WO1994021394A1

WO1994021394A1 - Methods for the biodegradation of environmental pollutants

Info

Publication number: WO1994021394A1
Application number: PCT/US1994/003079
Authority: WO
Inventors: James D. Stahl; Stevens D. Aust
Original assignee: Utah State University Foundation
Priority date: 1993-03-22
Filing date: 1994-03-22
Publication date: 1994-09-29
Also published as: AU6490494A

Abstract

A process for degrading and disposing of environmental pollutants by reacting those pollutants with fungal mycelia of a white rot fungus and causing the pollutants to be reduced to less toxic products. Initially, the white rot fungus is cultured in a nutrient medium unitl fungal mycelia is grown. This fungal mycelia is then contacted with the pollutant and the reduction process is allowed to begin. The reduction process can be performed in an aerobic environment. After reduction, degradation is allowed to proceed until there is a substantial amount of mineralization.

Description

METHODS FOR THE BIODEGRADATION OF ENVIRONMENTAL POLLUTANTS

BACKGROUND

1. Field of the Invention

The present invention is related to the biodegradation of environmental pollutants. More particularly, the present invention relates to the biodegradation of highly oxidized environmental pollutants in an aerobic environment.

2. Background Art

Many environmental pollutants have been disposed of in the biosphere. The term "environmental pollutants" is used herein to include those compounds which are only slowly degraded, if they are degraded at all, in the natural environment. Unfortunately, the effects of the environment, including the action of microorganisms, are not sufficient to degrade and fully dispose of these environmental pollutants.

One such category of pollutants includes ferricyanide and impermeable dyes. Another such category includes halobenzoates such as chlorobenzoate and bromobenzoate. A third such category includes nitrocompounds, such as 2,4,6-trinitrotoluene (TNT) and other explosives such as RDX, HMX, PETN, and EGDN.

As an example, the toxicity of TNT and its metabolites is well known. TNT is toxic to bluegill fish, unicellular green algae (Selenatrum capricornutum) , Microcvstis aeruqinosa. Chlamvdomonas reinhartii. tidepool copepods (Tiαriopus californicus. and oyster larvae (Crassostrea qiαus) . TNT is also a mutagen.

Attempts have been made to remove and dispose of environmentally persistent compounds such as TNT. For example, in the past, the disposal of TNT consisted of diluting TNT into streams or holding lagoons. However, at a solubility of only 125 mg/1, large volumes of water are required to remove residual explosives. As much as 500,000 gallons of waste water could be produced per day by a plant manufacturing and disposing of TNT. Unfortunately this situation has led to large areas becoming contaminated with TNT.

Substantial effort has also been directed to degradation of TNT by animals, plants, bacteria and fungi. Heretofore, such systems have involved a reductive process, as confirmed by the byproducts such as 4-amino-2,6- dinitrotoluene (4 AmDNT) , 2-amino-4,6-dinitrotoluene (2-AmDNT) , and 2,2',6,6^,-tetranitro-4,4 -diazoxytoluene. Further, while bacteria isolated from TNT contaminated soil or sludge and many fungi have been shown to reduce TNT, no more than 2% mineralization to carbon dioxide has been observed through these methods. One particular study, Parrish, F.W. Appl. Environ. Microbiol.. 34:232-233, reported that of 190 fungi representing 98 genera, 183 species were able to transform TNT to reduced metabolites, but none were able to mineralize TNT to carbon dioxide. Since the foregoing metabolites of TNT are also toxic and mutagenic, many bacterial and fungal systems which have heretofore been tested have not solved the serious problems posed of how to fully dispose of TNT.

Further, degradation of TNT by animals, plants, bacteria and fungi, has heretofore all begun with reduction in anaerobic environments. Because of this limitation, the process is limited to anaerobic areas. One problem with this is that use of the process in situ is not possible since most pollutants are found in an aerobic environment. Another problem with this is that systems for use in anaerobic environments can be both expensive and difficult to design and implement, making current systems impractical. BRIEF SUMMARY OF THE INVENTION The present invention relates to a process for bioremediation of environmental pollutants such as TNT, having high oxidation potentials, wherein the first step is reduction. It has been found that because of these high oxidation potentials, reduction, prior to oxidation, is an important step in degradation of compounds such as TNT.

The present invention also relates to a process for reducing environmental pollutants having very high oxidation potentials within an aerobic environment. The main advantage of the present invention over previous mechanisms for reducing chemicals is the ability to be performed in an aerobic environment. This allows in situ degradation of the environmental pollutants. Further, reductive systems for use in an aerobic environment are less expensive and easier to design and implement than reductive systems for use in an anaerobic environment.

The invention also provides a process in which the rate of biodegradation of chemicals is increased, and in which chemicals that would normally be toxic to microorganisms are biodegraded.

