N-Phosphonomethylglycine, known in the agricultural chemical art as glyphosate, is a highly effective and commercially important phytotoxicant, useful in controlling the growth of germinating seeds, emerging seedlings, maturing and established woody and herbaceous vegetation and aquatic plants. N-Phosphono- methylglycine and its salts are conveniently applied in an aqueous formulation as a post-emergent phytotoxicant for the control of numerous plant species. N-Phosphono- methylglycine and its salts are characterized by a broad spectrum activity, i.e., the control of a wide variety of plants. Numerous methods are known in the art for the preparation of N-phosphonomethylglycine. However, most of the N-phosphonomethylglycine that is manufactured today is made by two major processes. The first process is the phosphonomethylation of glycine, or a glycinate ester, such as that disclosed in UK Patent 2,034,313 which discloses the reaction of glycine, formaldehyde and triethanolamine in alcohol and then adding a dialkyl phosphite. The reaction product is hydrolyzed, and acidification yields N-phosphonomethylglycine in reasonable yields and purity. The other major process is the oxidation of N-phosphonomethyliminodiacetic acid using molecular oxygen or a peroxide. For example, U.S. Patent 3,969,398 to Hershman discloses a process for the production of N-phosphonomethylglycine by the oxidation of N-phosphonomethyliminodiacetic acid with a molecular oxygen-containing gas as the oxidant in the presence of a catalyst consisting essentially of activated carbon. U.S. Patent 3,954,848 to Franz discloses the oxidation

of N-phosphonomethyliminodiacetic acid with hydrogen peroxide in an acid, such as sulfuric acid. U.S. Patent 4,898,972 discloses the oxidation of N-phosphonomethyl¬ iminodiacetic acid with a molecular oxygen-containing gas in the presence of a water-soluble metal salt. U.S. Patents 5,023,369 and 5,043,475 to Fields discloses the formation of N-phosphonomethyliminodiacetic acid- N-oxide, and thereafter, the decomposition of the intermediate N-oxide to N-phosphonomethylglycine. Regardless of the process by which N-phosphono¬ methylglycine is prepared, all of these processes produce aqueous waste streams that contain small amounts of various by-products and unreacted starting materials. In particular, the processes which oxidize N-phosphono- methyliminodiacetic acid to N-phosphonomethylglycine produce waste streams that contain small amounts of N-phosphonomethyliminodiacetic acid, N-formyl-N-phos- phonomethylglycine, aminomethylphosphonic acid, formaldehyde and the like. Such waste streams should be kept to a minimum to help preserve the environment.

It is known that certain microorganisms will degrade N-phosphonomethylglycine over a period of time.

L. E. Hallas, et al. "Characterization of Microbial

Traits Associated with Glyphosate Biodegradation in Industrial Activated Sludge", Journal of Industrial Microbiology. 2 (1988) B 377-385, reports that microorganisms from two industrial activated sludges will degrade N-phosphonomethylglycine. However, no reference has been found which discloses that N-phosphonomethyliminodiacetic acid can be degraded using microorganisms.

U.S. Patent 4,859,594 discloses microorganisms separated from natural environments, purified and genetically modified, in a process for immobilizing these microorganisms by affixing them to substrates. The biocatalytic compositions are useful for the detoxification of toxin-polluted streams containing a wide class of toxicants.

Although the prior art discloses that certain microorganisms are effective for the degradation of N-phosphonomethylglycine, the prior art does not disclose that N-phosphonomethyliminodiacetic acid can be biodegraded. Now there is provided a mixture of microorganisms that have been conditioned to degrade N-phosphonomethyliminodiacetic acid and their use to degrade N-phosphonomethyliminiodiacetic acid in a short period of time and with a high degree of effectiveness. SUMMARY OF THE INVENTION

These and other advantages are achieved with microorganisms, suitable for biologically degrading N-phosphonomethyliminodiacetic acid having an American Type Culture Collection No. 55322, which are useful in a process to biologically degrade N-phosphonomethyl¬ iminodiacetic acid in an aqueous solution which comprises contacting the aqueous solution with immobilized microorganisms having an American Type Culture Collection Accession No. of 55322 for a sufficient time to degrade a substantial portion of the N-phosphonomethyliminodiacetic acid.

DETAILED DESCRIPTION OF THE INVENTION Colonies of microorganisms were obtained from various locations in and around manufacturing facilities to produce glyphosate owned by Monsanto Company. Such samples were obtained from sediments from waste treatment ponds, soil adjacent to processing units and sediment plus overlaying water from rivers downstream from the manufacturing facilities. Selective solid media were inoculated with such samples, and N-phos¬ phonomethyliminodiacetic acid (hereinafter referred to as PIA) was provided as the sole carbon source (1,000 mg/1) or as the sole phosphorus source (200 mg/1) . In accordance with the teachings of Leadbetter et al. "Studies on some Methane Utilizing Bacteria", Arch. f r. Mikrobiol. 30 (1958) . p 191-118, L salts were used as a source of nutrients in all media, which contain 15 mg/1 calcium chloride, 1 mg/1 ferrous sulfate, 200 mg/1

