PROCESS FOR THE DEGRADATION OF ENVIROMENTALLY PERSISTENT ORGANIC COMPOUNDS

Abstract: A process is described for degrading organic pollutant compounds using fungal enzymes containing a lignin de­ grading enzyme using hydrogen peroxide. The process par­ ticularly uses Phanerochaete chrysosporium as a source of the hydrogen peroxide and the enzymes. The process is par­ ticularly useful for degrading halogenated hydrocarbons with the halogens substituted in bicyclic rings, which are resistant to biodegradation and which occur in the environment as persistent pollutants.

Description: PROCESS-FOR THE DEGRADATION Of ENVIRONMENTALLY PERSISTENT ORG.NNIC COMPOUND5 BACKGROUND OF THE INVENTION (1) Field of the Invention The present invention proposes and demonstrate-s a method for degrading environmentally persistent organic compounds. In particular the present invention uses a common fungus Phanerochaete chrysosporium and its biodegrading enzymes for such degradation. This work was performed under Environmental Protection Agency Contract No. CR-811464-01-0. (2) Prior Art Large quantities of synthetic organohalides are preset in the biosphere as environmental pollutants. As a group, organohalides are often toxic and/or carcinogenic. bdditionally, many organohalides are persistent in the environment and tend to bioaccumulate in the body fat of animas oecupying higher-trouphic levels. One reason for their persistence is the fact that microorganisms which normally possess the ability to degrade or metabolize a wide spectrum of organic compounds are either unable to degrade these persistent chemicals or do so vr very slowly. The environment is contaminated with relatively persistent polyhalogenated bicyclic organic compounds. Polychlorinated biphenyls (PCB's) in the organic layer on the bottom of the Kalamazoo River and polybrominated biphenyls (PBB's) in the Gratiot County landfill in Michigan and many other areas are example. Frequently these contaminated sites are biologically inactive because they are essentially anaerobic or lack the necessary sicroorganisms for biodegradation. The most complete solution to environmental contamination by halogenated organic compounds proposed to date is incineration; however, this is usually impractical where there are large volumes of waste to be processed or where the matrix or material containing. the compounds is difficult to process. One possible alternative solution is a temporary aerobic landfill which would accomplish the degradation using microorganisms The problem is that most microorganisms are only able to metabolize the halogenated organic compounds partially-and selectively and are greatly affected by the intermediate compounds encountered in' the biodegradation. Lignin degradation is an enzymatic process which can be accomplished by some fungi. This is particularly shown by Forney, L. J. et al, Journal of Biological Chemistry 257, 11455-11462 (1982) and by Tien, M. et al, Science 221, 661-663 (1983). The process which occurs is a natural one. Lignin is one of the most abundant (250 million tons per year) and persistent natural chemicals. Its structure is variable and its molecular weight is relatively large. Lignin is not degraded by oxidase enzymes from most microorganisms; however, lignin is degraded by Phanerochaete chrysosporium, a white rot & fungus. In the article by Forney, L. J., et al it was suggested that the hydroxyl radical, an oxidant, degrades lignin and that the white rot fungi produced this radical. Tien et al subsequently showed that the fungi produce an extracellular oxygenase enzyme which uses hydrogen peroxide to generate the oxidant. They also showed that the oxidant radical is capable of degrading lignin as well as selected substructure model compounds which are used in experiments to simulate lignin. What was not recognized by Forney et al and Tien et al was that relatively low molecular weight halogenated (Br and C1) organic compounds, particularly with halogenated aromatic ring substituents which are bicyclic or other organic pollutants, which are man-made and which persist in the environment could be efflciently degraded by this fungus. At this point it is open to question as to whether a hydroxyl radical is responsible for the oxidation; however, it is clear that the oxygenase is involved in lignin degradation. Eriksson, K. et al Arch. Microbiol. 135:161-168 (1983) describes a mutant white rot fungi which has no cellulase activity and yet still degrades lignin. U.S. Patent No. 4,478,747 to Crawford et al discloses various white rot fungi used to degrade lignocellulose prior to acid polymerization. U.S. polymerization. U.S. Patent No. 3,617,436 describes various species of white rot fungi. Blanchette, R. t., Appi and Env. Microbiol. 48, 647-653 (1984) describes a large number of white rot fungi and as can be seen, there are many hundreds of them which are known and which can be used in the present invention. Leatham, F. G., et al Applied and Environmental Microbiol. 