Microbes and their use to degrade N-phosphonomethylglycine in waste streams

A process is provided for the degradation of N-phosphonomethylglycine. A mixed culture of microorganisms (ATCC 55050) is attached to an inert, immobile support, and then contacted with an aqueous solution of N-phosphonomethylglycine for a sufficient time to degrade the N-phosphonomethylglycine. One of the microorganisms in the mixed culture was identified as Moraxella anatipestifer (ATCC 55051).

BACKGROUND OF THE INVENTION 
This invention relates to microorganisms and their use to degrade 
N-phosphonomethylglycine in an aqueous solution, such as a waste stream, 
by biodegradation. 
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-phosphonomethylglycine and its salts are 
conveniently applied in an aqueous formulation as a post-emergent 
phytotoxicant for the control of numerous plant species. 
N-phosphonomethylglycine 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. For example, U.S. Pat. No. 3,969,398 to Hershman 
discloses a process for the production of N-phosphonomethylglycine by the 
oxidation of N-phosphonomethyliminodiacetic acid utilizing a molecular 
oxygen-containing gas as the oxidant in the presence of a catalyst 
consisting essentially of activated carbon. U.S. Pat. No. 3,954,848 to 
Franz disclose the oxidation of N-phosphonomethyliminodiacetic acid with 
hydrogen peroxide and an acid such as sulfuric acid. U.S. Pat. No. 
4,670,190 to Kleiner discloses a process for the preparation of 
N-phosphonomethylglycine by reacting aminomethylphosphonic acid and 
glyoxylic acid in a molar ratio of about 1 to 2 in an aqueous medium or 
aqueous organic medium at temperatures between 30.degree. and 100.degree. 
C. These references are only illustrative since there are many other 
methods known in the art for preparing N-phosphonomethylglycine. 
Regardless of the process by which N-phosphonomethylglycine is prepared, 
all of these processes produce aqueous, waste streams that contain small 
amounts of N-phosphonomethylglycine and various by-products and unreacted 
starting materials, such as N-phosphonomethyliminodiacetic acid, 
N-formyl-N-phosphonomethylglycine, aminomethylphosphonic acid, 
formaldehyde, and the like. Such waste streams should be kept to a minimum 
to help preserve the environment. (M. L. Rueppel, et al., "Metabolism and 
Degradation of Glyphosate in Soil and Water", Journal of Agriculture and 
Food Chemistry, Vol. 25 (1977) p. 517-522). 
It is known that certain natural microorganisms will degrade 
N-phosphonomethylglycine over a period of time. In addition, several 
microorganisms have been isolated which will degrade 
N-phosphonomethylglycine. For example, G. S. Jacob, et al., "Metabolism of 
Glyphosate in Pseudomonas sp. Strain LBr", Applied and Environmental 
Microbioloy, Vol. 54, No. 12 (Dec. 1988) p 2953-2958, reports the 
metabolism of glyphosate by Pseudomonas sp. Strain LBr. L. E. Hallas, et 
al., "Characterization of Microbial Traits Associated with Glyphosate 
Biodegradation in Industrial Activated Sludge", Journal of Industrial 
Microbiology, 3 (1988) p 377-385 reports that the microorganisms from two 
industrial activated sludges that treat N-phosphonomethylglycine waste 
streams were enumerated by microscopic examination. It was suggested that 
the degradation activity is not a universal trait, and its expression 
requires enrichment through specific selective pressures. T. M. Balthazor, 
et al., "Glyphosate-Degrading Microorganisms from Industrial Activated 
Sludge", Applied and Environmental Microbiology, Vol. 51 No. 2 (Feb. 1986) 
p 432-434, discloses a plating medium to isolate microorganisms that will 
degrade N-phosphonomethylglycine as the sole phosphorus source. One 
purified isolate metabolized N-phosphonomethylglycine to 
aminomethylphosphonic acid was identified as a Flavobacterium species. 
U.S. Pat. No. 4,859,594 discloses microorganisms separated from natural 
environments and purified and genetically modified, and 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, and that 
N-phosphonomethylglycine can be biodegraded in an industrial pond, such 
biodegradation requires a significant amount of time to achieve 
substantial degradation of the N-phosphonomethylglycine. Now there is 
provided a mixture of microorganisms that have been conditioned to degrade 
N-phosphonomethylglycine and their use to degrade N-phosphonomethylglycine 
in a very 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-phosphonomethylglycine having an American Type 
Culture Collection number 55050, which are useful in a process to 
biologically degrade N-phosphonomethylglycine in an aqueous solution which 
comprises contacting the aqueous solution with immobilized microorganisms 
having an American Type Culture Collection accession number of 55050 for a 
sufficient time to degrade a substantial portion of the 
N-phosphonomethylglycine. 
