MXPA00012390A - Methods for the removal of unwanted monomer amide compounds from polyamide preparations - Google Patents

Abstract

The present invention discloses methods for the removal of an unwanted amide monomer compound from a polyamide or polymerized amide preparation at a pH of about 2 to less than 6, by conversion of the amide monomer compound to the corresponding acid compound using a pure culture of an induced microorganism strain capable of converting an amide moiety to an acid moiety.

Description

METHODS FOR THE REMOVAL OF UNWANTED MONCMAMAZING CCT LKJESTOS OF POLYAMIDE PREPARATIONS 1 . C amp o de l a i nv in ion. The present invention describes the methods for the removal of an unwanted monomer-amide compound from a polymerized amide or a polyamide preparation, for example, the removal of the unwanted acrylamide monomer from a polyacrylamide preparation at a pH of about 2 to less than 6. , using a pure culture of an induced microorganism strain capable of removing the monomer compound from the polymer preparation. 2. Background of the invention. Nitriles are extremely versatile compounds that can be used in the synthesis of a wide variety of compounds, including amines, amides, amidines, carboxylic acids, esters, aldehydes, ketones, imines, and hetyclics. One of the most important commercial nitriles is acetonitrile which is a common solvent, other nitrile compounds used as herbicides or in the synthesis Ref. No. 125585 of detergents or antiseptics. Another of the most important commercial nitriles is acrylonitrile, which is used to produce acrylamide, acrylic acid, acrylic fibers, copolymer resins and nitrile gums. Another method of producing acrylonitrile is by using the SOHIO / BP process, which causes ain. direct propene oxidation (propylene a / k / a) by ammonia vapors in the air in the presence of a catalyst (see generally, Acr yloni tri le, 1979, Process Economics Program Report, Stanford Research International, Menlo Park, CA Weissermel and Arpe, 1978, Industrial Organic Chemistry, Verlag Chemie Weinheim New York, pp. 266-270). The waste stream from this process has a complex mixture of nitriles, including dinitriles, amides and acids at very high concentrations. More particularly, the waste stream generally contains nitriles such as acetonitrile, citrate, succinone, and rhenium as well as acrylamide. In addition, the cyanide (s) to c: ncent r aci ons and / or high this / they are sometimes present. The waste stream generally contains high and / or variable concentrations of ammonium sulfate. This effluent of hazardous waste can not be released to the environment due to its toxicity and in the United States it is usually eliminated due to the good injection in depth in the sub-surface formations. Such an arrangement can not be considered to be a "treatment" of the waste stream, but preferably is analogous to the process of the practice of pouring urban solid waste into land depressions. Outside the United States, it is common practice to "treat" the residual current of acrylonitrile production by diluting the residual stream to a low total nitrile concentration of about 250 ppm or less and treating it with biological waste stream systems from the aerated with encional, that is, sewage sludge activated, then wet oxidation / air; that removes volatiles and partially oxidizes many of the constituents organic Such a "treatment" method is not suitable for the efficient removal of a waste stream of easy nitrile production for the following reasons: (1) moisture / air oxidation causes volatile compounds to be stripped, creating a problem of air emission; (2) the dilution of the residual current requires a great treatment of the residual current that facilitates the flow management and the large residence time required to obtain the appropriate "treatment"; and (3) the combination of moisture / air oxidation with the biological treatment when activating the sludge results in high treatment costs. The depth in a nitrile production industry for an efficient, cost effective, environmentally friendly method to place the effluent from the nitrile production plants. It is widely known that certain microorganisms are useful for converting a nitrile compound to its corresponding amide or biologically acidic compound. Both the scientific or patent literature contains numerous references describing the use of microorganisms converting nitrile for the production of specialty chemicals, for example, acrylamide and acrylic acid or acrylonitrile acrylate. See generally, Kobayshi et al., 1992, Trends Biotechnol. 10: 402-408. The microorganisms that convert nitrile have been shown to have activities that include, the nitrilase, which converts a nitrile compound to its corresponding amide compound; and amidase, which converts an amide compound to its corresponding acid compound. To the knowledge of the present inventor, however, in all these specialty chemical productions using microorganisms that degrade the nitrile, only a single compound has been used to induce the relevant activity and only a single nitrile compound that has been converted to produce a compound of desired specialty. 2. 1 MICROORGANISMS WHICH CAN USE A NITRILE COMPOUND The literature contains certain references which describe a number of microorganisms which can use a nitrile or amide compound as the sole source of carbon and / or nitrogen. For example, Asano, et al., 1982, agrie. Biol.

Chem. 46: 1165-117, describes an isolated strain of Arthrobacter which is capable of growing using acetonitrile as a single source of carbon and nitrogen. Nawaz et al., 1989, 43rd Purdue Ind. Waste Conf. Proc., Pp. 251-256 (Nawaz, 1989), describes the isolation of a Pseudomonas s aeruginosa strain which is capable of using various nitrile compounds, including acetonitrile, as a single source of carbon and energy. However, this strain is incapable of using other nitrile compounds such as acrylonitrile, acrylamide, benzonitrile and malonitrile. Nowaz et al., 1992, Appl. Envirom. Microbiol. 58: 27-31 (Nawaz), describes a Klebsiela strain pn e um i a NCTRI which, after acclimation using benzoyl and some other nitrile selected from the but ironit rilo, acetonitrile, gl ut ar on i t r i 1, propi oni t r i 1, succinoni t r i lo and methacrylonitrile. In contrast to the present method for induction which does not require the presence of an aromatic nitrile, the benzonitrile required for the microorganisms in order to induce the ability to degrade mixtures of benzonitrile and some other nitrile. Furthermore, and more importantly, in order to achieve the degradation of any of the nitrile mixtures, the benzonitrile must be present. Since the nitrile waste stream of a nitrile production does not contain benzonitrile, this organism and the method described by Nawaz may be completely impractical and, in fact, not operative to treat the waste stream. Chapatwala et al., 1993, App. Biochem. Biotech 39/40: 655-666, describes the isolation of a strain that is capable of using acetonitrile as a single source of carbon and nitrogen. However, there is no description of the use of any other compound containing nitrile by the strain. Narayanasamy et al., 1990, Indian J. Exp. Biol. 28: 968-971, describes the use of acrylonitrile, acetonitrile, acrylamide and acetamide by an Arthrobacter sp. individually. There is no indication that the bacterial strain is capable of degrading the dinitriles or a mixture of nitriles. O'Grady and Pembroke, 1994, Biotech, Letters 16: 47-50, describes the isolation of an Agrobacteria sp. And the ability of the isolated strain to use or break a number of nitrile compounds differently individually. There is no indication that the strain is isolated, it may be able to use or break a mixture of the compounds n i t * ~ i 1 o. Martinkova et al., 1992, Folia Microbiol. 37: 373-376, describes the isolation of several bacterial strains, including the strain Coryneba cteri wn p. 3B and the strain Agrobacterium radioba cter 8 '4/1 which is capable of using acetonitrilc as a single source of carbon and nitrogen. There is no description that the strains are capable of using any other nitrile compound or a mixture of nitrile compounds. Nawaz et al., 1994, Appl Environ, Microbiol. 60: 3343-3348, describes the isolation of a bacterium, tentatively identified as a Rh or do c o c c u s sp. of dirt contaminated with the alachlor herbicide. This bacterium has shown to be adept at using acrylonitrile as a unique source of carbon and nitrogen. Nawaz et al., 1993, Can. J. Microbiol. 39: 207-212, describes the isolation of Ps e u dom on a s sp. and Xa n t om on a s matofila, which can use acrylamide as a single source of carbon and nitrogen. Armitage et al., International Patent Publication WO 97/06248, published on February 20, 1997, discloses methods for producing an amidase by culturing a suitable microorganism in the presence of an amide or an amide precursor, such as a nitrile, or a mixture of the same, under continuous cultivation, carbon limiting conditions where the amide or precursor forms amide at least 20% in mol and preferably all carbon. Suitable microorganisms include Pseudomonas, Rhodococcus, etc.