46: 191-197 July 1983 describe the degradation of various phenolic compounds. One compound 2-chloroisovanillic acid was only poorly degraded. There were no bicyclic compounds disclosed as being degraded, Objects It is therefore an object of the present invention, to provide a process for degrading organic compounds which are persistent environment pollutants. It is further an object of the present invention to provide a process which uses an oxygenase and hydrogen peroxide for the degradation of the organic compounds. Further tt is an object of the present invention to provide a preferred process which uses a fungus which generates the. oxygenase and hydrogen peroxide in situ during the degradation. Further still, it is an object of the pSesent invention to provide a preferred process which relies upon the use or Phanerochaete chrysosporium as the fungus for the b a & u generation of the oxygenase and hydrogen peroxide. Further still, it is an object of the present invention to provide a process which is simple and economic and which used not generate stable unwanted chemical intermediates, especially toxic intermediates. These and other objects will become increasingly apparent by reference to the following description and the drawings. In the Drawings Figure 1 is a graph showing 14C02 evolution from 14C-DDT in Nitrogen Deficient Cultures of P. chrysosporium. Cultures of P. chrysosporium were incubated at 370-390C as described by Kirk et al. (rch. Microbiol. 117, 277-285, 1978). Each culture contained 1.25 nmoles (50 nCi or 110,00-0 dpm) of 14C-labeled DDT. Figure 2 is a graph showing 14C02 Evolution by Nitrogen Deficient Cultures of-P. chrysosporium containing selected 14C-labeled Organohalides. Cultures of P. chrysosporium were incubated at 370-39"C as described by Kir et al. (Arch. Microbiol. 117, 277-285, 1978). Each culture contained 50 nCi (110,000 dpm) of a 14C-labeled organohalide corresponding to a mass of 0.93, 6.1, 0.34 and 1.85 nmoles of Lindane, TCDD, 2,4;5,2',4',5'-HCB and 3,4,3' ,4'-TCB, respectively. Figure 3 is a graph showing the effect of Nitrogen on the Evolution of 14cho2 from 14C-DDT in Cultures of P. chrysosporium. Cultures of P. chrysosporium were incubated at 370-390C as described by Kirk et al. (Arch. Microbiol. 117, 277-285, 1978). Each culture contained 1.25 nmoles (110,000 dpm) of 14C-labeled DDT. Nitrogen concentration (2.4 mM or 24 mM) refers to the amount of nitrogen present in the culture media as ammonium tartrate. Figure 4 is a graph showing ¯the effect of Nitrogen Concentration on the Evolution of 14cho2 from 14C-Lignin in Cultures of P. chrysosporium. Cultures of P. chrysosporium were incubated at 370-390C as described by Kirk et al. (Arch. Microbiol. 117, 277-285, 1978). Each culture contained 0.122 mg (110,000 dm) of 14C-labeled lignin. Nitrogen concentration (2.4 mM and 24 mM) refers to the amount of nitrogen present in the culture media as ammonium tartrate. Figure 5 is a graph showing the disappearance of DDT in Nitrogen Deficient Cultures of P. chrysosporium. Triplicate cultures of P. chrysosporium were incubated at 37'-39C as described by kirk et al. (Arch. Microbiol. 117, 277-285, 1978). The initial concentration of DDT was 1.7 ppm. Figure 6 is a graph showing the disappearance of Benzo(a)pyrene in Nitrogen Deficient Cultures of P. chrysosporium. Quintuplicate cultures of P. chrysosporium were incubated at 370--39-0C as described by Kirk et al. (Arch. Microbiol. 117, 277-285, 1978). Benzo(a)pyrene was extracted and quantitated as described by Selkirk et al. (Cancer Res. 34, 3473, 1974). The initial concentration of benzo(a)pyrene was 100 uM. Figure 7 is a graph showing evolution of 14C02 from 14C-Benzo(a)pyrene in Nitrogen Deficient Cultures of P. chrysosporium. Cultures of P. chrysosporium were incubated at- 370-390C as described by Kirk et al. (Arch. Microbiol. 117, 277-285, 1978). Each culture contained 1.0 umole (91 nCi or 199,800 dpm) of 14C-benzo(a)pyrene. General Description The present invention relates to a process for degrading organic pollutant compounds which comprises: providing organic pollutant compounds which persist in environmental materials; reacting fungal enzymes containing a lignin degrading oxygenase and hydrogen peroxide with the organic pollutant compounds in a reaction mixture under aerobic conditions; and degrading the organic pollutant compounds in the reaction mixture. The present invention also relates to a process for degrading organic compounds which occur as persistent environmental pollutants which comprises: providing fungal enzymes containing a lignin degrading enzyme and hydrogen peroxide with the otganiccompound in an aqueous medium under aerobic conditions; and degrading the organic compound with the enzyme and hydrogen peroxide. The present invention also relates to a process for degrading organic pollutant compounds which comprises: providing pollutant organic compounds which persist in .environmental materials admixed with the environmental materials in which they persist; reacting fungal enzymes containing a lignin degrading oxygenase and hydrogen peroxide with the organic compounds in the environmental materials in a reaction mixture under aerobic conditions; and degrading the organic pollutant compounds in the reaction mixture. The present invention also relates to a fungal composition which comprises: a biologically pure culture of a fungus which generates a lignin degrading oxygenase and hydrogen peroxide in the presence of a carbon source nutrient; a particulate cellulosic material, wherein the fungus is present in an amount sufficient to initiate decomposition of organic pollutants in environmental materials with which the composition is mixed; and an anaerobic package for the composition. In particular the present invention relates to a preferred process where the oxygenase and hydrogen peroxide are provided by a biologically pure culture of a lignin degrading fungus or fungi mixed with the pollutant organic compound. The fungus avoids the need for introducing the hydrogen peroxide and