DETAILED DESCRIPTION OF THE INVENTION 
Since it is known that certain microorganisms are effective in the 
degradation of N-phosphonomethylglycine, and particularly the 
microorganisms that exist in the waste treatment pond at Monsanto 
Company's N-phosphonomethylglycine manufacturing facility located at 
Luling, La., a colony of microorganisms containing approximately 40 
species of microorganisms was obtained from the waste treatment pond. 
These were obtained as sludge samples from the pond, and the sludge was 
used to establish a bioreactor which was continuously mixed, aerated, 
routinely titrated to pH 7, and fed nutrients in an aqueous solution 
containing increasing amounts of N-phosphonomethylglycine. Inorganic 
nitrogen was ammended into the bioreactor by the addition of ammonium 
nitrate. The degradation of N-phosphonomethylglycine, the production of 
aminomethylphosphonic acid and the pH were monitored in the bioreactor. 
When the N-phosphonomethylglycine was degraded, the bioreactor was allowed 
to settle for two hours, and 80% of the liquid volume was discarded. The 
bioreactor was then refilled by metering in fresh aqueous solution over a 
four-hour time interval containing up to 2200 milligrams per liter of 
N-phosphonomethylglycine until degradation of N-phosphonomethylglycine was 
achieved in the bioreactor. 
After the colony of microorganisms was conditioned to accept high loadings 
of N-phosphonomethylglycine, the colony was then placed on a immobilized 
carrier by techniques known to those skilled in the art. 
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. Pat. No. 
4,775,160 and U.S. Pat. No. 4,859,594. 
Solid surfaces to which the microorganisms can be affixed are, preferably 
an aminopolysaccharide surface such as chitan, 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, chromiaalumina, 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 provides 
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 N-phosphonomethylglycine 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 N-phosphonomethylglycine, which is usually about 10-30 minutes 
under optimum conditions, to achieve greater than 90 percent degradation 
of the N-phosphonomethylglycine. 
The microorganisms that have been conditioned to degrade 
N-phosphonomethylglycine are important in the process of the present 
invention. The culture of microorganisms containing approximately 40 
species obtained from the waste treatment pond at Luling, La., were 
conditioned to degrade 200 milligrams per liter of 
N-phosphonomethylglycine, and thereafter, a sample was submitted to the 
American Type Culture Collection and assigned ATCC 55050. The species of 
microorganisms remaining after conditioning is unknown, and all of the 
species that degrade N-phosphonomethylglycine is also unknown. The 
predominate characteristics of the culture are set forth in Table 1. 
TABLE 1 
______________________________________ 
PREDOMINANT METABOLIC CHARACTERISTICS IN 
ACTIVATED SLUDGE MICROORGANISMS (ATCC 55050) 
Trait % Presence* 
______________________________________ 
Fermentation 
D-Arabitol 93 
D-Turanose 96 
Trehalose 96 
Saccharose 96 
Maltose 96 
Mannitol 95 
Inositol 95 
L-Fructose 99 
D-glucose 99 
Adonitol 99 
Arginine 94 
Glycerol 100 
N-Acetylglucosamine 
95 
Enzymatic 
.alpha.-glucosidase 
97 
Gly Aminopeptidase 
100 
Glucosaminidase 96 
Arg Aminopeptidase 
100 
Leu Aminopeptidase 
100 
Alkaline Phosphatase 
99 
______________________________________ 
*A predominant characteristic was defined as occurring in &gt;90% of the 
microbes. 
One microorganism that had a high degree of degrading activity toward 
N-phosphonomethylglycine in the conditioned colony was isolated an 
identified on a Biolog, In. (Hayward, Calif.) GN Microlog plate. This gram 
negative, rod-shaped microorganism is characterized as Moraxella 
anatipestifer (ATTC 55051). The characteristics of this microorganism is 
set froth in Table 2. 