Also described are methods for producing a nitrilase by growing a microorganism in the presence of a nitrile or a nitrile precursor, or a mixture thereof, under limited culture conditions of continuous culture carbon. Suitable microorganisms include Nocardia, Rodococcus spp including Rodococcus ATCC 39484, etc. The induced enzyme is then used, inter alia, to convert a nitrile or amide to its corresponding acid. Although microorganisms which utilize a nitrile compound may possibly be useful for removing the unique nitrile compound from a nitrile-containing composition, the use of such nitrile to assist in the removal of the waste stream of equipment from a nitrile production is not expected since the use of nitrile is dependent on the expression of a specific nitrile hydrase or nitrile for the unique nitrile compound used. The expression of a specific nitrase or nitrile hydratase does not ensure that the microorganism will have the ability to convert the mixture of the nitrile compounds or the mixture of the nitrile and amide compounds present in high concentrations found in the waste stream of a nitrile production device . 2. 2 TREATMENT OF NITRILE WASTE THAT INCLUDES A RESIDUAL CURRENT OF A PLANT OF NITRILE PRODUCTION. A number of references describe attempts to provide a microbiological method for removing a residual nitrile, which includes the waste stream from a plant, from nitrile production. The U.S. Patent No. 3,940,332 for kato et al., (Kato) describes the use of an isolated bacterial strain, Nocardia rubr ope rti nc ta, ATCC accession No. 21930, in combination with activated sludge from a wastewater treatment plant to degrade a waste stream containing organic nitriles and cyanides. Kato also indicates that the bacterial strain is capable of degrading nitriles, which include acetonitrile, acrylonitrile, proto-ionyl, butyl 1 on itri 1, crotononi tri lo, smoking oni tri 1 or, valeronitrile, glu t aroni tri lo, and benzonitrile, although no indication is given of the amount of each of the nitriles or conditions under which the nitriles are degraded or if the nitriles can be degraded together. There is no indication that the strain described by Kato can degrade or degrade a mixture of nitrile compounds. In addition, the residual nitrile treated by kato was a residue of low hardness, 50-250 ppm of the total nitrile concentration. In addition, the present inventor has tested the strain described by kato and found that it does not remove acetonitrile from a mixture of nitriles with the same efficiency as can be realized by using the methods of the present invention. Sunarko and Meyer, 1989, DECHMA Biotech, conf. 3: 859-862, describes that the lyophilized cells of Myc oba c t e ri um UB T5, Ba c i l l u s UB T2, Coryn eba cte ri um UB T9, and Fl ex was ct er UB T4, which have been induced by the growth of cells in the presence of 2 -pent enoni tri lo, are able to degrade small amounts of acetonitrile found in the effluent from the HPLC column of the laboratory. Brown et al., 1980, Water Res. 14-775-778, discloses that acrylamide nails at concentrations of 0.5 ppm to 5 ppm in natural and contaminated water from the environment results in the degradation of acrylamide. Kincannon et al., 1983, Journal WPCF 55: 157-163, discloses that a mixture of microorganisms isolated from a municipal activated sludge water treatment plant is capable of degrading acrylonitrile after one month of acclimatization. In addition, the authors also show that acrolein can be similarly degraded by the mixture of microorganisms. Donberg et al., 1992, Environ. Toxicol Chem. 11: 1583-1594, describes that a mixture of microorganisms found in the sludge is capable of degrading acetonitrile under conditions aerobic Some mixtures are able to degrade from 10-100 ppm of acrylonitrile in the order of 2 days. However, at high concentrations of acrylonitrile (1000 ppm), degradation is inhibited. The authors speculate that the inhibition is due to inhibitory effects of the main acryl compound. Knowles and Wytt, European Patent No. 274, 856 Bl and Wyatt and Knowles, 1995, Biodegradation 6: 93-107, describe the degradation of a mixture of nicrilo and amide compounds from the waste stream of a production plant. of nitrile (using the BP / SOHIO process of the production of acrylonitrile) by a mixture of microorganisms. The use of a mixture of microorganisms preferably of. a pure crop, is a serious disadvantage of Knowles and Wyatt's method. It is difficult to maintain a mixed culture for the reaction change conditions, certain strains within the mixed culture will be favored at different times to grow so that the efficiency of the degradation can decrease.

There are a number of disadvantages associated with the above references. For example, many of the individual microbial strains described above have a single limited range of the nitrile or amide compounds in which they can degrade. The time required for the degradation / utilization of the nitrile and amide compounds is in the order of days or weeks. Furthermore, the removal by the treatment of traditional activated sludge has its own disadvantages, such as the large amount of biomass produced, which will eventually be eliminated. In addition, and more importantly, the use of a mixture of microorganisms preferably of pure cultures, makes it more difficult since it is difficult to keep the culture mixed. At the reaction change conditions, certain strains within the mixed culture will be favored at different growth times, as the time over the characteristics of the mixed culture will change and the degradation efficiency may decrease. The mixed culture is not easily reproduced or maintained. 2. 3 TREATMENT OF POLYACRILAMIDE PREPARATIONS TO REMOVE THE MONOMER ACRYLAMIDE. A number of references describe attempts to provide a microbiological method for removing an unwanted acrylamide monomer from a polyacrylamide preparation. Polyacrylamide polymers are widely used in several industries, including wastewater treatment, papermaking, mining industry, and biological and chemical research. However, their usefulness is restricted and can not be used in relation to foodstuffs since they are generally contaminated with the unreacted acrylamide monomer which is a cumulative of neurotoxins and a carcinogen. One method to reduce the amount of monomer that did not react is the "heat treatment" method. However, this treatment increases the cost of manufacturing and decreases the efficiency of polymer production due to unwanted branching of the polymers.

Another method is the use of the amidase obtained from a microorganism. For example, Carver et al., European Patent Publications EP 272,025 A2 and EP 2072,026 A2, describe the decomposition of acrylamide in polyacrylamide preparations by using an amidase obtained from an organism such as Me t hy lophi 1 is sp . which has been heated to a temperature in the range of 40 ° C to 80 ° C. However, the methods described by Carver et al., Does not allow for the removal of the acrylamide monomer from a cationic polyacrylamide preparation at its original pH value, that is, an original pH value of about 4. See Figure 6 in EP 272025 A2 which clearly shows that in order for the acrylamide monomer to be removed from the cationic polymer, the pH must be high from a pH of 4 to a pH of 6. US patents Nos. 4 687 807 and 4 742 114 to Westgrove et al. Describe the production of water-in-oil emulsions of the amidase for use in the removal of the unwanted acrylamide monomer from a preformed acrylamide polymer.

Farrar et al., European Patent Publication EP 329,325 A2, describes the removal of the acrylamide monomer from polyacrylamide using an aqueous amidase gel obtained from an amidase expression microorganism. Ferrar, International Patent Publication WO 92/05205, discloses the reduction of the residual methacrylamide monomer in a polymerized methacrylamide by incorporating the amidase into a polymerizable mixture for the exothermic polymerization of the polymethyl methacrylamide. Armitage et al., International Patent Publication WO 97/06248, published on February 20, 1997, also discloses methods for the conversion of methacrylamide to ammonium methacrylate in or after the polymerization of acrylamide using an amidase which has has been induced in a suitable microorganism obtained by culturing the microorganism in the presence of an amide or an amide precursor, such as a nitrile, or a mixture thereof, under a continuous culture, carbon limiting conditions wherein the amide or the amide precursor way at least % mol and preferably and substantially all the carbon. Suitable microorganisms include Ps e u dom on a s, Rh odo co c c u s, etc. However, examples are not provided where the enzyme induced effectively converts the monomer acrylamide into a polyacrylamide preparation, and furthermore, there is no statement regarding the pH range in which the conversion reaction can take place. The mention or identification of any reference in section 2 or in any section of this application may not be construed as an admission that such reference is available as the prior art to the present invention.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides methods for removing unwanted monomer amide from polyamide or polymerized amide preparations, for example by removing the acrylamide monomer from a polyacrylamide preparation. An illustrative method causes contact with a polyacrylamide preparation containing the monomer acrylamide, said polyacrylamide preparation having a pH of about 2 to less than 6, with a pure culture or a crude extract of a microorganism strain which has been induced multiple times for a sufficient period of time to reduce the amount of the acrylamide monomer by converting the acrylamide to the corresponding acrylamide. Preferably, the polyacrylamide preparation has a pH of from about 2 to about 5, more preferably a pH of from about 3 to about 4. Also preferably, the amount of the acrylamide monomer in the po-1-amide preparation is reduced to less than 100 ppm. A strain of microorganisms useful for removing an unwanted monomer from a polyamide preparation can be induced multiple times by cultivating a pure culture of a strain of my organism in a nutritionally complete medium containing a mixture of nitrile compounds or a mixture of nitrile and amide compounds. Alternatively, a strain of microorganism useful for removing amide monomer unwanted polyamide preparation can be induced multiplely by cultivating a pure culture of a microorganism strain in a minimal medium containing a mixture of nitrile compounds or a mixture of nitrile and amide compounds as the sole source of carbon and energy and / or nitrogen. The methods for multiple induction do not require the presence of an aromatic nitrile. The pure cultures of the microorganisms can be stored for prolonged periods of time or after induction, for example, at least 4 months under normal cooling temperatures, ie, close to 4 ° C, or more (years) under conditions of freezing temperature, that is, -20 ° C or less or when freeze drying or cr i ocons e rvac ion is employed, without the loss of amide removal activity. Certain microorganisms are capable of using at least one of the nitrile compounds as a single source of carbon and energy. Certain microorganisms are capable of using at least one of the nitrile or amide compounds as a single source of carbon and energy and nitrogen.

