PROTECTION AGENCY DECLARED TOXINS BY NATURALLY OCCURRING ANAEROBIC ORGANISMS

BACKGROUND OF THE INVENTION

One of the more important problems currently facing mankind is pollution caused from by-products of our modern society. In fact, the Environmental Protection Agency has listed many organic chemical by¬ products as being on the EPA priority pollutants list. Among those priority pollutants are many organic compounds such as certain chlorinated hydrocarbons, certain phthalate esters, certain polynuclear aromatic hydrocarbons, certain triazines, and many commonly occurring phenols. These contaminants when present in soil and water are difficult to remove and cause many potential risks such as carcinogenic properties, risk to human organs such as the liver (necrosis), and risk to domestic animals that inadvertently ingest sufficient quantities of the pollutants to interfere with their normal metabolic processes. In short the potential risks are not only to human health but also to the health of some of man\'s food sources.

One potential solution for environmental contamination may be the breakdown of toxic compounds by microorganisms. Just now microbial ecologists and microbiologists are beginning to unearth an array of microorganisms with certain unexpected abilities to biodegrade some of the toughest and most recalcitrant environmental chemicals. One such group of very hazardous environmental chemicals includes the pyrrolizidine alkaloids, which all commonly contain the alkaloid ring which has the following structure:

H

Pyrrolizidine alkaloids (PA) are known to be produced by some plants which grow in pasture lands used for domestic animals, particularly cattle and sheep. One of these commonly occurring plants is Tansy Ragwort (Senecio Jacobaea) . Tansy Ragwort in particular when concentrated in the food supply of grazing cattle can result in an accumulation of pyrrolizidine alkaloids within the organs of the animal, causing liver damage. Over the life of the animal it may decrease it\'s growth and overall efficiency.

It has in the past been reported that toxic plants account for approximately 9.7% of all livestock deaths in the United States. Forages containing plants with pyrrolizidine alkaloids are common and are distributed throughout this country, as well as the rest of the world. Cattle, horses and humans are highly susceptible to the toxic principle with chronic terminal hepatic disease produced after consuming as little as 5% of their body weight in plant material. On the other hand, it has been observed that sheep can eat quantities of many of these toxic plants, and in particular Tansy Ragwort up to as much as 300% of their body weight with no measurable abnormalities. This unusual quality of sheep in comparison with cattle and horses has led to

an investigation of the reasons why sheep have this apparent resistance.

Surprisingly it has been discovered that sheep rumen fluid contains certain microbial factors which are capable of detoxifying certain EPA priority pollutants, including pyrrolizidine alkaloids.

These initial observations of the nature and character of rumen microbial factors of sheep have led to investigatory research into the use of similar anaerobic bacterial factors for reducing pyrrolizidine alkaloid levels in silage contaminated with toxic plants, and have led to use of compositions containing these naturally occurring bacteria of sheep\'s rumen fluid for use as general EPA priority pollutant detoxifiers.

It can therefore be seen that there is the distinct possibility of genetically engineering the protective factors of shee \'s rumen fluid and adding these to silage inoculants, or to cow\'s rumen, or to simply use them in compositions as general detoxifying agents for EPA priority pollutants. The net result is that pollutants can be oxidized to less harmful compounds, that contaminated forages can be protected, and that pastures can be safely fed.

Accordingly, a primary objective of the present invention is to prepare detoxifying compositions which can be useful for detoxifying priority pollutants, and in particular PA, and to prepare detoxifying compositions which can be used in silage inoculants and/or in direct inoculation into the rumen of less resistant animals such as cattle and horses.

The method of accomplishing this as well as other objectives of the present invention will be apparent from the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph showing the ability of sheep\'s rumen fluid to metabolize pyrrolizidine alkaloid (PA) .

Fig. 2 is a graph showing ovine ruminal fluid detoxification of PA, after preconditioning of Tansy Ragwort.

Fig. 3 is an overlay graph showing TNT deterioration degradation over time when exposed to an enriched bacterial consortium derived from sheep\'s rumen fluid.

SUMMARY OF THE INVENTION

A culture media composition which contains detoxifying effective amounts of bacteria normally contained in sheep\'s rumen fluid. It is used as a detoxifying agent, as silage inoculant and for use in direct treatment of animals normally not resistant to toxic agents such as pyrrolizidine alkaloids.