TABLE 2 
______________________________________ 
Identifying Metabolic (Biodegradation) Characteristics of 
Moraxella Anatipestifer (ATCC 55051) 
______________________________________ 
Dextrin D-Galactonic Acid Lactone 
Glycogen D-Galacturonic Acid 
D-Gluconic Acid 
Tween 40 D-Glucosaminic Acid 
Tween 80 D-Glucuronic Acid 
Adonitol .alpha.-Keto Glutaric Acid 
L-Arabinose D,L-Lactic Acid 
D-Arabitol Propionic Acid 
Cellobiose Succinic Acid 
I-Errthyitol Bromosuccinic Acid 
D-Fructose Alaninamide 
L-Fucose D-Alanine 
D-Galactose L-Alanine 
Gentiobiose L-Alanylglycine 
.alpha.-D-Glucose 
L-Asparagine 
M-Inositol L-Aspartic Acid 
.alpha.-Lactose L-Glutamic Acid 
Maltose Glycyl-L-Aspartic Acid 
D-Mannitol Glycyl-L-Glutamic Acid 
D-Mannose Hydroxy L-Proline 
Psicose L-Ornithine 
L-Rhamnose L-Proline 
Sucrose D-Serine 
Turanose L-Serine 
Methylpyruvate L-Threonine 
Mono-Methyl Succinate 
D,L-Carnitine 
Cis-Aconitic Acid 
.alpha.-Amino,Butyric Acid 
Citric Acid 
______________________________________ 
A culture of the microorganism and the conditioned colony has been 
deposited in the American Type Culture Collection at Rockville, Md., and 
each culture assigned an identifying member, each as previously 
identified, this depository affording permanence of the deposit and ready 
accessibility thereto by the public on grant of a patent, and under 
conditions which assure (a) that access to the culture will be available 
during pendency of the patent application to one determined to be entitled 
thereto under 37 CFR 1.14 and 35 USC 122, and (b) that all restrictions on 
the availability to the public of the culture so deposited will be 
irrevocably removed upon grant of a patent.

The invention is further illustrated by, but not limited to, the following 
examples. 
EXAMPLE 1 
A culture of microorganisms was obtained in a sludge sample taken from the 
waste treatment pond at Monsanto Company's facility located at Luling, La. 
The sludge was used to establish a bioreactor which was continuously 
mixed, aerated, routinely titrated to pH 7, and fed an aqueous solution 
containing N-phosphonomethylglycine ranging from 500-2000 milligrams per 
liter. The degradation of N-phosphonomethylglycine, the production of 
aminomethylphosphonic acid and the pH were monitored in the bioreactor. 
When the N-phosphonomethylglycine was completely degraded, the bioreactor 
was allowed to settle for two hours, and 80% of the liquid volume was 
discarded. The bioreactor was then refilled by metering in fresh aqueous 
solution over a four-hour time interval containing up to 2200 milligrams 
per liter of N-phosphonomethylglycine. Inorganic nitrogen was ammended 
into the bioreactor by the addition of 50 milligrams per liter of ammonium 
nitrate. This was continued until degradation of N-phosphonomethylglycine 
was achieved in the bioreactor. 
After the culture of microorganisms was conditioned to accept high loadings 
of N-phosphonomethylglycine, the colony was then placed on an immobilized 
carrier contained in a cell column. The column consisted of an acrylic 
tube 60 centimeters (24 in.) long having an internal diameter of 8 
centimeters (3.25 in.) with a wall thickness of 0.625 centimeters (0.25 
in.). 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 (0.25 in.) 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 (0.625 in.) hole bored 
through the plastic tubing 12.5 cm (5 in.) above the glass frit. The hole 
was sealed with a rubber stopper containing a 20 cm length (8 in.) of 
0.625 cm (0.25 in.) stainless steel tubing containing a 90.degree. bend at 
the midpoint. This feed tube discharged waste 2.5 cm (1 in.) above the 
glass frit. A second and third hole were bored 35 cm (14 in.) and 60 cm 
(24 in.) up the column from the frit and used as direct waste discharge 
ports for the packed column. 
Immobilized cell columns were prepared by packing the plastic tubing to 
heights of 30 cm with a biocarrier, which was diatomaceous earth, 
identified as Manville R-635. 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 
was added to the column to form a bioreactor. The bioreactor was 
continuously mixed, aerated, routine titrated to pH 7, and fed batches of 
aqueous solutions containing N-phosphonomethylglycine concentrations 
ranging from 500-2000 mg per liter. Glyphosate degradation, 
aminomethylphosphonic acid production and pH were monitored in the 
bioreactor. 
An aqueous solution containing 400 mg per liter of N-phosphonomethylglycine 
was then pumped through the column at a rate of 3 ml per minute resulting 
in a retention time in the column of 350 minutes. Greater than 99% of the 
N-phosphonomethylglycine was degraded immediately. After 9 days of 
operating with this feedstock, the concentration of 
N-phosphonomethylglycine was increased to 1400 mg per liter, and the 
degradation activity dropped to about 85% after 5 days and returned to 
greater than 99% 2 days later. A significant increase in bacterial biomass 
was observed. 
On day 27, the pumping rate was increased to 25 ml per minute (retention 
time 42 minutes), and within 4 days greater than 99% degradation activity 
was observed. High flow studies were then conducted preliminary to Example 
2 pilot plant work. The biocarrier bed depth was reduced to 30 cm (12 in.) 
resulting in a lower retention time. The concentration of 
N-phosphonomethylglycine in the feedstock was lowered to 50 mg per liter 
in a stepwise fashion. Degradation activities of &gt;98% to 82% were achieved 
at pumping rates of 25 ml per minute (retention time 23 minutes) and 30 ml 
per minute (retention time 19 minutes), respectively. 