oxygenase periodically into the reaction mixture since the fungus produces both the extracellular enzyme and H2O2. White rot fungi are preferred. The most preferred fungus is Phanerochaete chrysosporium which produces a oxygenase having a molecular weight of about 42,000 daltons. The oxygenase enzyme from Phanerochaete chrysosporium or other fungi which degrade lignin can be used for the reaction with a pollutant organic compound. The oxygenase can be obtained in a purified pure form by taking a culture filtrate from the fungi, dialyzing it and then lyophilizing the dialyzate to prepare a concentrate of the enzyme. The oxygenase and the hydrogen peroxide can be reacted with the pollutant organic compound in aqueous solution or suspension. The hydrogen peroxide can be either provided directly or generated in situ using glucose and glucose oxidase, a H2 2 generating enzyme. This process is not preferred because of the cost ofisolating the enzyme which is an unnecessary step. The preferred Phanerochaete chrysosporium was grown in a low nitrogen containing growth medium which has been found to stimulate lignolytic activity. The cultures were preferably grown at 37-39 C for six (6) days in a source of carbon, nitrogen, minerals, and the vitamin thiamine as described by Kirk et al. (Arch. Microbiol. 117, 277-285, 1978). The pollutant organic compounds can be mixed with various aerobic matrices using a small volume of acetone which may then be allowed to evaporate. The matrices include lignin, cellulose (shredded paper), wood shavings, sawdust and humus. Water can be added to the matrix to give 50% by weight moisture and then the fungus can be added. The incubations are at 390C (preferably between about 37 and 40"C) in air or oxygen. EnvF nmental samples can be degraded, such as the PCB's in Kalamazoo, Michigan river sediments which may or may not require a cellulose matrix depending upon the amount of organic nutrients already present. Unexpectedly it was found that the secondary metabolism (which induces the oxygenase), resulted in complete degradation of the pollutant compounds to C02. This result is unprecedented as far as the inventors know, since in many instances there are halogenated organic compounds formed in biodegradative processes which are not degraded to C02 by microorganisms. Specific Description The Eollowing is a specific description of the process of the present invention. Example 1 In order to test for the preferred growth temperature for the Phanerochaete chrysosporium, various cultures were prepared and tested. Six (6) separate 10 ml liquid cultures of Phanerochaete chtysosporium were prepared using 1 ml of a spore suspension in 9 ml of a culture medium as described by Forney et al. The culture medium was low in nitrogen source which, based upon the results of Forney et al, appears to induce the activity of the fungal culture. Two of the cultures were incubated at 37C and four were incubated at room temperature. It was found that the fungus grew better at 370C, although significant growth did occur at room temperatures. Example 2 In this experiment 2,4,5,2',4',S'-hexabromo biphenyl (HBB) was used as a model halogenated bicyclic organic compound. This compound is a persistent man-made environmental pollutant. The compound was incubated in an aqueous solution containing four (4) day old cultures of Phanerochaete chrysosporium as described by Forney et al. Thiamine is a desirable ingredient for this fungus. There were fungal extracts in three (3) separate vials. The vials were incubated at 370C and the vials were tested at 0, 2 and 6 days post inoculation for the presence of the HBB. The contents of each vial upon testing was quantitatively transferred to a 15 ml graduated centrifuge. tube with rinses using acetone. The volume was adjusted to 10.0 ml in acetone. A 5.0 ml aliquot of each of these samples was evaporated to approximately 0.5 ml extracted with 5 ml of toluene and passed over a 9 centimeter x .7 centimeter Florisil column with elution by 10 ml of toluene. The samples were then adjusted to a final volume of 10,0'ml with toluene and subjected to gas chromatographic analysis. Bas°d upon the analysis the results are shown in Table 1. Table 1 Sample (Time) Percent Recovered 0 days 81.9 2 days 23.3 6 days 18.3 As can be seen from Example 1, the concentration of HBB was 5ubstantially reduced as a result of fungal degradation. Ove.r a period of time the RBB or intermediates thereof could be completely degraded by the process by repeated treatments. example 3 In this example DDT 1,1-BisX4-chlorophenyl)-2,2,2-trichloroethane) was assayed for degradation by Phanerochaete chrysosporium. This compound is also bicyclic. Two 10 milliliter cultures in 125 ml Erlenmeyer flasks received 3.4 microliters of DDT in acetone (50 mg/lO ml) which produced a concentration of 1.7 ppm DDT. At the time the DDT was added to the culture, the culture was four (4) days old. The cultures were incubated at 370C with the DDT. Eighteen (18) days later the incubation by the Phanerochaete chrysosporium was terminated by adding 25 ml of acetonitrile to the culture and the mixture was homogenized in a homogenizer (10-20 Stokes). The mixture was transferred to a separatory funnel and vigorously shaken (1 minute) with 2 milliliters of a saturated sodium chloride solution along with 60 ml of water. Approximately 60 ml of petroleum ether was then added to the mixture which was vigorously shaken in the sepaZ y funnel for about 1 to 2 minutes. The aqueous phase was rated and saved. The