According to the present invention, once induced, the culture of pure microorganisms will not have to be actively divided or they will be alive for the removal to occur. This decoupling of growth from the removal allows a rapid removal of the unwanted monomeroamide compounds under growth inhibitory conditions; for example, under very high concentration (s) of nitrile compounds, highly alkaline or with an acidic pH, high temperatures, for example, at 55 ° C, etc. Besides decoupling: (1) it seems that since the cells do not need to grow to remove the monomer amide, the cells (biomass) are not produced or their production is minimized, when the cells are growing, it becomes necessary to distribute with the residual biomass and it is created; (2) Since the cells are not growing, compounds that could not serve as growing substrates can be removed by chance; and (3) the process results in the production of less toxic intermediaries which can easily be degraded naturally (reduction in the rate of citric acid as well as in toxicity). 3. 1 DEFINITIONS As used in this application, a "nitrile" compound is intended to encompass an organic compound containing one or more nitrile radicals, ie, C = N and at least one carbon atom in addition to the radical C = N. As used herein, the term "nitrile" includes compounds such as acroleic cyanohydrin. It can be seen that acrolein in the presence of reactive cyanide exists in the form of cyanohydrin acrolein. The nitrile oxygenated microorganisms are capable of converting a nitrile which is a cyanohydrin to a corresponding acid. For example, the cyanohydrin acrolein is converted to acrylic acid. As used herein, the term "nitrile" includes, but is not limited to, acetonitrile, acrylonitrile, fumaric acid, succinone, or trichloride, adipone trile, benzonitrile, but i roni tri 1 or , ß-propriosulfononitrile, isovaleronitrile, valeronitrile, fenilni trilo, acroleina, cyanohydrin, etc. When used in this application, an "anionic" polyamide preparation is a polyamide preparation containing an anionic backbone and / or side chain group (s) and having a negative net charge. As it is used in this application, an amide "cathonic" is a polyamide preparation containing a cationic main chain and / or group (s) of side chains having a positive net charge. As used in this application, a "nonionic" polyamide preparation is a polyamide preparation that does not contain either an anionic and / or cationic backbone and / or side chain (s), or if one or more such groups is present with a neutral net charge. 3. OBJECTIVES OF THE INVENTION It is an object of the present invention to provide methods for the removal of a unwanted monomer amide of a polyamide preparation at a pH of about 2 to less than 6 by converting the monomer amide to the corresponding acid compound. Other objects and / or advantages of the present invention will be apparent to those skilled in the art. 4. DETAILED DESCRIPTION OF THE INVENTION The present invention includes methods for the removal of an unwanted amide monomer from a polyamide or polymerized amide preparation at a pH of about 2 or less than 6 by the conversion of the monomer amide to the corresponding acid compound using a culture. Pure of a strain of microorganism induced to be able to convert the monomeroamide radicals to acidic redicales. The removal of an amide monomer from the preparation can be monitored by evaluating the fading of the monomer amide compound and / or the concurrent appearance of the corresponding acid compound by any method known to those skilled in the art, by example, when using gas-liquid chromatography with a flame ionization detector (GLC-FID) to detect the amide and high pressure liquid chromatography (HPLC) to detect the corresponding acid compound. The conversion to the corresponding acids results in the stoichiometric production of ammonia for each original amide group present. The area of amide compound conversion can be monitored by measuring the release of ammonia used in the technique of Fewcett and Scott, 1960, J. Clin. Pathol. 13: 156-159. If the release of ammonia can not be measured due to the presence of ammonia or an ammonia salt, the concentration of the amides present can be monitored by methods known to those skilled in the art, including but not limited to, GLC. -FID, etc. alternatively, the fading of the amide compounds can be measured by valuing the production of the corresponding: acid compounds which can be monitored by derivatizing the acid compound and by detecting the product derivatized when using GLC-FID. The amides and acids can be derivatized by analysis when using GLC-FID if it is first alkylated esterified or salicylated. (see generally, Supelco, Chormatography Products Catalog, 1997 on pages 653-656 (Supelco Inc., Bellefonte PA)). The acid and other non-polyamide compounds can then, if desired, be degraded to C02, H20 and bromas. For clarity of the description and not by way of limitation, the detailed description of the invention is divided into the following sections: (1) Methods for the induction and identification of strains of microorganisms that have an ability to remove the amide; (2) Characterization of the isolated microorganism strain; and (3) Applications or methods of use of the strains of microorganisms induced for the emotion. 4. 1. METHODS FOR THE INDUCTION AND IDENTIFICATION OF STRAINS OF MICROORGANISMS THAT HAVE ABILITY TO REMOVE AN AMIDA Strains of microorganisms useful for the removal of an unwanted amide monomer compound from a polymerized amide or polyamide preparation can be isolated and obtained by the growth of a strain of microorganisms in the presence of a mixture of nitrile compounds or a mixture of Amide and nitrile compounds. Surprisingly, it has been discovered that, in addition to inducing a broad amide removal ability, that is, the ability to remove a variety of monomer amides, the culture medium has been supplemented with more than one nitrile compound. More particularly, it has been discovered that the ability to remove a broad spectrum of monomer amides can be induced using a mixture of amide and nitrile compounds. In this way the strains are "multiple induction". After multiple induction, the strains, in the pure culture, are able to removing an unwanted amide monomer compound from a polymerized amide or polyamide preparation. Strains of microorganisms selected for sustained-induction are selected from known sources or can be recently isolated microorganisms, and they can be thermophilic or other ex remo roat, such as halophilic or acidophilic. Advantageously, the multiple induction methods do not require an aromatic nitrile, such as benzonitrile. It has been noted that microorganisms which can be of multiple induction have a monomer amide that removes the activity appears for growth slowly in a medium containing a nitrile or amide as the isolated source of carbon or as the isolated source of carbon and nitrogen when compared to growth in a medium containing an easily usable carbon source such as a carbohydrate, etc. Preferably, a strain of microorganism which has been induced by multiple induction having a monomer-amide removal activity is cultured in a medium containing a source of readily usable carbon (such as glucose, maltose, sucrose, acetate, benzoate, etc.). The carbon and nitrogen source is added incrementally and continuously as the carbon and nitrogen levels in the reactor are below 1%. In this way large amounts of cells are produced quickly and easily. When the desired cell mass has been carried out, then the multiple induction microorganisms according to the methods described in section 4.1.1 or 4.1.2 below, for example, for at least 20 hours of the production cycle. 4. 1.1 INDUCTION USING NITRILE OR AMID COMPOUNDS AND NITRILE AND A COMPLETE MEDIUM NUTRITIONALLY. A method for the induction of the monomer-amide removal activity comprising using nutritionally complete culture medium supplemented with a mixture of nitrile compounds or a mixture of amide and nitrile compounds. More particularly, the method comprises a microorganism in a complete medium nutritionally supplemented with a mixture of nitrile compounds or a mixture of amide and nitrile compounds and collect the culture of microorganisms. When growing on agar plates, the microorganisms are cultured for about 24 to 48 hours in the presence of a mixture of nitrile compounds or a mixture of amide and nitrile compounds. When grown in a fermentor, the microorganisms are cultured, in a complete nutritional medium, from 1 to 48+ hours, before the addition of a mixture of nitrile compounds or a mixture of amide and nitrile compounds; then the collection of 4 to 5 hours after the addition of the mixture of nitrile compounds or mixtures of amide and nitrile compounds. If a large biomass is desired, the microorganisms can be cultured in the fermenter for long periods of time before the addition of the nitrile mixture of the mixture of amide and nitrile compounds. As is known to those skilled in the art, one can add additional nutrients, as necessary for growth. In an alternative method, the microorganism is induced in a complete medium nutritionally supplemented with a mixture of nitrile compounds. Useful mixtures of nitrile compounds include the following: acetonitrile from about 50 to about 5C3 ppm; acrylonitrile from about 50 to about 500 ppm; and succinonitrile from about 25 to about 100 ppm. Optionally, about 1-10 ppm of KCN or NaCN can be added to the mixture. Although not intended to be limited to any particular mechanism, the inventor believes that the presence of KCN or NaCN during induction acclimates microorganisms to inorganic cyanide. Also optionally, cobalt is added to the mixture in a concentration of about 1-25 ppm. Also optionally, urea in a concentration of about 1-10 g / 1 may be added to the mixture. Preferably, a complete nutritional medium is supplemented with a mixture of nitrile compounds containing a mixture of therefore, an acetonitrile and an acrylonitrile in a concentration of about 150 ppm each and succinoni trilo and smoking oni t r i 1 o at a concentration of about 50 ppm each. More preferably, the nitrile mixture comprises acetonitrile and acrylonitrile of about 150 ppm each and succinone t r i 1 o at a concentration of about 50 ppm. Optionally, KCN and cobalt in a concentration of about 10 ppm each and urea in a concentration of about 7 g / 1 are added to the culture medium. Equally more preferable, a nutritionally complete medium which is a BACTO ™ R2A medium (Difco, Deitroit, Michigan) or the YEMEA medium or a medium containing the proportion of YEMEA components without agar is supplemented with acetonitrile and acrylonitrile of about 150 ppm each and ucc i non itri lo in a concentration of about 50 ppm, KCN and cobalt of about 10 ppm each, and urea of about 7 g / 1. In another alternative method, the microorganism is induced in a complete medium nutritionally supplemented with a mixture of amide and nitrile. Useful mixtures of amide and nitrile compounds include the following: (1) at least one of succinonite from about 50 to about 150 ppm and acrylonitrile from about 50 to 150 ppm; and (2) acetamide of about 50 to 500 ppm and acrylamide of about 50 to 500 ppm. Optionally, KCN or NaCN of about 1 to 10 ppm can be added. Also optionally, cobalt from about 1 to about 25 ppm may be added and urea from about 1-10 g / 1 may be added. Preferably, a complete nutritional medium is supplemented with 50 ppm of s ucc i noni t r i 1 o and acetamide and acrylamide of 150 ppm each. A complete nutritional medium is a growth medium which provides the microorganisms with all the necessary nutrients required for their growth, for example, carbon, and / or nitrogen. For example, and not for limitation, the BACTO ™ R2A medium (Difco, Detroit, Michigan) which contains glucose, peptone, KH2P04, MgSO4, casamino acids, yeast extract, soluble starch and sodium pyruvate. it is a complete nutritional means. Another nutritional complete medium for use in the present invention is the YEMEA medium which contains glucose, malt extract and yeast extract without agar. Another nutritionally complete medium for use in the present invention is a nutritionally complete medium containing glucose, peptone, KH2P0, MgSO4, soluble starch and sodium pyruvate. Any complete means nutritionally known to those skilled in the art can be used. The microorganism is cultured under conditions that include a pH between 3.0 and 11.0, preferably between about 6.0 and 8.0; and the temperature between 4 ° C and 55 ° C, preferably between about 15 ° C and 37 ° C. In addition, the dissolved oxygen tension should be between 0.1% and 100%, preferably between about 4% and 80%, more preferably between about 4% and 30%. The dissolved oxygen tension can be monitored and maintained in the desired range by complementary oxygen in the form of air, pure oxygen, peroxide, and / or other peroxy compositions which release oxygen. At the end of the cultivation period, the cultivated microorganisms are collected and concentrated, for example, by schami ent, centrifugation, filtration, etc. or by any method known to those skilled in the art. In an exemplary method, the cells are collected at 4 ° C, prepared as cell concentrates and then rapidly frozen (dry ice and acetone) and then stored at -20 ° C or less. The frozen cell concentrate can be used or can then be immobilized. The activity of ammonium monomer removal of the microorganisms, once of multiple induction according to the methods described above, can be stabilized by the addition of one or more substrates to the cultured microorganisms. Further, it is not intended to be limited to any mechanism, the inventor's notes that are well known to those skilled in the art that the Amidase enzymes are generally more stable in the presence of a substrate. Thus, for example, the addition of an acidic compound, such as isobutyric acid, can stabilize an amidase such that the activity is retained for long periods of time. The stabilization can also be carried out by the immobilization of the microorganism induced in acrylamide or polyacrylamide cubes or in an alginate which has been degraded with a polyethylene imide. Preferably, the cells are stabilized by immobilization in a degradable alginate with a polyethylene imide. 4.1.2. INDUCTION USING NITRILE OR AMID AND NITRILE COMPOUNDS AND A MINIMUM HALF. Another method for the induction of the monomer-amide removal activity comprises the use of a minimum culture medium supplemented with a mixture of nitrile compounds or a mixture of amide and nitrile compounds. More particularly, the method comprises culturing a microorganism in a minimal medium supplemented with a mixture of nitriles or a mixture of nitrile compounds and amide and collect the cultured microorganisms. When grown on plates with agar, the microorganisms are grown for about 24 to 48 hours. When grown in a fermentor, microorganisms are grown in a minimal medium supplemented with a mixture of nitrile compounds or a mixture of nitrile and amide compounds for 1 to 48+ hours, preferably 1 to 20 hours, more preferably from 16 to 23 hours; then it is collected from 4 to 5 hours after the addition of the mixture of nitrile compounds or the mixture of nitrile and amide compounds. If a large biomass is desired, the microorganisms can be grown in the fermenter for long periods of time. Useful mixtures of the nitrile compounds include the following: acetonitrile from about 50 to about 500 ppm; acrylonitrile from about 50 to about 500 ppm; and succinonitrile from about 25 to about 100 ppm. Optionally, they can be added to a mixture of 1-10 ppm of KCN or NaCN, and optionally, can be added to the cobalt mixture in a concentration of about 1-25 ppm. Also optionally, urea in a concentration of about 1-10 g / 1 may be added to the mixture. Preferably, the minimal medium is supplemented with a mixture of nitrile compound containing at least one acetonitrile and acrylonitrile at a concentration of about 150 ppm each and succinoni tri lo and smoking oni tri lo at a concentration of about 50 ppm each . More preferably, the nitrile mixture comprises acetonitrile and acrylonitrile of about 150 ppm each and succinct it at a concentration of about 50 ppm. Optionally, KCN and cobalt in a concentration of about 10 ppm each and urea in a concentration of about 7 g / 1 are added to the culture medium. In an alternative method, the microorganisms are induced in a minimal medium supplemented with a mixture of nitrile and amide compounds. Useful mixtures of nitrile and amide compounds include the following: (1) at least one of succinoni is from about 25 to about 100 ppm, acetonitrile from about 50 to about 150 ppm and acrylonitrile from about 50 to 150 ppm; and (2) acetamide of about 50 to 500 ppm and acrylamide of about 50 to about 500 ppm. Optionally, KCN or NaCN of about 1 to 10 ppm can be added. Also optionally, cobalt of about 1-25 ppm may be added and urea of about 1-10 g / 1 may be added. Preferably, a minimum medium is supplemented with 50 ppm of succinoni tri lo and acetamide and acrylamide of 150 ppm each. The minimum medium is an incompletely nutritional medium which does not supply the microorganisms with organic carbon for their growth. Preferably, the minimum medium can be supplemented with compounds wherein the microorganisms can be used as a source of carbon and / or energy. For example, and not by any limitation, the Stanier minimum medium (Stanier et al., 1966, J. Gen. Microbiol. 43: 159-271) and saline buffer phosphate (PBS) is accepted as the minimum medium for use in the methods of induction.

