammonia or the like or by previously adding a buffer solution to the system. pH values outside the above range would lead to further hydrolysis of the produced and accumulated acrylamide or methacrylamide to form by-products or would lead to reduction in stability of the cell enzyme. Thus, acrylamide or methacrylamide can be produced and accumulated with almost 100% conversion.

These microorganisms are used as immobilized cells; in particular, immobilized cells entrapped by a polyacrylamide and related polymer gels, are preferred.

The cell immobilization can be conducted by suspending the aforesaid microorganisms in a suitable aqueous medium (e.g., water, a physiological saline, a buffer solution, etc.) containing an acrylamide series monomer and a cross linking agent, adding a suitable polymerization initiator and a polymerization accelerator to the suspension, and conducting polymerization and gellation at about 0 to 300 C, preferably 0 to 1 50C, at a pH of about 5 to 10, preferably about 6 to 8. The content of microorganisms in the polymerization reaction solution depends upon the kind and the form of the microorganisms used, but it is usually about 0.1 to 50 wt %, preferably about 1 to 20 dry wt %.

The acrylamide series monomers used to immobilize the cells in the present invention include, for example, acrylamide, methacrylamide, etc. and, if necessary, ethylenically unsaturated monomers copolymerizable with them may be used in combination. The concentration of such monomers in the reaction should at least be at a level high enough to form gels as a result of the polymerization, and is usually about 2 to 30 wt %, preferably about 5 to 20 wt %, based on the reaction solution.

The cross-linking agents include N,N'-methylenebisacrylamide, 1 ,3-di-(acrylamidomethyl)-2- imidazolidone, etc. As the polymerization initiator and the polymerization accelerator, those which least jnhibit the activity of microorganisms are selected. Usually, potassium persulfate, ammonium persulfate, etc. are used as the initiator, and dimethylaminopropionitrile, triethanoiamine, etc. are used as the accelerator, each in an amount of about 0.01 to 10 wt %.

Thus, there can be obtained polymer gels containing bacterial cells. i.e., immobilized cells.

The reaction of the present invention using the above-described immobilized cells and the continuous column process described hereinafter enables one to obtain a highly concentrated acrylamide aqueous solution with extremely good industrial advantages through relatively simple procedures while stably maintaining the activity of cell enzyme for a long time.

That is, the continuous column process in accordance with the present invention comprises using one or a plurality of columns connected to each other in series, which are filled with the aforesaid immobilized cells in a density of about 0.3 to 0.5 g immoblized cells/cc. having been crushed to a suitable size (about 0.5 to 5 mm, preferably about 1 to 3 mm), continuously feeding an aqueous solution of acrylonitrile or methacrylonitrile via column inlet and, at the same time, continuously feeding acrylonitrile or methacrylonitrile at an intermediate stage or location before completion of the reaction in an amount soluble in the reaction solution. In more detail, where one column is used, a so-called sectional column having one or more feed inlets provided betwen the column inlet and the column outlet (one feed inlet per section), and which usually comprises a few sections, is preferable.An aqueous solution of acrylonitrile or methacrylonitrile is continously fed via the column inlet and, at the same time, acrylonitrile or methacrylonitrile is continuously fed via all the fed inlets. A suitable feed rate is usually about 0.1 to 1.5 g AN or MAN/g cell.hr., preferably 0.3 to 0.8 g AN or MAN/g cell.hr. The amount of acrylonitrile or methacrylonitrile fed via each feed inlet is such that the acrylonitrile or methacrylonitrile added is soluble in the reaction mixture. It is preferable that the concentration of acrylonitrile or methacrylonitrile in the reaction system is limited to a level of not higher than 2 wt % since as explained above at higher concentrations it begins to have a toxic effect and inhibit the enzymatic reaction.Feeding rates in respective inlets are not necessrily the same due to the differences in the rate of consumption of acrylonitrile or methacrylonitrile during the progress of the reaction, i.e., the rates will vary depending on whether the acrylonitrile or methacrylonitrile is soluble at that particular level.

Where two or more columns are used, they are connected to each other in series, and an aqueous solution of acrylonitrile or methacrylonitrile is fed via the column inlet of the first column, and acrylonitrile or methacrylonitrile is fed via the subsequent and successive column inlets(s) in the same manner as described above. Thus, there can be obtained a highly concentrated acrylamide or methacrylamide solution as an eluate from the second or final column.