organic phase was collected in g 100 ml volumetric flask. Approximately 40 ml of petroleum ether was used to rinse all the glassware, the aqueous layer was re-extracted with this volume of petroleum ether, the organic layer was then drained into the 100 ml flask to the 100 ml mark. A control culture was spiked with 3,4 microliters of DDT (5.0 milligrams/milliliter acetone) and extracted in the same manner. In the same manner 1,1 milliliters of DDT (5.0 milligrams/milliliter acetone) in petroleum ether was used as a further control The culture extract, the extracted &commat;trol and the DDT petroleum ether were assayed by gas ctOgraph as in Example 2. Extracts were diluted 1:10 for gas chromatographic analysis to give a 100 final dilution. The result was that 70.0* of the original concentration of DDT remained after 21 days of incubation with the fungus at an initial concentration of 1.7 ppm of DDT. Example 4 The experiment of Example 3 was repeated, except that the cultures were incubated with the DDT in an atmosphere of oxygen and at 390C. The cultures were treated with oxygen for one (1) minute at 3 day intervals prior to testing, The results are shown in Table 2 for two separate tests. Table 2 Day % of DDT Remaining 15 39.9 40.5 21 20.9 25.9 33 26.4 20.9 As can be seen from this Example, the DDT is in fact degraded over a period of time. Example 5 In this experiment 14C labeled DDT was used. Triplicate cultures were prepared with Phanerochaete crysosporium fungus. The triplicate cultures were prepared using 125 ml Erlenmeyer flasks as described by Kirk et al Archives of Microbiology 117 277-285 (1978). The purpose was to determine whether 14cho2 would be produced. - Each culture contained 110,000 dpm of 14C-DDT. The culture was assayed for carbon dioxide evolution every three (3) days. Day three (3) samples were collected for the carbon dioxide evolution studies. Oxygen was introduced into the closed culture vessel. Gases were expelled through latex tubing equipped with a 22 gauge needle and bubbled into a carbon dioxide trap (5 ml scintillation fluid, 1 ml ethanolamine and 4 ml methanol). The time for gassing was 15 minutes per sample. In the same manner, the day six (6) samples were collected and so on every three (3) days for a total of 57 days. The results are shown in Table 3. Table 3 C02 Trapping From 14C-DDT DPN DPX DPM A-O 10.4 B-O 4.5 C-O 1.4 3 15.8 3 8.1 3 3.2 6 43.7 6 396.7 6 6.7 9 300.8 9 965.3 9 36.7 12 518.5 12 1290.2 12 193.5 15 909.3 15 1582.6 15 471.5 18 1224.1 18 1963.0 18 900.8 21 1826.7 21 2568.3 21 1313.3 24 2274.6 24 3013.0 24 1623.1 27 2630.3 27 3656.6 27 1810.7 30 2992.3 30 4030.4 30 2037.3 33 3370.3 33 4525.4 33 2238.1 36 3793.5 36 4929.9 36 2375.2 39 4172.8 39 5189.9 39 2522.5 42 4527.2 42 5301.6 42 2641.3 45 4915.9 45 5399.8 45 2699.0 48 5507.3 48 5478.9 48 2746.9 51 5659.5 51 5528.8 51 2793.0 54 5745.9 54 5578.8 54 2834.7 57 5855.7 57 5638.3 57 2892.3 The samples were assayed for radioactivity on a Packard" Tricarb Liquid Scintillation Spectrometer. The results are graphically shown for each of the three samples in Figure 1. It appeared that considerable variation occurred in the identical cultures in the rate of 14cho2 production. Once carbon dioxide evolution began in each culture, the slope, i.e. the rate of carbon dioxide evolution was the same for all three cultures. It was observed that no carbon dioxide evolution occurred in any culture until day 6. This result coincides with that found in the literature for lignin degradation by Tien et al and Forney et al. Example 6 The degradation of other 14C-labeled halogenated organic compounds by Phanerochaete chryrosporium was performed. Thus 14 C-2, 4, 5, 2', 4', 5'-hexachlorobiphenyl; 14C 3, 4, 3', 4'-tetrachlorobiphenyl; 14C-lindane and 14C-2,3,7,8, tetrachlorodi-benzo-p-dioxin were also metabolized to 14CO2 by Phanerochaete chrysosporium. At the same time it was to be determined whether 14CO2 evolution from 14C lignin coincides with the 14CO2 evolution from the halogenated organic compounds. Finally experiments were conducted to determine if high nitrogen concentration inhibited carbon- dioxide evolution from 14C-DDT. This phenomena of high nitrogen inhibition of CO2 evolution is observed when 14C-labeled lignin is used as substrate. Protocol: Cultures were prepared in quintuplicate for 14C-DDT, 24C-2,4,5,2',4',5'-8CB, 3,4,3',4'-TCB, 14C-Lindane.and 14C-TCDD. The culture medium was the same as that used in Example 5. An additional set of five (5) cultures were prepared for 14C-lignin. Still another five t5) cultures were prepared for 14C-DDT. These differed from the others in that they contained 12.0 mM diammonium tartrate (24 mM nitrogen) and were designated "high nitrogen". All cultures were equipped for 14C02 evolution studies as described by Kirk et al (Arch. Microbiol. 117, 277-285 (1978). All cultures were incubated at 37-390C for 3 days under ambient atmosphere. At day 3, the first time pofPts were taken by flushing with 99.7% 2 as described by Kirk et al (Arch. Microbiol. 