The microorganism is cultured under conditions that include a pH between 3.0 and 11.0, preferably between about 6.0 and 8.0; and the temperature between 4 ° C and 55 ° C,. preferably between about 15 ° C and 37 ° C. In addition, the dissolved oxygen tension should be between 0.1% and 100%, preferably between about 4% and 80%, more preferably between about 4% and 30%. The dissolved oxygen tension can be monitored and maintained in the desired range by complementary oxygen in the form of air, pure oxygen, peroxide, and / or other peroxy compositions which release oxygen. At the end of the culture period, the cultivated microorganisms are collected and concentrated, for example, by discarding, centrifugation, filtration, etc. or by any method known to those skilled in the art. The removal activity of the monomer amide of the collected microorganisms, once induced multiply according to the methods described above, can be stabilized by the addition of one or more substrates to the cultivated microorganism. Although it is not intended to be limited to any mechanism, the inventor's notes are well known to those skilled in the art that amidase enzymes are generally more stable in the presence of a substrate. Thus, for example, the addition of an acidic compound, such as isobutyric acid, can stabilize an amidase such that the activity is retained for a long period of time. The stabilization can also be carried out by the immobilization of the microorganism induced in polyacrylamide and acrylamide cubes or in alginate which has been degraded with polyethylene imide. Preferably, the cells are stabilized by immobilizing a degradable alginate with a polyethylene imide. 4. 1.3 IDENTIFICATION OF USEFUL MICROORGANISMS According to one aspect, the present invention provides a method of microorganisms selected to identify and isolate microorganisms useful for removing monomeric unwanted amide compounds from polymerized amide or polyamide preparations. The method, in general, involves exposing a microorganism to be tested to the conditions, described above in sections 4.1.1 and 4.1.2, which are used for the removal ability of the amide induced mu 11 ipl i each and then to value the ability of the putatively induced "test" microorganisms to remove an unwanted amide monomer from a polyamide preparation. The method of selecting for microorganisms useful for removing an amide monomer from a polyamide preparation comprises culturing a test microorganism in a minimal or complete medium nutritionally supplemented with a first mixture of nitrile compounds (see sections 4.1.1 and 4.1. 2, above) of about 24 to 48 hours in dishes with agar under favorable growth conditions to obtain a causatively induced microorganism; and value the ability of the microorganism puttically induced to remove an amide monomer from a polyamide preparation by contacting said microorganism with a polyamide preparation containing an amide monomer, and monitoring the fading of the amide monomer, wherein the fading of the monomer of amide in the preparation indicates that the test microorganism has the ability to remove a monomer amide compound from a polyamide preparation. Preferably, the polyamide preparation comprises a polyacrylamide and a monomer acrylamide and the fading of the entire amide monomer in about 30 minutes indicates that the test microorganism has the ability to remove an amide monomer from the polyamide preparation. More preferably, the fading of all the amide monomer in the preparation of the polyamide in about 10 minutes indicates that the microorganism .i = ne the desired ability. If it is desired that the microorganism has the ability to remove an amide monomer in the presence of Ammonium sulfate, ammonium sulfate is included in the second mixture of about 1-8% ammonium sulfate. According to a preferred type of this aspect, the minimum or complete nutritional medium is complemented with a first mixture of nitrile compounds containing a mixture of at least one of the acetonitrile and acrylonitrile in a concentration of about 150 ppm each and succinoni tri 1 o and smoking oni tri 1 or at a concentration of about 50 ppm each. More preferably, the first mixture of nitriles comprises acetonitrile and acrylonitrile of about 150 ppm each and s uccinoni t r i 1 o at a concentration of about 50 ppm. Optionally, KCN is added at a concentration of about 10 ppm to the culture medium. According to another preferred way of this aspect, the minimum or complete medium is optionally supplemented with a first mixture of amide and nitrile compounds (see sections 4.1.1 and 4.1.2, above).