Additionally, a more concentrated acrylamide or methacrylamide aqueous solution or crystals of acrylamide or methacrylamide can be obtained from the thus obtained acrylamide or methacrylamide aqueous solution of the present invention using conventional techniques. For example, the aforesaid reaction solution is treated, if necessary, with active carbon, ion-exchange resin, etc., and then concentrated under reduced pressure to obtain a more concentrated acrylamide or methacrylamide aqueous solution or crystals thereof.

The present invention will now be described in more detail by the following examples of preferred embodiments of the present invention which, however, should not be construed as limiting the present invention. Additionally, all parts and percents in the following examples are by weight. The reaction products such as acrylonitrile and methacrylonitrile, and by-products like methacrylic acid and acrylic acid were determined by means of gas chromatography.

EXAMPLE 1 40 parts of the washed cells of the strain N-771 (water content: 75%) prepared by aerobic culture using a culture medium (pH: 7.2) containing 1% glucose, 0.5% peptone, 0.3% yeast extract and 0.3% malt extract, 4.5 parts of acrylamide, 0.5 part of N,N'-methylenebisacrylamide and 40 parts of physiological saline were mixed to prepare a uniform suspension. To this suspension were added 5 parts of a 5 /0 dimethylaminopropionitrile aqueous solution and 10 parts of a 2.5% potassium persulfate aqueous solution, and maintained at 1 00C for 30 minutes to polymerize. The thus obtained massive, cell-containing gels were crushed into small particles and washed well with physiological saline to obtain 100 parts of the immobilized cells.

5 jacketed columns, 3 cm inside diameter, 25 cm in length, each filled with 40 g of the immobilized cells were connected to each other in series, and a 4.5% acrylonitrile aqueous solution (using a 0.05 M phosphate buffer; pH: 8.0) was allowed to flow down via the top of the column No. 1 at 1 00C at a flow-down rate of 50 ml/hr (SV .0.5 hr~'). Subsequently, 100 parts of the eluate was mixed with 4.5 parts of acrylonitrile, and allowed to flow down via the top of column No. 2 at a flow-down rate of 52.3 ml/hr (SV =. 0.53 hr-1). The eluate was then similarly allowed to consecutively flow down through column No. 3 while controlling the flow-down rate at 54.5 ml/hr (SV = 0.54 hr~t), column No.

4 at 56.8 ml/hr (SV =. 0.57 her~1), and column No. 5 at 59 ml/hr (SV = 0.59 her~1) for 48 hours. Thus, there was continuously obtained an eluate at a reaction ratio of 100%. Additionally, the acrylamide concentration in this eluate was 25.5%.

EXAMPLE 2 7 jacketed columns, 3 cm inside diameter and 25 cm in length, filled with 40 g of the immobilized cells prepared in the same manner as in Example 12 were connected to each other in series, and a 2.5% methacrylonitrile aqueous solution dissolved in 0.05 M phosphate buffer (pH: 8.0) was allowed to flow down through column No. 1 via the top thereof at a flow-down rate of 100 ml/hr (SV ='. 1.00 hurl). Then, 100 parts of the eluate was mixed with 2.5 parts of methacrylonitrile and allowed to flow down through column No. 2 via the top thereof at a flow-down rate of 103 ml/hr (SV =. 1.03 hr~').

The eluate was then similarly allowed to consecutively flow down through column No. 3 while controlling the flow-down rate at 105 ml/hr (SV 1.05 hurl), column No. 4 at 108 ml/hr (SV .1.08 hr'), column No. 5 at 110 ml/hr (SV 1.10 hr1), column No. 6 at 113 ml/hr (SV = 1.13 hr -1), and column No. 7 at 115 ml/hr (SV =. 1.15 hf1) for 48 hours.The reaction ratio in this eluate was 100%, and the concentration of methacrylamide in the eluate was 19.3% EXAMPLE 3 45 parts of the washed cells of the strain N-775 (water content: 78%) prepared by aerobic culture using a culture medium containing 1% of glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, 0.1% acetonitrile, 0.1% KH2PO4 and 0.05% MgS04.7H2O, 4.5 parts of acrylamide, 0.5 part of N,N'-methylenebisacrylamide and 40 parts of physiological saline were mixed to prepare a uniform suspension.To this were added 5 parts of a 5% dimethylaminopropionitrile aqueous solution and 10 parts of a 2.5% potassium persulfate aqueous solution, and maintained at 1 00C for 30 minutes. The thus obtained massive, cell-containing gels were crushed into small particles, and washed well with physiological saline to obtain 100 parts of the immobilized cells.