117, 277-285 (1978). Subsequent time points were taken at three (3) gy intervals. In addition to test flasks, a control was run in which all the components of the culture media were present except the fungus. RESULTS TABLE 4 Lignin Day dpm/3 days Running Total (dpm) 3 44.3 + 20.6 44.3 6 10,888.5 + 2191 10,932.8 9 19,257 t 2056 3'0,189.8 12 9559.7 t 1076 39.749.5 15 5t575.2 t 600.9 45,324.7 18 2,-144.3 t 609.3 47,469.0 21 983.0 + 223.9 48,452.0 24 501.9 + 112.9 48,953.9 27 426.7 t 112.1 49,375.6 The results are shown in Figure 4 TABLE 5 DDT High Nitrogen .Regular" Nitrogen Levels Day dpm/3 days Running Total dpm/3 days Running Total 3 0 0 1.7 + 1.5 1.76 6 50.5 + 13.8 50.5 811.7 + 127 813.4 9 374.0 +166.6 424.5 1525.5 t 197.3 2338.9 12 172.0 + 25.4 596.5 1566.9 t 285 3905.8 15 119.4 + 17.5 715.9 1930.8 t 341.5 5836.6 18 156.8 +100.8 872.7 1179.3 + 266.3 7015.9 21 118.8 + 44.2 991.5 296.2 + 83.7 7312.1 24 85.2 + 15.3 1076.7 164.0 t 44.0 7476.1 27 50.0 + 26.0 1126.7 140.3 + 40.6 7616.4 The results are shown in Figure 3. TABLE 6 3,4,7,8,-TCDD Day - dpm/3 days Running Total 3 7.74 + 4.0 7.74 6 631.1 +111.3 639.5 9 816.5 t134.9 1456.0 12 591.2 +182.0 2047.2 15 560.3 +185.6 2607.5 18 395,3 +141.8 3002.8 21 138.3 + 50.6 3141.1 24 71.4 t 28.3 3212.5 27 57.0 t 29.7 3269.5 The results are shown in Figure 2. TABLE 7 Lindane Day dpm/3 days Running Total 3 77.8 + 14.3 77.8 6 1957.5 +208.3 2035.3 9 1850.2 +205.7 3885.5 12 1185.1 +182.0 3070.6 15 887.5 +175.9 5958.1 18 603.5 t 76.6 6561.6 21 202.0 + 30.4 6763.6 24 89.6 t 21.3 6853.2 27 75.4 + 14.7 6928.6 The results are shown in Figure 2. TABLE 8 TCB and HCB Day dpm/3 days Running Total dpm/3 days Running Total 3 4.5 + 1.6 4.5 0 0 6 211.0 + 42 215.5 167.1 t13 167.1 tl3 9 226.1 t 59.7 441.7 369.9 +36.9 537 12 163.3 + 46.7 605.0 335.8 +20.1 872.8 15 144.8 + 39.3 749.8 287.4 +17.6 1160.2 18 135.9 + 92 885.7 321.3 +23.6 1481.5 21 47.0 + 20 932.7 81.5 t20 1563.0 24 26.1 + 6.8 958.8 39.5 t 4 1602.5 27 16.9 t 12.9 975.7 43.4 + 8.4 1645.9 The results are shown in Figure 2. Comments and Conclusions 14CO2 evolution from 14C-DDT by Phanerochaete chrysosporium was a reproducible phenomena. In addition to 14C-DDT, Phanerochaete chrysosporium was able to metabolize 14C-2,4,5,2'4',5',-hexachlorobiphenyl (HCB), 14C-3,4,3',4'-tetrachlorobiphenyl (TCB), 14C-lindane, and 3,4,7,8-tetrachlorodibenzo-p-dioxin (dioxin) to 14CO2. 14CO2 evolution from 14C-lignin coincides with 14CO2 evolution from 14C-2,4,5,2',4',5',-hexachlorobiphenyl, 14C-3,4,3'4', tetrachlorobiphenyl, 14C-lindane an 2,3,7,8,-tetrachlorodibenzo-p-dioxin. This suggested that the lignin degrading system of Phanerochaete chrysosporium was required for degradation of these organohalides. High nitrogen concentration suppressed 14cO2 evolution from 14C-DDT. This was the same phenomena observed with 14CO2 evolution from lignin (Kirk et al., Arch. Microbiol. 117, 277-285, 1978). This, fqrthçg indicates that the lignin degrading system of Phanerochaete chrysosporium is involved in degradation of they organohalides. example 7 Purposes To demonstrate reproducibility of initial finding shown in DDT degradation. Protocol: Eight cultures of Phanerochaete chrysosporium containing 1.7 ppm DDT were set up in triplicate. In addition, two control cultures (minus Phanerochaete chrysosporium) were set up in duplicate. Cultures were sealed with a t5 rubber stopper and incubated in the dark at 370C. On days 3, 6, 9, 12, 15, 21 and 30, three cultures and two control cultures were analyzed for DDT concentration. Because the final concentration of the standard was 170 ppb (3.4 microliter DDT) (5.0 microgram/milliliter diluted to 100 ml with petroleum ether), this value was chosen for calibration. Note, 170 ppb. is also the concentration one would expect if no DDT was metabolized. An exception to the procedure previously described was that after the extract was collected in a 100 ml flask, no further dilution was required for gas chromatographic analysis. A standard was injected before and after each assay. The average of the two standards was used to determine the % DDT remaining. Each culture was assayed three times for DDT concentration and the average was determined. The values obtained for each of the three cultures were averaged and the average S.D. was calculated. TABLE 9 Day (1) Culture (2) Culture (3) Culture Ave + S.D. 3 88.2 85.1 87.9 87.1+ 1.4% 6 87.7 76.7 84.5 83.0 t 4.6% 9 74.2 63.2 74.7 70.7 t 5.3% 12 61.4 70.9 65.7 66.0 + 3.9% 15 68.0 57.7 44.1 56.6 + 9.8 21 85.2* 45q5 46.1 58.9 #18.6 *believed to be an error in reading The study was repeated and the results are shown in Figure 5. The results were reproducible and comparable to the earlier studies. Example 8 Figures 6 and 7 show the results for the degradation of benzo(a)pyrene with Phanerochaete chrysosporium in the manner of Examples 6 and 7. As can be seen, this resistant chemical is degraded to CO2. The studies using 14C-labeled compounds demonstrated that all the compounds tested (DDT, 2,4,5,2',4',5'-HCB, 3,4,3',4'-TCB, TCDD, Lindane, and benzo(a)pyrene) were degraded to carbon dioxide by nitrogen deficient cultures of P. chrysosporium. Because all of the radiolabeled compounds were labeled exclusively in ring carbons, it was concluded that P. chrysosporiu has the ability to degrade halogenated aromatic rings of environmental pollutants which are normally difficult to degrade using microorganisms. It is also apparent: that P. chrysosporium is able to dechlorinate alkyl chlorides as evidenced by the fact that Lindane, a compound which La chlorinated on every carbon atom, is degraded to carbon dioxide. Collaborating evidence was obtained in DDT metabolite studies in which it was demonstrateQ that DDT is converted to DDD, as a result of reductive dechloinOtion Although all radiolabeled compounds assayed could be degraded to 14CO2, this degradation was a relatively slow process. This is not a problemtl environmental degradations. The highly chlorinated PCB congener 3,4,3',4'-TCB and 2,4,5,2',4',5'-E1CB exhibited rates (pmoles of 14cm2 