The nutritionally useful complete medium and minimum medium are described above in Sections 4.1.1 and 4.1.2. the test microorganism is cultured under conditions that include the pH between about 2.0 and 11.0, preferably between about 2.0 and less than 6.0; and the temperature between about 4 ° C and 55 ° C, preferably between about 15 ° C and 37 ° C. The extent of the removal of the monomer-amide compounds can be monitored by measuring the release of ammonium using the technique of Fawcett and Scott, 1960, J. Clin. Pathol. 13: 156-159. If the release of ammonium can not be measured by the presence of ammonium or an ammonium salt, the concentration of amides present can be monitored by methods well known to those skilled in the art, including but not being limited to, GLC-FID, etc. Alternatively, the fading of the amide compounds can be measured by evaluating the production of the corresponding acidic compounds which can be monitored by derivatization of the acidic compound and the detection of the derivative product when using GLC-FID. Amides and acids can be derived by GLC-FID analysis if first alkylated, etherified or silylated (see generally, Supelco Chromatography Products catalog, 1997, pages 653-656 (Supelco Inc., Bellefonte PA). MICROORGANISM STRAINS Strains of microorganisms, described below, isolated or obtained from known sources have been discovered by having the ability to remove a monomer-amide compound from a polyamide preparation by converting the monomer-amide compound to the corresponding acid compound after multiple induction as described above in section 4.1 Tables I and II below show certain characteristics of the specific strains of two strains Rh odo coccus, DAP 96622 and DAP 96253, derived from two strains obtained from American Type Culture Collection, Rockville, MD , ATCC Access No. 33278 and ATCC Access No. 39484, respectively, and which is discovered by having the ability to to remove a compound of amide monomer of a polyamide preparation by converting the amide monomer to the corresponding acid compound after multiple induction as described above. In an illustrative example, the two strains of Rh odo c or c c u s are multiply induced by using 50 ppm of succinoni t r i 1 or with or without 50 ppm KCN. Tests for carbohydrate utilization are improved using protocols described in Manual or f Methods for General Bacteriology, 1981, Philip Gerhardt, ed., Am. Soc. Microbiol., Washington, D.C. nitrile utilization tests are performed after the chains are induced by culturing the microorganism in a complete medium nutritionally supplemented with 150 ppm each of acetonitrile and acrylonitrile and 50 ppm of succinic acid. The current utilization test is performed in a minimal medium supplemented with a particular test compound (s) as a single source of carbon and / or energy.

Table I Strain Rhodococcus rhodochrous DAP 96622 USE OF CARBON AND NITROGEN Minimum Stanier medium with nitrile as the only carbon and nitrogen source USE OF CARBON SOURCES Minimum Stanier medium containing 1 g / 1 of ammonium sulfate USE OF CARBON SOURCES Minimum Stanier medium containing 1 g / 1 of ammonium sulphate but with 10 ppm of KCN USE OF CARBON SOURCES Minimum Stanier medium without ammonium sulphate but with 10 ppm of KCN Table II Strain Rhodococcus sp DAP 96253 * I means "intermediary" between sensitive resistant. USE OF CARBON AND NITROGEN Minimum Stanier medium with nitrile as the only carbon and nitrogen source USE OF CARBON SOURCES Stanier minimum medium containing 1 g / 1 of ammonium sulfate USE OF CARBON SOURCES Minimum Stanier medium containing 1 g / 1 of ammonium sulphate but with 10 ppm of KCN USE OF CARBON SOURCES Minimum Stanier medium without ammonium sulphate but with 10 ppm of KCN The following strains of microorganisms, characterized in the tables below, have been discovered by removing an unwanted amide monomer compound from a preparation upon conversion. the monomer of amide to the corresponding acid after multiple induction as described above. The isolation of these microorganisms is described in W096 / 18724. Briefly, over 200 separate isolated pure microorganisms are cultured from contaminated sludge from the same industrial site. All these pure isolates are combined and cultivated, aerobically, with a sludge-like material containing a mixture of aromatic compounds, no trio- romatics, halo-aromatics, aliphatics, and 1 to 1 . A culture mixture of microorganisms is recovered from the cultivated material and has been maintained in a BACTO ™ R2A medium (Difco, Detroit, Michigan). The mixed culture designated DAP-2, which is deposited with the American Type Culture Collection and assigned ATCC access No. 55644, degrades at least the following compounds or mixtures thereof: benzene, toluene, xylene, ethylbenzene , naphthalene, chlorobenzene, phenol, cresol, or tr-benzene, aniline, anthracene, tell me 1-f-ene-1, styrene, halonaphthalene, 2-, 3- or 4-chlorotoluene, 2-, 3- or 4-chlorobenzoate or, 1,3-di chlor oben zoa to, 1,2-, 1,3- or 1, 4 -dini t robbenzene, 1-chloro-3-nitrolbenzene, 1-chloro-4-n-t-benzene, 1- or 2-methyl-1-ene-1, pyrene, acenaphthalene, fluoranthene, phenanthrene, benzo- (b) - fluorant ene, diben zofuran, chrysene, catechol, m-toluic acid, cinnamyl acetate, vanillin, tr ans cinma ldehyde, mesitylene, salicylate, melamine, cyanuric acid, d - (-) - liminene, hexadecane, methanol, formaldehyde , and chlorine form. The following pure cultures are isolated and characterized from the mixed culture designated DAP-2 by the isolation of single colonies in a BACTO ™ R2A medium supplemented with 150 ppm each of neither t-benzene, naphthalene, and toluene.

Microorganisms DAP 623: DAP 623 is a mobile Gram negative rod, generally the only small rods, although some pairs are observed. The fouling may be different and there is some flocose mass formation. In addition, this body can use next: mesitylene, lactate, succinate, limonene, m-toluic acid, chlorobenzene, salicylate, 2-, 3-, and 4-chlorotoluene, 2-, 3-, and 4-chlorobenzoic acid, and 1,3-di chlor Obencene as a unique source of carbon and energy. The DAP 623 is deposited with the American Type Culture Collection and assigned ATCC Access No. 55722 and is also characterized as a coir. it is shown in Table III.

Table III DAP 623 Microorganism DAP 626: The DAP 626 is a variable gram rod which varies in size and occurs simple and in pairs. The growth in dishes with flagella is seen which indicates flagellar mobility. In addition, this organism can use the following: methysylene, lactate, succinate, limonene, cinnamyl acetate, catechol, m-toluic acid, chlorobenzene, 2-, 3-, and 4-chlorotoluene, acid 2-, 3-, and 4 - chlor oben zoi co, and 1,3-dichlorobenzene as a single carbon source and Energy. The DAP 626 is deposited with the American Type Culture Collection and assigned ATCC Access No. 55723 and is further characterized as shown in Table IV.

TABLE IV DAP 626 Microorganisms DAP 629: The DAP 629 is a mobile rod of negative gram, for little cocco-baci 11 to r and. Colonies appear white with a slight fluorescence when grown on a BACTO ™ R2A medium with agar. In addition this microorganism can use the following: f 1 uo rant r ene, methysilene, lactate, succinate, limonene, m-toluic acid, chlorobenzene, 2-, 3-, and 4-c 1 orot or luene, acid 2-, 3 -, and 4-chlorobenzoic, and 1, 3 -di c 1 or robenzene as a sole source of carbon and energy. The DAP 629 is deposited with the American Type Culture Collection and assigned ATCC Access No. 55726 and is also characterized as shown in Table V.

TABLE V DAP 629 Microorganism DAP 632: The DAP 632 is a thin mobile rod of variable gram, seen both simple and in pairs. The colonies have a creamy to yellowish appearance when growing on BACTOtm R2A agar. In addition, this organism can use the following: f luorant reno, acenaf taleno, methysilene, lactate, limonene, m-toluic acid, chlorobenzene, 2-, 3-, and 4-chloro-oluene, acid 2-, 3-, and 4 -chlorobenzoic, and 1, 3-di c 1 orobenzene as a sole source of carbon and energy. The DAP 632 is deposited with the American Type Culture Collection and is assigned ATCC access No. 55727 and is further characterized as shown in Table VI.

TABLE VI DAP 632 Microorganism DAP 115: The DAP 115 is a mobile rod of negative gram. The growth is observed in dishes with flagellum, which indicate if the mobility is flagellar. The colonies appear white when they grow on a BACTO ™ R2A agar, but appear yellow in the nutrient broth. In addition, this organism can use the following: benzo- (b) - f luorant reno, f luoran t reno, diben zof ur anus, aceña ft a loño, salicylate, lactate, succinate, glyoxylate, mesitylene, vanilino, limonene, acetate cinnamyl, catechol, m-toluic acid, chlorobenzene, 2-, 3-, and 4-cl or ot-oluene, 2-, 3-, and -chlorobenzoic acid, and 1,3-di-chloride or -benzene as a single source of carbon and energy. The DAP 115 is deposited with the American Type Culture Collection and is assigned ATCC access No. 55724 and is further characterized as shown in Table VII.

TABLE VII DAP 115 Microorganism DAP 120: The DAP 120 is a mobile rod of negative gram. The growth is observed in dishes with flagellum, which indicate if the mobility is flagellar. In addition, this organism can use the following: chrysene, pyrene, lactate, succinate, glyoxylate, salicylate, mesitylene, vanillin, limonene, cinnamyl acetate, catechol, m-toluic acid, chlorobenzene, 2-, 3-, and 4-chlor oto 1 ueno, acid 2-, 3-, and 4-cl oroben zoi co, and 1,3-dichlorobenzene as a sole source of carbon and energy. The DAP 120 is deposited with the American Type Culture Collection and is assigned ATCC access No. 55725 and is further characterized as shown in Table VIII.