DETAILED DESCRIPTION OF THE INVENTION

This invention involves the recent discovery that certain anaerobic organisms biodegrade certain EPA priority pollutants, and more specifically pyrrolizidine alkaloids. These naturally occurring anaerobic microorganisms were derived from sheep\'s rumen fluid. The investigation began after making the initial observation that domestic sheep seem to be unusually resistant to Tansy Ragwort poisoning whereas cattle and horses grazing on the same land were not so resistant. The cattle and horses showed classic pyrrolizidine alkaloid toxicity symptoms such as liver necrosis and hardening of the blood vessels, coupled with general over-all decreased animal function efficiency.

While sheep\'s rumen fluid contains many different bacteria, it has been discovered that critical to the detoxifying properties of sheep\'s rumen fluid are the necessity for a combination of bacteria of the following genera: Streptococcus sp., Peptococcus sp., Lactobacillus sp., Bacteroides sp., and Ruminococcus sp. and preferably but not essential Veillonella sp. and Eubacterium sp.

Compositions containing this combination of enumerated bacteria, have the capability of degrading of pyrrolizidine alkaloids. It is not certain how this biodegradation works, but it is believed that metabolites of the organisms oxidize the pyrrolizidine alkaloids by splitting the alkaloid ring. For pyrrolizidine alkaloids it is preferred that the composition contain streptococcus sp. and peptococcus and most preferrably also contains at least two additional gram positive bacteria.

Surprisingly, the combination of anaerobic microorganisms earlier specified is not only effective against pyrrolizidine alkaloids, but it is also effective against numerous EPA recognized priority pollutants. These EPA priority pollutants fall into five major categories: phenols, triazines, polynuclear aromatic hydrocarbons, phthalate esters, and chlorinated hydrocarbons. Amongst the phenols which may be biodegraded by the bacteria compositions of the present invention are the following: 2,4,6- trichlorophenol, 4-chloro-3-methylphenol, 2- chlorophenol, 2,4-dichlorophenol, 2,4-dimethylphenol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 2- Methyl-4,6-dinitrophenol, pentachlorophenol, and phenol. Amongst the polynuclear aromatic hydrocarbons

which may be biodegraded by the bacterial compositions of the present invention are the following: acenaphthene, fluoranthene, naphthalene, benzo(a)- anthracene, benzo(a) yrene, benzo(b)fluoranthene, benzo(k)fluoranthene, chrysene (93%), acenapthylene, anthracene, benzo(ghi)perylene, fluorene, phenan- threne, dibenzo(a, )anthracene, indeno(1,2,3cd)pyrene, and pyrene. Finally, amongst the chlorinated hydrocarbons which may be biodegraded by the compositions of the present invention are the following: 1,2,4-trichlorobenzene, hexachlorobenzene, hexachloroethane, 2-chloronaphthalene, 1,2- dichlorobenzene, 1,3-dichlorobenzene, 1,4- dichlorobenzene, and hexachlorobutadiene.

For phenols it has been noted as preferred to have Ruminococcus sp. present and for Triazines additional spore forming bacteria such as Clostridia sp. However all of these are present in sufficient amounts in sheep\'s rumen fluid.

Use of bacterial metabolism to biodegrade these EPA pollutants offers a significant, natural means of pollution clean up. It offers the potential for many of mankind\'s concerns for chemical carcinogens to be eliminated, and the potential for rendering many of these pollutants less persistent and less toxic in the environment.

The detoxification of pyrrolizidine alkaloid from Tansy Ragwort in sheep\'s rumen fluid has led directly to the discoveries of the present invention. In short, it has been definitively shown that the biodegradation of pyrrolizidine alkaloids (PA) from the plant Tansy Ragwort can be reduced or eliminated by sheep\'s rumen fluid. Moreover, as demonstrated by

the examples shown below this ruminal fluid has been shown to biodegrade a number of other priority pollutants.

Common to all of the sheep\'s rumen fluid demonstrated to exhibit this property are a combination of bacteria of the following genera: Streptococcus sp., Peptococcus sp., Lactobacillus sp., Bacteroides sp. and Ruminococcus sp. Generally each of these should be present in approximately similar organism concentrations. In particular the concentrations of each should be from 10 8 to 1012. In the preferred composition there may also be bacteria of the following additional genera: Veillonella sp. and Eubacterium sp. and some spore forming bacteria.

These preferred organisms when present should be present in the same earlier expressed concentration levels.