The immobilized cells were filled in a section column comprising several sections each of which had a volume of 100 ml and contained 40 g immobilized cells. A 2.5% methacrylonitrile solution (using a 0.05 M phosphate buffer; pH: 8.0) was continuously fed at a rate of 100 ml/hr via the top of the uppermost section while maintaining the temperature inside the column at 1 50C, and methacrylonitrile at a rate of 3 ml/hr was added via the tops of each of sections Nos. 2 to 7 for 48 hours to conduct the reaction. In this case, no methacrylonitrile was detected in the eluate from the bottom of the 7th section of the column. Thus, the reaction was 100%. The content of methacrylamide in this eluate was 19.3%.

EXAMPLE 4 40 parts of the washed cells of the strain CBS 717.73 (the strain described in Examples of USP 4,001,081) obtained by culturing in the same manner as in Example 12 (water content: 75%), 4.5 parts of acrylamide, 0.5 part of N,N'-methylenebisacrylamide and 40 parts of physiological saline were mixed to obtain a uniform suspension. To this were added 5 parts of a 5% dimethylaminopropionitrile aqueous solution and 10 parts of a 2.5% potassium persulfate aqueous solution, and the system was maintained at 1 00C for 30 minutes to polymerize. The thus obtained massive, cell-containing gels were crushed into small particles, and well washed with physiological saline to obtain 100 parts of immobilized cells.

5 jacketed columns, 3 cm inside diameter, 25 cm in length, each filled with 40 g of the immobilized cells were connected to each other in series, and a 4.5% acrylonitrile aqueous solution (using a 0.05 M phosphate buffer; pH: 8.0) was allowed to flow down through column No. 1 via the top thereof at a temperature of 1 00C at a flow-down rate of 50 ml/hr (SV = 0.5 hut1). Subsequently, 100 parts of the eluate was mixed with 4.5 parts of acrylonitrile, and allowed to flow down through column No. 2 via the top thereof at a flow-down rate of 52.3 ml/hr (SV .0.53 hurl).

The eluate was then similarly allowed to consecutively flow down through column No. 3 while controlling flow-down rate at 54.5 ml/hr (SV =0.54 hurl), column No. 4 at 56.8 ml/hr (SV 0.57 hr-l), and column No. 5 at 59 ml/hr (SV '.0.59 he1). Analysis of the eluate from column No. 5 48 hours after the initiation of the flowing down of the solution revealed an existence of a slight amount of acrylonitrile, and the concentration of acrylamide was determined to be 24.8%.

Claims (11)

1. A process for continuously producing a highly concentrated acrylamide or methacrylamide aqueous solution which comprises passing an aqueous solution of acrylonitrile and methacrylonitrile thrpugh one or more columns filled with immobilized bacterial cells having a nitrilase activity at a te çnperature of from the freezing point of the medium to 300C at a pH of 6 to 10 while feeding acrylonitrile and methacrylonitrile via one or more inlets intermediate the column inlet and outlet in an amount soluble in the reaction mixture.

2. A process as claimed in Claim 1, wherein at least two columns connected in series are used and an aqueous solution of acrylonitrile and methacrylonitrile is fed to the first column inlet while acrylonitrile and methacrylonitrile are fed via the second and subsequent column inlets.

3. A process as claimed in Claim 1 or 2 wherein said bacteria are selected from the genus Corynebacterium, the genus Nocardia, the genus Bacillus, the genus Bacteridium in the sense of Prevot, the genus Micrococcus and the genus Brevibacterium in the sense of Bergey.

4. A process as claimed in Claim 3, wherein said bacteria are of the genus Corynebacterium.

5. A process as claimed in Claim 3, wherein said bacteria are of the genus Nocardia.

6. A process as claimed in any one of Claims 1 to 5, wherein said bacteria are immobilized with a polymer gel.

7. A process as claimed in Claim 6, wherein said immobilized bacteria are entrapped with a polyacrylamide and related polymer gel.

8. A process as claimed in any one of Claims 1 to 7, wherein the reaction is conducted at a temperature of from the freezing point of the medium to 1 50C.

9. A process as claimed in Claim 1, wherein said bacteria are the strains N-771 or N-774 belonging to the genus Corynebacterium or the strain N-775 belonging to genus Nocardia.

10. A process for producing acrylamide or methacrylamide substantially as hereinbefore described in any one of Examples 1 to 4.

11. Acrylamide or methacrylamide when produced by the process as claimed in any preceding claim.