TABLE VIII DAP 120 Table IX below shows that the pure cultures characterized above, isolated from the culture of mixtures designated DAP 2, are capable of growing in a minimal Stanier medium supplemented only with 150 ppm of each of acetonitrile and acrylonitrile. The cultures grow at 25-27 ° C, the colony size is determined after 14 days. The values presented were any of the 5 replicate colonies for each determination.

TABLE IX USE OF ACETO- AND ACR I LON I TR I LO growth mark as ++++ leafy, +++ good, ++ considerable, + modest, +/- scarce, - no growth. * growth of strain DAP 120 is very thin but rapidly expanded, therefore, precise quantification is not possible.

As demonstrated in Table IX, the strains are capable of using acetonitrile and acrylonitrile as sole sources of carbon and nitrogen. 4. 3. APPLICATIONS OR METHODS OF USE OF MICROORGANISMS FOR THE REMOVAL OF COMPOUNDS FROM MONOMERO AMIDA FROM POLYAMIDE PREPARATIONS. According to one aspect of the invention, a method is provided for removing a monomer amide in a polyamide or a polymerized amide preparation, wherein the preparation contains an unwanted or undesirable monomer amide, by converting the monomer amide to the corresponding acid compound monomeric, wherein the acid compound can be easily removed from the preparation, eg, by any chemical separation means known to those skilled in the art. For example, acrylamide can be converted to an acid Acrylic which is easily removed as ammonium salt. The method comprises putting into contact a polyamide or a polymerized amide preparation in contact with an unwanted monomer-amide compound with a pure culture of a useful microorganism strain., of multiple induction as described in section 4.1.1 and 4.1.2, for a period of time sufficient to convert the monomer amide to the corresponding acid. This method is particularly useful for purifying a polyamide or a polymerized amide preparation, such as a polyacrylamide preparation or an acrylamide containing a co-polymer. Any polyamide or polymerized amide preparation containing an unwanted monomer can be treated according to this aspect of the invention, which includes anionic, cationic, and nonionic polyamide preparations at a pH of from about 2 to less than 6, preferably , at a pH of about 3 to about 5, more preferably at a pH of about 3 to about 4. Advantageously, a polyamide cationic or polymerized amide containing a monomer amide can be treated at a pH of less than 6.0, preferably at a pH of about 2 to 4 and more preferably at a pH of about 3 to about 4, such that the groups of cationic side chains do not lose their functionality as possible if the pH is adjusted to a pH of about 6 or before removing the desired monomer. The strains of induced microorganisms may be growing, that is, actively divided or they may be latent, that is, not actively divided or not active. When the method links the use of an actively growing culture of microorganisms, the conditions for contact with a polyamide or a polymerized amide preparation should be such that bacterial growth is sustained. Such conditions include, for example, a pH between 2.0 and 11.0, preferably between about 2.0 and less than 6.0; the temperature between 4 ° C and 55 ° C, preferably between about 15 ° C and 37 ° C; the dissolved oxygen tension between 0.1% and 100%, preferably between about 4% and 80%, more preferably between about 4% and 40% of saturation wherein the oxygen can be supplemented by using an oxygen containing an oxygen release composition. Oxygen containing an oxygen release composition can be air, pure oxygen, peroxide, or other peroxy chemicals which can release oxygen or mixtures thereof. In addition, the culture medium can be stirred or can not be stirred by providing positive or negative dissolved oxygen tension. When the method causes the use of a culture of microorganisms which are not actively divided, the conditions for contact with a polyamide preparation containing the unwanted monomer amide may be such that the amide (enzymatic) removal activity is supported. . For example, the temperature is maintained between about 25 ° C and 55 ° C. The pH can be alkaline or acidic and is optimally maintained in the range of about 2 to less than 6. This particular aspect is possible since the removal of the monomer amide is not growth dependent, that is, once the activity of removal of the amide is induced, the microorganism does not need to grow to remove the monomer amide. This particular mode can be carried out under anaerobic conditions. The pure culture of a strain of microorganisms can be encapsulated or immobilized rather than allowing its free movement to allow collection and use again. In a preferred aspect, the microorganism strain is immobilized. The pure culture can be immobilized by absorption, electrostatic binding, covalent bonding, etc., on a solid support which helps in the recovery of the microorganisms from the reaction mixture. Suitable solid supports include, but are not limited to, granular activated carbon, compost, wood waste products, (eg, wood chips, chunks, shredded pallets or trees), alumina, ruthenium, iron oxide, ion exchange resins. (for example Amberlite ™ IRA-93 or IRA-96 (Rohm &Haas), DOWEX ™ (Dow Chemical Co., Inc.), DEAE cellulose, DEAE-SEPHADEX ™ (Pharmacia, Inc.), ceramic buttons, buttons of degradable polyacrylamide or cubes or other forms of gel, alginate buttons, K-carrageenan cubes as well as solid particles that can be recovered from aqueous solutions due to the inherent magnetic ability. The alginate buttons can be Ca ++ alginate buttons or hardened alginate buttons. K-carrageenan cubes can be hardened K-carrageenan cubes. As an illustrative example, a pure culture of an induced microorganism can be mixed with a solution of sodium alginate and calcium chloride to immobilize the microorganism in alginate buttons. In a preferred aspect, the induced microorganism is immobilized on alginate beads that have been degraded with the polyethylene imide or immobilized in a polyacrylamide type polymer. According to another aspect of the present invention, a method for the removal of an unwanted monomer amide from a polymerized amide preparation or polyamide comprises contact with an extract of a pure culture of a strain of microorganism useful, multiple induction of according to the present invention, with a polyamide or a polymerized amide preparation containing an unwanted monomer amide for a sufficient amount of time to convert the monomer amide to the corresponding acid. Preferably the extract is a crude extract of an individual microorganism. The extracts of the microorganism are prepared by ii.-. all known to those skilled in the art including but not limited to the following: cells from a sample of a pure culture of an induced microorganism are interrupted, for example, by sonication by compression, by using a French press, etc. . to produce cell lysate. The lysate is filtered and cefuged to remove the cell waste and unused cells, to produce a crude extract. The lysate can also be immobilized as previously described for all cells or immobilized by using known techniques for the use of enzyme immunoassays. In a particular illustrative aspect of the application of the invention described above the removal method is used to convert the unwanted acrylamide monomer to the acrylic acid in a polyacrylamide preparation. A pure culture, or an extract, of a strain of microorganism useful, of multiple induction and immobilized as shown here, is contacted with a polyacrylamide preparation containing the unwanted monomer acrylamide, said preparation having a pH of about 2 to less of 6, for a sufficient period of time, to reduce the amount of the acrylamide monomer in the polyacrylamide preparation, preferably to less than 100 ppm of the monomer acrylamide. Alternatively, a pure culture, or an extract of a multi-induction, useful microorganism strain is immobilized as shown herein, is contacted at a pH of about 2 to less than 6, with a monomer preparation of acrylamide simultaneously with an agent of polymerization (an agent that causes the monomer amide polymerizes acrylamide) so that the residual monomer acrylamide (after the polymerization reaction) is converted to the corresponding acrylic acid. Such agents of Polymerization include, but are not limited to agents such as persulfate, sulfate, bromate, chlorate, peroxide, or a mixture thereof, etc. The concetion of the acrylamide monomer in a polyacrylamide preparation can be as high as 100,000 ppm and is generally from less than 10,000 ppm to about 40,000 ppm when using the methods of the present invention, the acrylamide monomer levels are reduced to less than 1000 ppm, preferably less than 100 ppm. Preferably, the extract is a crude extract of an individual microorganism strain. The methods for preparing the extracts of the strains of the microorganisms are described above in this application. Preferably, a pure culture, or an extract of a microorganism described in more detail in section 4.2, is employed, as shown herein. Alternatively, a pure culture or an extract of a microorganism capable of removing a monomer-amide compound from a polyamide preparation that has been identified using the method (s) of The selection described above in section 4.1.3 is employed. The crude extract may optionally be further purified by purification methods of known enzymes in the field such as ion exchange column chromatography before contact. In a preferred aspect, the strain of the microorganism, or an extract thereof, is immobilized. The methods for immobilizing the strains of the microorganism, or extracts thereof, are described above in this application. More preferably, the induced microorganism, or extract thereof, is immobilized on alginate buttons that have been degraded with the polyethylene imide or immobilized on a polyacrylamide-type polymer or on Dowex ™ (Dow Chemical Co., Ine an ion exchange resin Alternatively, in another preferred aspect the microorganism or extract is immobilized on a flat surface of a material. A 'polyamide, for example, the polyacrylamide the preparation is passed over the flat surface that has the immobilized microorganisms or extracts from the same. In one aspect, the polyamide preparation is in an aqueous preparation. In an illustrative aspect the flat surface is in the form of a sheet or series of sheets of a material wherein an induced microorganism or extract is immobilized. In a preferred aspect the immobilized multiple induction microorganism or extract can be easily again by simply flowing a new preparation onto the flat surface and there is no need to recover the multiple induction microorganism or polyamide product extract. The immobilization allows a maximum retention and the use again of the microorganism or extract of the same, to remove more monomer, in this way decreases the amount of microorganism or extract thereof, when using and in this way, reducing costs. In addition, the need for mixing, i.e., in imparting the additional energy and the amount of microorganism or extract thereof, in the preparation of the polyamide is minimized. Also alternatively, the microorganism or extract thereof, is immobilized by contacting it with a preparation of the monomer amide together with the polymerization agent and a degradation agent, such that the microorganism, or extract thereof, becomes immobilized in the resulting polyamide preparation, where it removes the residual monomer amide. In addition, in the manufacture of the polyacrylamide preparations, the unwanted residual monomer-nitrile compounds may also be present and the removal method detailed above also allows the conversion of these monomer-nitrile compounds, in addition to the monomer-amide compounds present, to the corresponding compounds acids, which are removed in their salt form. Any method for contacting the strain of the induced microorganism with a composition comprising polyamides and monomeroamide compounds can be used according to the present invention. Such contact methods include, but are not limited to, a closed container, a container with an apparatus containing the induced strains, etc.