The bacteria may be contained in a conventional nutrient carrier system. Preferably the system should be designed to simulate and maintain the same ecological conditions as in the sheep rumen, in particular pH within the range of from about 6.8 to about 7.2. While sheep\'s rumen fluid will function effectively as the medium, certain synthetic media also can be used in conjunction with the organisms earlier specified. In particular, mediums that simulate the habitat of the rumen from other aspects as well as pH can be employed. Included in the successful habitat simulating medium was RCGA (rumen fluid, cellobiose, glucose, and auger, Bryant and

Burkey, 1953) and 98.5 (a modification of RCGA,

Bryant and Robinson, 1961). Both of these media contain 30% of sterilized ruminal fluid and are

similar in concentrations of salt and minerals; however, the 98-5 medium contains less sugars. Another useful medium is Pyrrolizidine alkaloid enrichment media modified from one reported by Shelton and Tiedje, 1984, which contains a basal medium of salts, trace minerals and vitamins to which rumen fluid (5% of supernatants) is added. So far it appears that the detoxifying bacteria are able to survive in a variety of mediums which function effectively as nutrient-carriers, with the most important criteria being the successful pH control within the range earlier specified. One particular media designed to simulate the composition of sheep\'s saliva is McDougall\'s buffer composed of the following in grams per litre: NaHCO-, - 9.8; KC1 - 0.57; Na ₂ HP0 ₄ :12 H ₂ 0 - 9.3, NaCl - 0.47; MgCl ₂ (anhyd) - 0.06; CaCl ₂ (anhyd) - 0.04. In addition, (NH ₄ ) ₂ S0 ₄ at 2.64 g/1 was added to the solution).

The amount of consortium of bacteria to be added is a detoxifying effective amount. This amount can be one cell of each organism in the consortium. These organisms can multiply and, thus, as little as one cell can be effective. It will be readily apparent to one skilled in the art that, for practical purposes, an optimum concentration may be obtained by\' pretesting. A small scale concentration test could involve, for example, providing for a predetermined amount of the waste material to be detoxified in test tubes, and varying amounts of concentration of the consortium added. One tube, for example, might contain .1% consortium to the total volume of waste, the second tube contain 1%, and the third tube 10%. It will typically be found that less than 5% by volume

will be necessary in order to bioremediate. If the waste product has an inhibitory effect upon the bacteria, a larger volume can be used for more optimum effect.

If a bioreactor is being used, in many circumstances the optimum amount to be added is equal to or greater than 2% of the initial bioreactor volume.

Thus, one skilled in the art will readily appreciate that an amount as small as one cell of each of the organisms, would be effective to bioremediate. The optimum volume to be applied to the waste material can be quickly found by standard testing.

The following examples are offered to illustrate but not limit the process of compositions of the present invention.

EXAMPLE 1

A series of in-vitro experiments were conducted comparing the degradation capabilities and rates of ovine versus bovine ruminal fluid utilizing a modified Hungates artificial rumen technique with serum bottles.

In these experiments ruminal fluid was removed manually through a permanent rumen fistula in sheep and cattle. . The ruminal fluid was immediately filtered through nylon mesh into a prewarm (38° C) thermos bottle for transport back to the laboratory. The rumen fluid was cut 50/50 with pre-warmed and carbon dioxide flushed McDougall\'s buffer and crystalline pyrrolizidine alkaloid was added to the solution at a concentration of 10 milligrams per ml (pyrrolizidine alkaloid in phosphate buffer at pH of

6.8). One aliquot of ruminal fluid was pasteurized in each trial prior to incubation. All bottles were flushed with carbon dioxide and incubated at 38° C in a shaking incubator. Samples were taken at 0 time, 12, 24 and 48 hours and the pyrrolizidine alkaloid extracted and quantitated using high performance liquid chromotography (HPLC) . Fig. 1 shows the results.

In summarizing, as can be seen from Fig. 1 utilizing a number of sheep (n = 15) and cattle (10) trials, it was evident that the sheep ruminal fluid contained microorganisms that metabolized the pyrrolizidine alkaloid in 12 to 24 hours while only a minor portion of the pyrrolizidine alkaloid was degraded by the bovine microorganism. Pasteurization eliminated the microorganism population with the resultant persistence of the pyrrolizidine alkaloid concentrations.