evolved per culture per 30 days) of 16 and 91, respectively. DDT and TCDD showed rates of 10 and 34, while Lindane, a non-aromatic polychlorinated pesticide and benzo(a)pyrene, a non-halogenated polyaromatic hydrocarbon showed the highest rates of 58 and 7, respectively. In all cases, 14cm2 evolution was induced between the third and sixth day of incubation and showed a maximum rate between the third and eighteenth day of incubation after which 14cho2 evolution continued at decreasing rates until the end of the thirty day incubation. Using 14C-DDT demonstrated that 14cho2 evolution was dependent on the presence of glucose. After the 30 day incubation period, increased rates of 14cho2 evolution could be induced simply by adding glucose to the glucose depleted culture. It was also shown that the rate of 14C02 evolution was dependent on the absolute concentration of glucose in the culture medium. For example a two-fold increase in the glucose concentration (from 56 mM to 112 mM) was shown to more than double the amount of 14C02 evolved over the 30 day incubation period. It is important to note that even in stationary phase cultures constitutive levels of 14cho2 evolution continued to occur. In other experiments, the rate of DDT disappearance was monitored by GLC. It was shown that 50-60% of the DDT initially present in culture wa-s metabolized during the 30 day incubation period. DDD dicofol and DBP were identified as major metabolites. Upon addition of more glucose and continued incubation for 18 more days, it was demonstrated that more than 90% of the DDT initially present had been metabolized. Furthermore, there was no significant accumulation of identifiable DDT metabolites. By comparison, lignin degradation in P. chrysosporium was induced in nitrogen deficient cultures after approximately 3 days of incubation, after which 14cm2 evolution from 14C-lignin was observed. Maxima rates of lignin degradation, as assayed by 14CO2, were observed between day 3 and day 18 of the incubation. Lignin degradation continues to occur, albeit at successively lower rates, until the glucose concentration was depleted. The presence of a suitable carbohydrate source was critical in this system because hydrogen peroxide, a required cosubstrate for lignin degradation, was generated in actively respiring cultures. The same pattern of induction at day 3, maximal activity between days 6 and 18, and subsequent metabolism until the glucose supply was depleted was observed in 14cm2 evolution studies in which ring labeled 14C-DDT was presented as substrate. Whereas nitrogen deficiency induces 14C-lignin degradation to 14cho2, nitrogen sufficient cultures suppressed lignin degradation. Again, the same pattern was seen for the degradation of 14-DDT. Previous efforts using microorganisms have focused on a number of strategies to demonstrate or enhance their relative abilities to degrade various persistent xenobiotics. For example, there are a number of studies in which microorganisms have been adapted to grow on carbon sources whose structure resembles the organic pollutant under study. When the microorganism is presented with the structurally similar organic pollutant, it too is often metabolized. This is known as analog induced metabolism. Still other investigators have attempted to culture microorganisms using the organic pollutant itself as the sole carbon source with the intent to isolate strains that could be used to inoculate areas in the environmeq that have been contaminated with the pollutant in question, Other studies have focussed on the use of anaerobio organisms. This strategy has been adopted because many organic pollutants are found in lake and river sedl entg and landfills, sites where oxygen availability is limiting. Suflita et al (Science 18, 1115 1982), for example have demonstrated that a methanogenic bacterial consortium from sewage sludge possess the ability to dehalogenate and sometimes metabolize a variety of halobenzoates to methane and carbon dioxide. The present invention has focused on the use of P. chrysosporium as a useful organism in aerobic biodegradation. Fungi in general are the principle degraders of bio-organic molecules in nature. Because most organic nutrients are presented to fungi as large, insoluble macromolecular complexes, the organism must first degrade these large complexes to smaller substituents which may then be internalized and used as a source of nutrients. This is accomplished by the secretion of a battery of stable extracellular enzymes which may include proteases, lipases, cellulases and, in the case of P. chrysosporium, ligninases. Since many organic pollutants are physically absorbed in soils and sediments, thus making them unavailable for metabolism, these extracellular enzymes provide a mechanism whereby organic pollutants are released from these barriers to metabolism. The enzyme system of P. chrysosporium is deserving of special note because it is non-specific and non-stereoselective in its action. This is precisely the types of mechanism which one would select for site decontamination especially when one considers that contamination by only one compound is likely to be the exception rather than the rule. It has been postulated that selective pressure to induce the evolution of microorganisms capable of degrading recalcitrant man-made compounds may occur only in ecological niches where the organopollutant is present in concentrations high enough to cause enzyme induction and subsequent metabolism of the compound in question. Therefore