The methods of the present invention may further comprise monitoring the conversion of the monomer-amide compound to the corresponding acid compound by assessing the disappearance of the monomer-amide compound and / or the concurrent appearance of the corresponding acid compound by any method known to those skilled in the art. J .. technique, for example by using gas-liquid chromatography with a flame ionization detector (GLC-FID) to detect the amide, and liquid high-pressure chromatography (HPLC) to detect the corresponding acid compound. The conversion of the corresponding acid results in the stoichiometric production of the ammonia for each amide group originally present. The conversion stress of the amide compounds can be monitored by measuring the release of ammonia using the technique of Fawcett and Scott, 1960, J. Clin. Phatol. 13: 156-159. If the release of the ammonia can not be measured due to the presence of ammonia or an ammonium salt, the concentration of the amide present can be monitored by methods known per se. those with experience in the art, including, but not limited to, GLC-FID, etc. Alternatively, the disappearance of the amide compounds can be measured by assessing the production of the corresponding acid compounds which can be monitored by derivatizing the acid compound and by detecting the derivatized product when using GLC-FID. Amides and acids can be derivatized by analysis when using GLC-FID if they are first alkylated, esterified or silylated (see generally Supelco Chromatography Products catalog, 1997, at pages 653-656 (Supelco Inc., Bellefonte PA).

The polyamide or the polymerized amide preparations contain an unwanted monomer amide which may be in the form of solid, liquid and / or gaseous. When a polyamide preparation is in the gaseous and / or liquid form it can be absorbed onto a material such as a solid. The energy can be imparted, for example, by imparting mechanical energy, for example when mixing; when imparting acoustic energy, for example, by establishing a permanent sound wave in the fluid; or by imparting an electric or electrostatic field. At different time points one can remove the samples and measure the concentrations of the monomer amide or polyamide or polymerized amide compounds by methods known to those skilled in the art; for example, GLC-FID, etc. 4. 3.1. MODES OF OPERATION Methods for the removal of an unwanted monomer-amide compound from a polyamide preparation can be operated in a variety of modes, including batch mode, sequential batch mode, or continuous or semi-continuous mode, and flow through a biofilter.

In all modes of operation, samples of the contents can be periodically removed to monitor the removal of the compounds of interest. Additionally, agitation and / or mixing of the contents of the reactor can induce foaming. In In these cases, the anti-foam agent can be added to prevent the formation of foam. Suitable anti-foam agents include silicon containing an anti-foaming emulsion (e.g., Dow ANTIFOAM-A®, a silica based on an antifoaming agent). 4. 3.1.1. TYPE LOT OPERATION. The batch operation involves placing a fluid containing a monomer-amide compound in a container, such as a bioreactor, by inoculating with induced microorganisms as described in section 4.1 and incubating the mixture to culture the microorganisms so that the monomer compound Unwanted amide is removed. After a predetermined time, the incubation stops and the contents are removed and the solids, if any, are separated from the liquid by filtration.

The samples can then be taken both in the liquid and solid phases and examined, for example, by GLC-FID, to assess the level of the monomer amide compounds and to confirm that the monomeroamide compounds have been removed. The reactor solids are subsequently washed out and can be further processed in, for example, a controlled discharge or they can be used as a bacterial inoculum for the next batch. In the batch type the washed out solid residue is again added in about 2% to 40% by weight or volume, preferably in about 5% to 20%. Air or oxygen can be pumped into the reactor and the contents stirred, mechanically in the bioreactor. 4. 3.1.2 SEQUENTIAL LOT-TYPE OPERATION The sequential batch type is operated the same as the batch type except that after the incubation period has ended, the reactor is allowed to settle for a time, usually about 15 minutes, and 60% - 95% of the higher reactor contents are removed the established salient solids, if any, at the bottom as inoculum for the next batch of neutralized fluid. Preferably between 70% and 90% of the contents are removed. The sequential batch type in a preferred aspect for remove the fluids that contain an unwanted monomer amide since the acclimatization phase is reduced high levels of biomass are retained in the reactor, the variability in the composition of the residual feed is better accommodated, and the residual solids remaining after the biot are potentially reduced. 4. 3.1.3 SEMI-CONTINUOUS / CONTINUOUS TYPE The semi contiguous / continuous type is similar to the batch and sequential lot types. However, rather than stopping the incubation after a predetermined time, a fresh fluid containing an unwanted monomer amide is pumped into the bioreactor in a fixed amount over a given period of time as a treated fluid is removed from the bioreactor. This provides for a continuous fluid treatment without having to stop the removal process. 4. 3.2 BIOFILTERS Biofilters can be used for the removal of a monomer-amide compound from a Polyamide or polymerized amide preparation in effluents such as air, vapors, aerosols and water or aqueous solutions. For example, if the volatile monomer amide components are present, the volatiles can be stripped of the solid or aqueous solution where they are located and the interruption can be carried out so that lu. volatile are trapped in a biofilter. Once entrapped, the volatiles can be contacted with a pure culture or an extract of a multiple induction microorganism strain to remove the monomer-amide compound. A classic biofilter can be used, where air containing a polyamide preparation containing an unwanted monomer amide is passed through the biofilter. A biofilter of the percolator can be used, where the aiie and aqueous polyamide solutions containing an unwanted monomer amide are passed through the biofilter apparatus. The biofilLrcs may comprise an apparatus having a pure culture of a microorganism induced according to the methods described above, or an extract thereof, immobilized on a solid support. The microorganisms can be actively divided or not actively divided. The microorganisms can also be hydrolysed before the combination with the biofilter device. Suitable solid supports include, but are not limited to, granular activated carbon, manure, wood waste products (e.g., wood chips, gold nuggets, shredded pallets or trees) alumina, iron oxide, ruthenium, exchange resins ionic (for example, Amberlite ™ IRA-93 or IRA-96 (Rohm &Haas), DOWEX ™ (Dow Chemical Co, Inc.), DEAE cellulose, DEAE-SEPHADEX ™ (Pharmacia, Inc.), polyacrylamide button buttons, alginate buttons, K-ca rr ageenan cubes as well as solid particles that can be recovered from the solutions watery due to its inherent magnetic ability. Preferably, the solid support are alginate buttons that have been degraded with the polyethylene imide. Alternatively, the microorganism or the extract thereof can be immobilized in a flat surface of a material. The biofilter apparatus can have inlet and outlet holes, so that the material to be treated can flow through the apparatus. Preferably, the microorganism attached to the solid support is selected from the group consisting of microorganisms having ATCC access Nos. 55899, 55898, 55722, 55723, 55726, 55727, 55724, and 55725, which has been induced as described above. The biofilters can be used in a method comprising the flow of an effluent, containing a polyamide or a polymerized amide preparation and an unwanted monomer-amide compound, through a biofilter comprising an apparatus having a pure culture of a microorganism induced as described above, or an extract thereof, immobilized on a solid support. The method may also include monitoring the effluent to determine that the monomer-amide compound has been removed. Preferably, the microorganisms attached to the solid support are selected from the group consisting of microorganisms having ATCC access Nos. 55899, 55898, 55722, 55723, 55726, 55727, 55724, and 55725, which have been induced as described above. The following examples are presented for purposes of illustration only and are not intended to limit the scope of the invention in any aspect.

. EXAMPLE: IMMOBILIZATION OF THOSE CELLS IN POLYACRILAMIDE. Those cells of the Rh or do co c c u s strain, the DAP96253 is immobilized in po 1 i acr i a lmi da to fix the cells in a matrix. In addition, the polyacrylamide provides for mechanical integrity and strength which results in more effective retention of the cells in the reactor vessel and serves to stabilize the amidase activity in the cells. 10 grams of DAP 96253 of multiple induction cells, wet weight, are suspended in 40 ml of distilled water. The 40 ml of cells are added to 40 ml of distilled water containing 4.5 g of acrylamide and 0.5 g of sacrificial and 1 amide of N-, N-methylene. To the 80 ml of the solution, add 5 ml of α-dime thi 1 aminopropioni t and 10 ml of a 2.5% potassium persulfate solution and the resulting mixture is stirred vigorously and then placed on ice. After the solution is polymerized, the polymerized solution is cut into small cubes which are washed with distilled water to remove any unpolluted monomer. 6. EXAMPLE: REMOVAL OF CONTAMINATED ACRYLAMIDE MONOMER FROM A PREPARATION OF CATIONIC POLYACRILAMIDE. 6.1 USE OF AN IMMOBILIZED EXTRACT In a series of experiments, a multiple-induction crude cell extract of DAP 96253 cells, obtained as described in section 4.1 above, by culturing the cells in a complete medium nutritionally supplemented with 150 ppm acrylonitrile , 150 ppm of acetonitrile and 50 ppm of succinoni tri lo, are immobilized in resin of DOWEX ™ ion exchange (Dow Chemical Co., Inc.). The immobilized extract is added directly to a commercial cationic polyacrylamide preparation having a pH of about 3.5. The reaction is carried out by mixing the preparation with the immobilized extract for 2 hours at 25-27 ° C. The concentration of the residual acrylamide monomer in the preparation of the cationic polyacrylamide is reduced to about 50 to 60% after 2 hours. In another series of experiments, the multi-induction crude cell extract of DAP 96253 cells is obtained as follows. The cells are multiply induced as described in section 4.1, above, in a complete medium nutritionally supplemented with 150 ppm of acetonitrile, 150 ppm of acrylonitrile and 50 ppm of succinonium. Cells are harvested, washed and re-suspended in buffered saline phosphate. The multiple induction cells are used and a crude extract of the cells is obtained as described in section 4.1.