In every instance the sheep\'s rumen fluid was examined and found to contain the following genera of bacteria: Streptococcus sp., Peptococcus sp., Lactobacillus sp. and Bacteroides sp. In some in¬ stances they also contained Veillonella sp. and Eubacterium sp.

EXAMPLE 2

It has been found that the microorganisms in the sheep\'s rumen can be environmentally induced with a resultant increase in the rate of detoxification of pyrrolizidine alkaloid. This was accomplished by feeding the previously unexposed sheep 5% Tansy Ragwort plant in their daily diet. In these trials, the time of detoxification was decreased from the mean of approximately 48 hours to between 12 and 24 hours,

depending on the particular animal evaluated. This is illustrated in Fig. 2 showing the effect of Tansy Ragwort conditioning on PA conversion.

Additional in vitro tests have induced the bacteria to further decrease the detoxification time. In particular re-spiking of in vitro rumen fluid daily for four days with 10 micrograms per milliter of pyrrolizidine alkaloids decreased the mean of 48 hours to within the range of 6 to 10 hours. This indicates that the bacteria and/or enzymatic pathway that metabolizes the alkaloid can be induced over several days.

EXAMPLE 3

A third set of experiments were designed to isolate the type of microorganisms in the sheep\'s ruminal fluid that were responsible for the pyrrolizidine alkaloid detoxification. Differential centrifugation of full ruminal fluid was done and the resultant supernatants tested for their ability to metabolize pyrrolizidine alkaloids compared to their respective uncentri-fugated control fluid. The rates of detoxification from 15 separate trials were analyzed. Analysis of the data combined with microscopic evaluation of the supernatants determined that the prime degrading microorganisms were a consortium of bacteria of the following genera: Streptococcus sp., Peptococcus sp., Lactobacillus sp., Bacteroides sp., Ruminococcus sp..

Gas chromatography-mass spectro photometer data on spiked whole ruminal fluid as well as the supernatants of centrifugation indicated that the pyrrole ring that is the backbone of the pyrrolizidine alkaloids was broken down during the degradation process.

If similar experiments are conducted on other EPA priority pollutants, particularly phenols and triazines and perhaps polynuclear aromatic hydrocarbons, phthalate esters and chlorinated hydrocarbons similar results may be obtained. In particular for phenols and triazines in each instance similar decreases in the level of toxicants in use of whole sheep rumen fluid were noted. Namely GC/MS analysis of rumen fluid reveals lack of structure of ring compounds characteristic of toxicant peaks.

The ability of this consortium of bacteria to degrade certain EPA priority pollutants offers the opportunity for many uses. In particular the bacteria consortium in a conventional nutrient-carrier medium can itself be used as a detoxifying composition. Alternatively it may be used as a silage inoculant. It also, can be combined with rumen fluid of other animals, generally at a range of about 50/50 and then used to treat for example cattle and horses to provide them the same unique toxic resistance that sheep rumen fluids naturally provides, if used as a probiotic, the composition may contain 70% sheep\'s rumen fluid and 30% cow\'s rumen fluid for use with bovine. Ruminal fluid levels of from 50:50 sheep:cow may range from 50:50 to 70:30. It may also be possible to simply add the isolated organisms directly to the bovine rumen and achieve similar results.

EXAMPLE 4

The purpose of this example was to determine the length of time required for complete disappearance of trinitrotoluene (TNT) and its intermediates as measured by thin layer chromatography (TLC) analysis. A consortium of bacteria derived from sheep rumen fluid and stored in a glycerol medium was used.

Ten milliliters of anaerobic minimal medium amended with a carbon source e.g. (glucose) was inoculated with a mixed culture of the earlier described organisms obtained from a freezer storage file. The culture was incubated overnight at 37°C, producing a very turbid culture. Again, late the following day, 1.0ml of the turbid culture was transferred to 9.0ml of the minimal medium with added glucose. The transfer culture was spiked with TNT dissolved in 95% ethanol to a concentration of 100 parts per million and incubated overnight at 37°C. The following morning 10ml of inoculum culture was transferred to 490 ml of the amended minimal medium. The medium was then spiked with TNT dissolved in 95% ethanol to a concentration of 100 ppm. The flask was closed with a rubber stopper and inverted several times to mix cells and TNT throughout the flask. Three types of samples were taken. A 2.0ml sample was taken for a measurement of absorbance and pH and was then filter sterilized and frozen for analysis of glucose concentration at a later time. A 1.0ml sample was taken for a delution series to estimate colony forming units (CFU) per milliliter of medium. A 0.5ml sample was taken for TLC analysis, acidified with 50 microliters of twelve normal sulfuric acid and frozen until analysis. All three samples were not taken at all sampling times, as indicated in the sampling table schedule below. The flask was closed with a rubber stopper that allowed a temperature probe to be inserted into the flask and the temperature inside the flask to be monitored. Though the incubator had a digital display and was set at 37°C a thermometer was placed inside the incubator to monitor the incubator temperature. Room temperature was also monitored.