magnesium sulfate, 40 mg/1 potassium chloride, 250 mg/1 ammonium chloride, 1000 mg/1 sodium nitrate, 210 mg/1 disodium hydrogen phosphate, 90 mg/1 sodium dihydrogen phosphate, 5 μg/1 copper sulfate, 10 μg/1 boric acid, 10 μg/1 magnesium sulfate, 7 μg/1 zinc sulfate and 10 μg/1 molybdenum trioxide at a pH of 7.0. The media containing PIA as the sole source of phosphorus were amended with 1000 mg/1 gluconate and pyruvate as alternate carbon sources. The media were incubated at 25 ^β C and microbial growth was monitored for three months. Out of the 120 samples that were taken, none of the samples showed PIA degrading activity as determined by using carbon-14 labeled N-phosphonomethylimino¬ diacetic acid. A 3-liter glass vessel was inoculated with a mixture of sludges, soil and sediments obtained from in and around Monsanto's manufacturing facility at Luling, Louisiana. The inoculum consisted of 10 grams of sediment from the waste treatment ponds, 5 grams of Mississippi River sediment, 5 grams of soil near the manufacturing facility, 240 milliliters of sludge from the decanting pond and 840 milliliters of digester sludge. The inoculum was brought to a volume of 2 liters with L salts adjusted to pH 6.8. The mixture was aerated by air forced through a porous frit near the bottom of the glass vessel. After 24 hours, the mixture in the glass vessel was allowed to settle, l liter of the supernatant liquid was withdrawn, and 1 liter of fresh feed was added at a flow rate of 4.0 milliliters per minute. The feed contained a 50/50 (vol/vol) glyphosate waste water (2% PIA) and domestic sewage (Grand Glaze, St. Louis, Missouri) supplemented with trace elements of the L salts solution. The final concentration of PIA averaged 600-700 mg/1. The microorganisms established the activity to degrade N- phosphonomethylglycine after one month. However, PIA remained recalcitrant for three months under these conditions. Accordingly, all alternative carbon sources

were removed from the feed, and the acclimated microbial population was provided with PIA as the sole source of carbon and energy. PIA as the sole carbon source remained persistent for three weeks. However, during the fourth week, the concentration of PIA began to decrease. The disappearance of PIA was associated with the production of aminomethylphosphonic acid. Analysis by HPLC revealed the consumption of 1.78 illimoles of PIA which resulted in the formation of 1.81 millimoles of aminomethylphosphonic acid.

To confirm that the degradation of PIA was occurring, carbon-14 labeled PIA was introduced into the biologically active samples. In this radiochemical assay, over 97% of the carbon-14 carboxymethyl-labeled PIA was converted to carbon-14 labeled carbon dioxide, indicating that over 97% of the PIA was degraded by the microorganisms.

The PIA degrading activity component sludge in the 3-liter glass vessel was split into two equal halves. Thereafter, experiments were conducted to understand the nutritional requirements of the sludge. Formate and acetate (20 mg/1 each) were periodically supplied to one half as a source of "soft carbon", which are readily biodegradable carbon sources. Soft carbon was omitted from the feed for the other sludge. The addition of acetate and formate to the sludge increased the rate of PIA removal compared to that of the half not receiving the soft carbon. Also, the addition of yeast extract (100 mg/1) reduced the lag period associated with PIA degrading activity. Ammonia added as ammonium carbonate (40 mg/1) also enhanced PIA degrading activity. The biomass increased in the sludge which was fed PIA supplemented with soft carbon, yeast extract and ammonia. Under this operation strategy, PIA degrading activity was established and enhanced, and the cycle time required to degrade PIA concentrations up to 1.6 g/1 was reduced from about 3 weeks to less than 3 days. A culture of the microorganisms from the portion

fed soft carbon was obtained and a sample was submitted to the American Type Culture Collection and assigned ATCC No. 55322 on May 22, 1992. Species of microorganisms remaining after conditioning is unknown, and all of the species that degrade PIA is also unknown. It was determined, however, that the culture contained gram negative, rod-shaped microorganisms, and that the culture readily exhibited nitrification of ammonia to nitrate. The PIA degrading activity of the culture is enhanced by the addition of yeast extract (10-100 mg/1) , potassium formate (20-250 mg/1) , sodium acetate (20-250 mg/1) and ammonia (40-100 mg/1) .

Inasmuch as the culture has been deposited in the American Type Culture Collection in Rockville, Maryland, and assigned an identifying number as previously identified, this depository will afford permanence of the deposit and ready accessibility thereto by members of the public, since such microorganisms are not readily available to the public. Access to the cultures will be readily available to the public if a Patent Office signatory to the Budapest Treaty certifies one's right to receive, or if a U.S. patent is issued citing the strains. It is further certified that the strains will be maintained for a period of at least 30 years after the date of the deposit or for a period of five years after the most recent request for a sample, whichever is longer. In the unlikely event that the deposit is no longer viable, then Applicants will redeposit a sample of the microorganisms to insure that adequate deposits are available to the public upon the issuance of a U.S. patent or a foreign patent application as described in the Budapest Treaty.