pollutants present in low concentrations may not be metabolized. In P. chrysosporius, we have shown that, like lignin degradation, organohalide degradation is induced, not by the substrates presence, but by nitrogen deficiency. Thus high levels of substrate need notbe present to induce the enzymes required to initiate their biodegradation. Numerous strategies have been used in the aerobic treatment of contaminated waste effluents, sludges, sediments and landfills. Activated elude processes, aerated lagoons, aerobic digestion, trickling filters, rotary biological contactors and aerobic composts are examples. The effectiveness of all of these systems is ultimately dependent upon the microorganisms present in the system. Thus it is critical that the most appropriate organisms (i.e., those with a demonstrated inherent ability to degrade a wide range of environmental pollutants) be selected for inclusion in these processes. Biotreatment systems inoculated with P. chrysosporium and fortified with a suitable carbohydrate source under nitrogen limiting conditions, can provide an effective and economical means for the biological detoxification and disposal of hazardous chemical wastes. The feasibility of using Phanerochaete chrysosporium to degrade pollutant organic compounds is clear based upon the Examples. The mechanism of biodegradation is particularly advantageous. The oxidant is generated extracellularly and it appears to have no adverse affect on the fungus. The result is that the pollutant organic compound being degraded does not have to be absorbed by the fungi in order to be degraded. The oxygenase does not have to recognize the organic compound as a substrate in order to provide the usual enzyme-substrate complex necessary for biodegrzdatlon. The enzymes secreted by the white rot fungi attack many recalcitrant organic chemicals by breaking carbon carbon bonds. Additionally, this organism causes debalgenation of organic compounds. Thus, less stable metabol are formed which may be metabolized to C02 by more convontional enzyme systems. The fungi are of the class Basidimycet and are collectively known as white rot fungi and are ubiquttous in nature. This means that the fungi can be released in concentrated form in soil or wastes containing the pollutant organic compounds since no new organisms representing a potential danger to the environment are used. The nutrients for the fungi are abundantly available and are generally present in the substrates containing the pollutant organic compounds. Examples are contaminated sediments in rivers and lakes, These sediments are generally of plant origin and highly contaminated with PCB for example in the Hudson River ia New York and, the Kalamazoo River and the Great Lakes in Michigan. All of these pollutants can be degraded. The fungi require aerobic conditions in order to generate- the oxidant. This means that various systems for aerating the environmental materials being treated need to be used. Equipment for this kind of treatment is well known. Sediments only require dredging from the bottom of a river or lake to be placed in a temporary aerobic compost. High rate composting of organic wastes would be an economic means of treatment of toxic compounds prior to ultimate usage or disposal of the waste. Hydrogen peroxide is commercially available. Thus the process of the present invention can be conducted chemically in the absence of the fungi by continuously mixing hydrogen peroxide and the enzymes from the white rot f-ungi with the pollutant organic compound over a period of time until it is degraded. However, the use of the Phanerochaete chrysosporium or other white rot fungi for this purpose is preferred since this fungus produces the oxygenase and hydrogen peroxide naturally. Further, the presence of the fungus is required if it is desired that degradation go completely to CO2. It will be appreciated that the process of the present invention can be conducted in an open system in the environment or alternatively in a closed system in a reaction vessel. In the closed system the oxygen or air can be introduced by various known means. It is preferred where environmental samples are being treated such as soils and sediments from rivers or lalces that they be combined with sawdust or other particulate cellulosic material as a source of nutrients for the Phanerochaete chrysosporium. The cellulosic materials provide glucose for the fungi which facilitates hydrogen peroxide generation. Glucose can also be added directly. Mixing in particulate cellulosic material may also physically facilitate aeration of the materials being treated, a condition which is necessary for the degradation. Since the enzyme systems secreted by P. chrysosporium attack carbon-carbon and carbon-halogen bonds, the process of the present invention can be used to degrade pollutant organic compounds. The process is particularly advantageous for halogenated bi- or di-aromatic or ring compounds which resist natural degradation in the environment. Such compounds are those described in the preceding examples. The fungi are packaged for shipment and use in a manner so that they are not activated. Generally this is accomplished using dry and anaerobic packaging conditions. It will be appreciated that the foregoing description is merely illustrative of the present invention and that there are numerous variations which will occur to those skilled in the art. It is intended thAt all of these variations be included within the scope of the present invention.