The crude extract is immobilized on a DOWEX ™ ion exchange resin (Dow Chemical Co., Inc.) as follows. One gram of the DOWEX ™ anionic rinsin is prewashed with 50% ethanol and then 150 μg of crude extract is contacted, letting it react and dosed twice.The immobilized crude extract is tested by the amidase activity by valuing the ability of the immobilized extract to convert acrylamide to acrylic acid at a pH of 7 and at 30 ° C. The immobilized extract exhibits an amidase activity of 20 units per gram (one unit equal to 1 μmol of acrylamide converted per minute at a pH of 7). , 30 ° C.) Approximately, 0.5 grams of the immobilized extract (10.3 units) are added directly to two preparations of sepedated commercial cationic polyacrylamide (Cytec Industries, Stamford, CT), each one having a pH of less than 4 and each one has a residual concentration of 1000-1100 ppm of onomeric acrylamide.The reaction is carried out by mixing the preparation with the immobilized extract and samples are taken at various time points for the determination of a Acrylamide concentration using gas liquid chromatography. The concentration of the residual acrylamide monomer in the first cationic polyacrylamide preparation is reduced to less than 1 ppm in 360 minutes. In the second preparation, the concentration of the monomer acrylamide is reduced to approximately 400 ppm in 360 minutes. In still yet another series of experiments, when this particular extract is not immobilized, this particular extract does not show a removal activity of monomer amide at a pH of less than 4. 6. USE OF IMMOBILIZED MICROORGANISMS The multiple induction cells, DAP 96253, are obtained as described in section 13 above, and are immobilized on DOWEX ™ ion exchange resin (Dow Chemical Co., Inc.). The immobilized cells are added directly to a commercial cationic polyacrylamide preparation, which has a pH of about 3.5. The reaction is carried out when mixing the preparation with the immobilized cells for a sufficient time, for example, 2 hours at room temperature, for example, 25-27 ° C, and the concentration of the monomer amide is monitored. 7. DEPOSIT OF MICROORGANISMS The following microorganisms are deposited on December 10, 1996 with the American Type Culture Collection (ATCC), Rockville, MD, and the indicated access numbers have been designated: The following microorganisms are deposited on November 30, 1995 with the American Type Culture Collection (ATCC) Rockville, MD, and have been assigned the access numbers indicated: The invention described and claimed herein is not limited in the field by the specific aspects described herein since these aspects are understood as illustrative of various aspects of the invention. Any of the equivalent aspects is understood to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art of the foregoing description. Such modifications are also understood to fall within the scope of the invention of the appended claims.

A number of references are cited here, the entire descriptions of which are incorporated herein, in their entirety, by reference.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property

Claims (27)

1. CLAIMS 1. A method for removing unwanted amide monomers from a polyamide preparation containing said monomer by converting the monomer amide to the corresponding acid, characterized in that it comprises the contact of a pure culture or an extract of a multiple induction microorganism, with a Preparation of polyamide containing said amide monomer, the polyamide preparation has a pH of about 2 to less than 6, for a sufficient time to convert the monomer amide to the corresponding acid.
2. 2. The method according to the rei indication 1, characterized in that the pure culture or extract is encapsulated or immobilized.
3. 3. The method according to claim 2, characterized in that the pure culture or extract is immobilized on a solid support selected from a group consisting of granular activated carbon, wood chips, alumina, ruthenium, iron oxide, buttons ceramics, alignato buttons, _ carragemna cubes, and ion exchange resins.
4. 4. The method according to claim 2, characterized in that the culture or extract is immobilized on a flat surface.
5. 5. The method according to claim 1, characterized in that the preparation of polyamide is a polyacrylamide preparation.
6. 6. The method according to claim 1, characterized in that the microorganism is selected from a group consisting of microorganisms having ATCC access Nos. 55899, 55898, 55722, 55723, 55726, 55727, 55724 and 55725 and which have been induction 11 ip 1 e.
7. 7. The method according to claim 1, characterized in that the Multiple induction microorganism is obtained by a method comprising culturing a pure culture of a microorganism in a culture medium supplemented with a mixture of nitrile compounds containing at least one of acetonitrile and acrylonitrile at a concentration of about 150 ppm each and of succinoni tri lo yf urna roni tri lo in a concentration of about 50 ppm each.
8. 8. The method according to claim 7, characterized in that the culture medium is supplemented with a mixture of nitrile compounds containing acetonitrile and acrylonitrile of about 150 ppm each and is about 50 ppm.
9. 9. The method according to claim 7, characterized in that the culture medium is further supplemented with about 1-10 ppm KCN or NaCN.
10. 10. The method according to claim 1, characterized in that the multiple induction microorganism is obtained by a method comprising culturing a pure culture of a microorganism in a culture medium supplemented with a mixture of nitrile and amide compounds containing: at least one s iuci i non itri lo of about 50 ppm, acetonitrile of about 150 ppm and acrylonitrile of about 150 ppm and (b) acetamide and acrylamide of about 150 ppm each
11. The method according to claim 10, characterized in that the culture medium is supplemented with a mixture of nitrile and amide compounds containing s ucc i non itri 1 or about 50 ppm and acetamide of about 150 ppm and acrylamide of about 150 ppm.
12. The method according to claim 10, characterized in that the culture medium is further supplemented with about 1-10 ppm of KCN or NaCN.
13. 13. A method for removing unwanted amide monomers from a polyamide preparation that it contains said monomer by converting the monomer amide to the corresponding acid, characterized in that it comprises contacting at a pH of from about 2 to less than 6, of a pure culture or extract of a multiple induction microorganism, with a monomer amide with polymerizable agent.
14. 14 The method according to claim 13, characterized in that the pure culture or extract is encapsulated or immobilized.
15. The method according to claim 14, characterized in that the pure culture or extract is immobilized on a solid support selected from a group - consisting of granular activated carbon, wood chips, alumina, ruthenium, iron oxide, ceramic buttons, alignato buttons, K-car r ageeana cubes, and ion exchange resins.
16. 16 The method according to claim 14, characterized in that the culture or extract is immobilized on a flat surface
17. The method of con fi rmity with claim 13, characterized in that the monomer amide is acrylamide.
18. 18. The method according to claim 13, characterized in that the microorganism is selected from a group consisting of microorganisms having ATCC access Nos. 55899, 55898, 55722, 55723, 55726, 55727, 55724 and 55725 and which have been induction 11 ip 1 e.
19. 19. The method according to claim 13, characterized in that the multiple induction microorganism is obtained by a method comprising culturing a pure culture of a microorganism in a culture medium supplemented with a mixture of nitrile compounds containing at least one of acetonitrile and acrylonitrile at a concentration of about 150 ppm each and of succ inoni t r i lo and smokes r oni t i o in a concentration of about 50 ppm each.
20. 20. The method according to claim 19, characterized in that the culture medium is supplemented with a mixture of nitrile compounds containing acetonitrile and acrylonitrile of about 150 ppm each and s ucc i non i t r i 1 of about 50 ppm.
21. The method according to claim 19, characterized in that the culture medium is further supplemented with about 1-10 ppm of KCN or NaCN.
22. The method according to claim 13, characterized in that the multiple induction microorganism is obtained by a method comprising culturing a pure culture of a microorganism in a culture medium supplemented with a mixture of nitrile and amide compounds containing: at least a ucc i noni tri lo of about 50 ppm, acetonitrile of about 150 ppm and acrylonitrile of about 150 ppm and (b) acetamide and acrylamide of about 150 ppm each.
23. The method according to claim 22, characterized in that the culture medium is supplemented with a mixture of nitrile and ce. placed amide containing succinoni t r i 1 or about 50 ppm and acetamide of about 150 ppm and acrylamide of about 150 ppm.
24. The method according to claim 10, characterized in that the culture medium is further supplemented with about 1-10 ppm KCN or NaCN.
25. The method according to claim 1, characterized in that the polyacrylamide preparation contains the monomer acrylamide in a concentration of about 1,000-40,000 ppm.
26. The method of consistency with claim 1, characterized in that the polyacrylamide preparation also contains an unwanted nitrile monomer and which is removed by the conversion of the monomer to the corresponding acid.
27. 27. The method according to the rei indication 13, characterized in that the polyacrylamide preparation also contains an unwanted nitrile monomer and which is removed by the conversion of the monomer to the corresponding acid.