The solid substrate of the carrier to which the microorganisms of this invention are attached is porous, and preferably of pore volume of at least 0.2 microns/gram of solids material. Preferably, the pore volume ranges from about 0.2 microns/gram to about 45

microns/gram, more preferably from about 5 microns/gram to about 15 microns/gram of solids material. Particle sizes range generally from about 0.5 mm to about 2.0 mm, preferably from about 0.75 mm to about 1.0 mm, in diameter. Biocatalyst formed on such substrates are employed as fixed beds. The biocatalyst particles are sized in accordance with accepted engineering principles to provide good contact between the effluent and the carrier. Greater details on such solid substrates are described in U.S. 4,775,160 and U.S. 4,859,594.

Solid surfaces to which the microorganisms can be affixed are, preferably an aminopolysaccharide surface such as chitin, chitosan, n-carboxychitosan, cellulose, or a porous inorganic oxide, such as alumina, silica, silica-alumina, clay, diatomaceous earth or the like. A preferred support is one wherein the chitin, or chitosan is dispersed upon a second solid support, e.g., a porous substrate. The classes of useful porous substrates is quite large, exemplary of which are, e.g., (1) silica or silica gel, clays, and silicates including those synthetically prepared and naturally occurring, for example, attapulgus clay, china clay, diatomaceous earth, fuller's earth, kaolin, kieselguhr, etc.; (2) ceramics, porcelain, crushed firebrick, bauxite; (3) synthetic and naturally occurring refractory inorganic oxides such as alumina, titanium dioxide, zirconium dioxide, chromium oxide, zinc oxide, magnesia, thoria, boria, silica-alumina, silica-magnesia, chromia-alumina, alumina-boria, silica-zirconia, silica carbide, boron nitride, etc.; and (4) crystalline zeolitic alumino- silicates such as naturally occurring or synthetically prepared mordenite and/or faujasite. Diatomaceous earth granules provide satisfactory results, and this is what we prefer to use. The solid support surface to which the microorganisms are affixed can be used advantageously in the method of this invention in any configuration, shape, or size which exposes the microorganism disposed

thereon to the effluent to be treated. The choice of configuration, shape, and size of the refractory inorganic oxide depends on the particular circumstances of use of the method of this invention. Generally, the support surface can be conveniently employed in particulate form, as pills, pellets, granules, rings, spheres, rods, hollow tubes, and the like. Granules are readily available commercially, and these are preferred. As will occur to those skilled in the art, the vessel containing the carrier with the deposited microorganisms can be of any size or shape, depending on factors such as the volume of liquid to be treated, the concentration of PIA in the aqueous stream, and the like. It is only necessary that the vessel is designed to permit contact of the aqueous stream with the microorganisms for a sufficient time to degrade the PIA, which is usually less than 3 hours under optimum conditions, to achieve greater than 90 percent degradation of the PIA. The invention is further illustrative by but not limited to the following examples.

Example 1 A column was prepared for the degradation of PIA using the mixed colony of microorganisms having ATCC No. 55322. The column consisted of an acrylic tube 60 centimeters long having an internal diameter of 8 centimeters with a wall thickness of 0.625 centimeters. An acrylic collar was fused onto the bottom of the tube and an identically sized acrylic collar was placed around a 350 ml buchner funnel containing a glass frit of medium porosity (Corning Glass Works No. 36060) . The glass funnel was attached to the plastic tubes by using C-clamps on the collars and tightening until the upper lip of the glass funnel sealed into a 0.625 cm thick rubber gasket. The glass frit served as the lower support for the biocarrier in the columns, and air was forced into the column through the funnel from the bottom.

A port for the inflow of waste consisted of a 1.6 cm hole bored through the plastic tubing 12.5 cm above the glass frit. The hole was sealed with a rubber stopper containing a 20 cm length of 0.625 cm stainless steel tubing containing a 90° bend a the midpoint. This feed tube discharged waste 2.5 cm above the glass frit. A second and third hole was bored 35 cm and 60 cm up the column from the frit and used as direct waste discharge ports for the packed column. The immobilized cell column was prepared by packing the plastic tubing to heights of 30 cm with diatomaceous earth, identified as Manville R-635 as the biocarrier. The biocarrier was soaked overnight in an acidic chitosan solution, then rinsed in water, and the pH adjusted to 7.0 before addition to the column. Then, the culture of microorganisms which had been conditioned in the series of progressive steps as described above (ATCC No. 55322) was added to the column to form a bioreactor. An aqueous solution containing PIA (250 mg/1), yeast extract (10 mg/ml), potassium formate (120 mg/1) , sodium acetate (120 mg/1) , L-salts and at pH 8 was introduced into the above column at about 25 ^β C and pH 8 at a rate of about 2.4 ml/min. to provide a hydraulic retention time in the column of about 3 hours. Analysis of the effluent showed that the aqueous solution had a pH 8, and greater that 99% of the PIA was degraded.

Although the invention has been described in terms of specified embodiments which are set forth in considerable detail, it should be understood that this by way of illustration only, and that alternative embodiments and operating techniques will become apparent to those skilled in the art in view of the disclosure. Accordingly, modifications can be made without departing from the spirit of the described invention.