EXAMPLE 2 
A culture (37.8 liters, 10 gallons) of microorganisms was obtained from a 
sludge sample taken from the Luling waste treatment pond. The sludge was 
used to establish a N-phosphonomethylglycine degrading activity similar to 
that described in Example 1. When the activity was established, the 
enriched sludge was transferred to a drum (208 liters, 55 gallons) 
containing 45.5 kg, 100 pounds of R-635 solid support as in Example 1. A 
center well was created in the middle of the drum using a washing tube (10 
cm ID) with a perforated bottom. The biocarrier, sludge and aqueous 
solution containing N-phosphonomethylglycine (500 mg per liter) surrounded 
the center tube. The aqueous solution was circulated through the carrier 
bed by pumping liquid from the bottom of the center well to the top of the 
drum; an air sparger provided oxygen. When N-phosphonomethylglycine 
degradation was complete, the solution was drained from the drum and fresh 
solution was added. 
A pilot plant containing an equalization tank (1900 liter) and a packed bed 
column (2.74.times.7.3 meters) configured in an upflow mode was prepared. 
Separate lines sparged air and fed an aqueous solution containing 
N-phosphonomethylglycine and ammonium nitrate to the tank. The pH was 
controlled in the equalization tank as in Example 1. Approximately 900 kg 
(2000 pounds) of fresh carrier was intermingled with the acclimated 
carrier containing N-phosphonomethylglycine degrading microorganisms. 
Steps were taken to promote microbial growth throughout the biocarrier as 
in Example 1. Initially, the aqueous solution was recirculated (18.9 
liters per minute) in the column with pH control. After 
N-phosphonomethylglycine disappeared, fresh aqueous solution containing 
yeast extract (25-50 mg per liter) was added. Treatment performance was 
monitored by analyses of oxygen, pH, and temperature. Mechanical 
performance was monitored by analyses of water and air flow rate, and pump 
operation. 
Continuous flow operation was begun after the biocarrier acclimated to 500 
mg/l N-phosphonomethylglycine degradation. Initially, an aqueous solution 
containing 50 mg/l of N-phosphonomethylglycine and 25 mg/l of inorganic 
nitrogen was pumped through the column at 3.78 liters per minute (100 
minute retention time). The detection limit for N-phosphonomethylglycine 
was 3-5 mg/l so 90-95% degrading activity could be confirmed. Optimal 
performance was established over the next 35 days through several 
operational and mechanical changes. The flow rate was increased to 19 
liters per minute (20 minute retention time). Yeast extract was 
occasionally added to promote microbial growth. It was also found that a 
pH increase of between 1-1.5 units was critical to good degrading 
activity. Finally, a fluidization of the column was accomplished using a 
378 liter per minute pump. This removed excess sludge and stabilized 
N-phosphonomethylglycine degradation. 
A second 30-day period was used to establish a maximum loading rate for 
N-phosphonomethylglycine. Three step flow rate increases were accomplished 
resulting in 15, 10, and 8 minute retention times. The 8-minute retention 
time produced some degradation activity. However, the 10-minute retention 
time (144 hydraulic turnovers per day) maintained a consistent &gt;90% 
N-phosphonomethylglycine degrading activity. 
A viability and surge test was also performed to test the resilience of the 
immobilized microorganisms. The flow rate was slowed to 3.78-11.3 liters 
per minute, and no chemical amendments on pH control occurred for 21 days. 
At that time, the flow rate was increased to 37.8 liters per minute 
(10-minute retention time). The N-phosphonomethylglycine level was 
increased to 50 mg/l over 5 days. Two days were required to initiate a 
degrading activity and an additional 2 days were needed before &gt;90% 
N-phosphonomethylglycine removal was seen. 
A sample of the conditioned sludge was taken from the solid support. It was 
placed in a mineral salts medium (as described by T. M. Balthazor, et al.) 
containing approximately 200 mg/l of N-phosphonomethylglycine. After the 
compound was biologically degraded, the sample was split. One-half of the 
sample was submitted as a mixed culture to the American Type Culture 
Collection and assigned ATCC 55050. The other half of the sample was 
separated into individual microorganisms using standard techniques. One 
culture exhibiting high degrading activity was identified and submitted to 
the American Type Culture Collection. It was Moraxella anatipestifer (ATCC 
55051). 
Although the invention has been described in terms of specified embodiments 
which are set forth in considerable detail, it should be understood that 
this is 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.