Claims: WE CLAIM: -1 A process for degrading organic pollutant compounds which comprises: (a) providing organic pollutant compounds which persist in environmental materials; (b) reacting fungal enzymes containing a lignin degrading oxygenase and hydrogen peroxide with the organic pollutant compounds in a reaction mixture under aerobic conditions; and (c) degrading the organic pollutant compounds in the reaction mixture -2 The process of Claim 1 wherein the enzymes are derived from a white rot fungus. -3 The process of Claim 1 wherein some of the degradative enzymes are extracellular. -4 The process of Claim 1 wherein the enzymes and hydrogen peroxide are provided in the reaction mixture by a biologically pure culture of a fungus. -5 The process of Claim 4 wherein the culture contains a stationary or log growth phase fungus. -6 The process of Claim 4 wherein the fungus is living. -7 The process of Claim 4 wherein the fungus is Phanerochaete chrysosporium. -8 The process of Claim 4 wherein the reaction mixture includes a carbon source which facilitates the generation of hydrogen peroxide by the fungus. -9 The process of Claim 1 wherein the mixing and degradation are at a temperature of less than the temperature which results in destruction of the enzyme. -10- The process of Claim 4 wherein the fungus is grown in a growth medium containing a minimal nitrogen level which, after growth of the fungus, increases the rate at which the organic pollutants are degraded. -11 The process of Claim 10 wherein the fungus is Phanerochaete chrysosporium. -12- A process for degrading organic compounds which occur as resistant environmental pollutants which comprises: (a) providing fungal enzymes containing lignin degrading enzyme and hydrogen peroxide with the organic compound in an aqueous medium under aerobic conditions; and (b) degrading the organic compound with the enzymes and hydrogen peroxide. -13 The process of Claim 12 wherein the enzymes and, hydrogen peroxide are provided in the aqueous medium by growing the fungus in a growth tedium to the stationary phase prior to mixing the fungus with the organic compound in the aqueous medium. -14- The process of Claim 12 wherein the fungus is Phanerochaete chrysosporium. -15- The process of Claim 12 wherein the lignin degrading enzyme has a molecular size of about 42,000 daltons. -16- The process of Claim 12 wherein the degradation occurs at a temperature that is not lethal to the organism. -17- The process of Claim 13 wherein the growth medium includes sources of carbon, nitrogen, thiamine and minerals for the cells. -18- The process of Claim 17 wherein the cells are grown in a growth medium containing a minimal nitrogen source level which increases the rate at which the halogenated organic compound is degraded. -19- The process of Claim 18 wherein the fungus is Phanerochaete chrysosporium. -20 The process of Claim 12 wherein cellulose or other suitable carbohydrate source is provided with the organic compound. -21- The process of Claim 12 wherein the aqueous medium is supplemented with a carbon source which facilitates the generation of hydrogen peroxide by the fungus. -22 The process of Claim 21 wherein the carbon source is a carbohydrate. -23 A process for degrading organic pollutant compounds which comprises: (a) providing pollutant organic compounds which persist in environmental materials admixed with the environmental materials in which they persist; (b) reacting fungal enzytnes containing a lignin degrading oxygenase and hydrogen peroxide with the organic compounds in the environmental materials in a reaction mixture under aerobic conditions; and (c) degrading the organic pollutant compounds in the reaction mixture. -24 The process of Claim 22 wherein the enzymes and hydrogen peroxide are provided in the reaction mixture by a biologically pure culture of a fungus. -25 The process of Claim 24 wherein the environmental materials contain a carbon source which facilitates the generation of hydrogen peroxide by the fungus. -26 The process of CLaim 25 wherein the enviromental material is supplemented with the carbon source. -27 The process of Claim 25 wherein the carbon source is a carbohydrate. -28 The process of Claim 23 wherein the environmental materials are soils, sludges or sediments or mixtures thereof. -29 A fungal composition which comprises: (a) a biologically pure concentrate of a fungus which generates a lignin degrading oxygenase and hydrogen peroxide in the presence of a carbon source nutrient; (b) a particulate cellulosic material, wherein the fungus is present in an amount sufficient to initiate decompo$1tion of organic pollutants in environmental nzater with which the composition is mixed; and (c) a package for the composition which prevrWts the fungi from being activated.

Inventors: AUST, STEVEN D., BUMPUS, JOHN A., TIEN, MING
Applicants: UTAH STATE UNIVERSITY FOUNDATION
Application year: 1986
Main classification: A62D3
IPC codes: C12N1, B09C1, A62D3, C02F3, C12P1, C12R1