Plates from the dilution series were incubated at 37°C and counted after 24 hours incubation. TLC samples were analyzed according to the TLC protocol outlined in Applied and Environmental Microbiology, June 1976, P. 949-958, Vol. 31, No. 6, McCormick et al, "Microbial Transformation of 2,4,6-Trinitrotoluene and Other Nitroaromatic Compounds".

Table of Sampling Schedule

The Fig. 1 graph shows TNT concentration, culture growth, and changes in pH. It reveals that after 24 hours, there was no trinitrotoluene detected, and only degradation products. The pH initially decreased during the first 24 hours and then gradually rose. At the same time the organism count increased up to 24 hours, decreased slightly from 24 to 48 hours, and then increased. These results are indicative of successful degradation of trinitrotoluene by the organisms present, indicating usefulness of the consortium for remediation of contaminated materials containing contaminates such as TNT.

EXAMPLE 5

A set of experiments was run to determine the degradation of a radiolabelled C..TNT (trinitro¬ toluene) . This radioactive label was in each carbon position of the toluene ring structure. The TNT was incubated with whole rumen contents and the sequentially produced degradation products were identified, especially those with known toxicity. The metabolites and breakdown products were separated by high performance liquid chromatography (HPLC) and detected by UV absorbance as well as a radioactive detector on the eluent stream of the HPLC column.

External standards were trinitrotoluene, aminodinitrotoluene, diaminonitrotoluene, and triaminotoluene. Since these reductive intermediates could not be commercially obtained, compounds that were used were those that had nearly the same elution profile and additionally synthesized a 2,6-diamino-4- nitrotoluene as one of the intermediates.

The HPLC program used two solvents, one being a phosphate buffer and the other being a trisolvent of

70% chloroform, 20% methanol, and 10% methylene chloride. This solvent was ramped in a 30-minute program. The program ramped for the first 10 minutes from 100% phosphate buffer to 40% phosphate buffer and 60% trisolvent, then held isocratic for five minutes, and continued to ramp to 80% trisolvent and 20% buffer solution. This program showed good separation of all the different isomers of toluene and aminotoluenes as they degraded. The detectors for the HPLC were in¬ line detectors, the first being UV absorbance followed by the radioactive decay counter. The UV detector was set at 254 nanometers and a sensitivity of .01-.05 full scale. The radiolabel detector took the eluent from the HPLC and tested it through a solid phase decay detector with sensitivity of 1 ppb. A standard mixture was injected as an external control at the beginning and end of each run.

Nitro and amino substituted toluenes were extracted from the reaction mix using a chloroform:methanol solvent extraction protocol. One ml of aqueous rumen fluid was added to this chloroform:methanol mixture. An 84% efficiency of extraction of the trinitrotoluene and similar extraction efficiencies out of the aminotoluenes was observed.

The data from these experiments, where rumen fluid was taken from a sheep, spiked with radiolabelled trinitrotoluene, and analyzed on a time course of 0, 3, 4, and 24 hours, was observed and recorded.

The control samples revealed that the system was operating and should be capable of detection of labeled TNT and its degradation products. At 0 time HPLC labeled TNT was noted present and most of the

counts observed were TNT, although some counts were observed as the monoamino peak. At 3.0 hours no peak was observed for TNT and the only peaks noted were the amino dinitro-toluene and the diamino nitrotoluene as indicated by both ultra violet (UV) absorbance and radioactivity. At 4.0 hours the same two intermediates were observed and the predominate form was the diaminonitrotolune. At 24.0 hours both compounds were still detectable, but almost all of these intermediates were gone. Most of the radioactive material detected at this time appears to have been triamino toluene and there was very much less quantitatively than would be expected for the amount of TNT originally spiked. This example indicates that whole sheep rumen fluid can be used to successfully degrade or bioremediate contaminants such as TNT.

It can be seen that the invention accomplishes at least all of it\'s stated objectives.