According to the process of the present invention, white rot fungi, such as Phanerocaete chrysosporium. are used . for successful bioremediation of pollutant contaminated environmental sites. Reduction of the pollutants is believed to be accomplished by contact of the pollutants with the plasma membrane redox system of white rot fungi. Since the plasma membrane redox system functions in aerobic environments, the process can occur in any aerobic environment where the fungus can be grown and where reduction of chemicals is desired.

It has been discovered that using fungal mycelium within the contaminated sites rather than inoculating the sites with fungal spores results in a successful biodegradation process. Many environmental pollutants are toxic to fungal spores. The fungal spores do not reduce the toxicity of the pollutants. Fungal mycelia, in contrast, can detoxify the chemicals rapidly so that the reaction can proceed to mineralization. Therefore, the first step of the reduction process is to grow fungal mycelia in a nutrient solution for about 5 days. Once the fungal mycelia is grown, it is then placed into the contaminated site where it can reduce the toxic chemicals.

Once reduction has occurred, the white rot fungus continues the degradation process through oxidation reactions so that a significant level of mineralization to carbon dioxide results.

It should be noted that the membrane potential reductive system, unlike other prior art reductive systems, is operative during any growth phase of the fungus.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more fully understand the manner in which the above-recited and other advantages and objects of the invention are obtained, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope, the invention will be described with additional specificity and detail through the use of the accompanying drawings in which:

Figure 1 is a graph illustrating the effect of potassium ferricyanide on TNT reduction;

Figure 2 is a graph illustrating reduction of potassium ferricyanide to ferrocyanide by washed fungal mycelia;

Figure 3 is a graph illustrating the effect of membrane impermeable dyes on TNT reduction;

Figure 4 is a graph illustrating the effect of 2 , 4 -dinitrophenol , carbonyl cyanide p- trifluoromethoxyphenyl hydrazone (FCCP) , and sodium azide on TNT reduction; Figure 5 is a graph illustrating the effect of pH on proton excretion and TNT reduction rates;

Figure 6 is a graph illustrating TNT metabolism in nutrient nitrogen deficient cultures of P__±. chrysosporium; Figure 7 is a graph illustrating TNT metabolism in nutrient nitrogen sufficient cultures of P_j_ chrysosporium: Figure 8 is a graph illustrating peroxidase activity in nutrient nitrogen deficient cultures of P. chrysosporium; Figure 9 is a graph illustrating TNT toxicity to P. chrysosporium when TNT was added on day 0;

Figure 10 is a graph illustrating TNT toxicity to P. chrysosporium when TNT was added on day 2;

Figure 11 is a graph illustrating TNT toxicity to P_s_ chrysosporium when TNT was added on day 6;

Figure 12 is a graph illustrating the effect of the amount of £^ chrysosporium mycelia on TNT reduction; and

Figure 13 is a graph illustrating the effect of TNT concentration on TNT reduction by P^ chrysosporium mycelia.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to methods for degrading environmental pollutants, such as those having very high oxidation potentials. The presently preferred embodiment of the process of the present invention comprises reacting a environmental pollutant with fungal mycelia of a white rot fungus and allowing reduction to take place. The reduction reaction may take place under aerobic conditions as well as under anaerobic conditions. The reaction is allowed to continue until the persistent environmental compound has been reduced to a non-toxic compound as desired. The white rot fungus then continues degradation so that mineralization to carbon dioxide occurs after reduction. It should be appreciated that the present invention may take place in an aqueous medium, a soil matrix, or by mixing the fungus directly with the pollutants. According to the present invention, a white rot fungus, such as I\_ chrysosporiu . degrades compounds such as TNT through a reductive process which can occur during the primary growth phase as well as during any other growth ^L phase of the fungus. As Pj. chrysosporium is the experimentally accepted standard of white rot fungi, the data presented herein was produced using that particular fungus. TNT will be mainly discussed as the environmental pollutant to be reduced by the P_-_ chrysosporium. It should be realized, however, that other white rot fungi and other environmental pollutants such as ferricyanide, impermeable dyes, and other explosives such as RDX, HMX, PETN, and EGDN, are within the scope of the present invention.

The present invention includes a process for substantially complete bioremediation of the persistent environmental pollutants comprising reduction of the pollutant, then oxidation by peroxidase activity, and then further degradation by ligninases, such that the entire process yields reduction and a substantial level of mineralization. In prior art processes, only a small amount of mineralization was achieved.

A. Plasma Membrane Redox System

The first step of the process of the present invention, the reduction of the pollutants, is believed to occur by action of the plasma membrane redox system of the fungus. As this plasma membrane redox system is operable within an aerobic environment, it is operable within any growth phase of the fungus. According to the present invention, the plasma membrane redox system of £-_ chrysosporium is a system capable of catalyzing the reduction of TNT and other environmental pollutants and then allowing further mineralization. The process of the present invention begins with the step of placing the pollutant to be reduced into contact with the white rot fungi. This can be done in an aerobic environment where the fungus can be grown and where reduction of chemicals is desired. For example, a fungus can be placed into a contaminated site, and the chemicals can be reduced in situ. When placing the fungus into a contaminated site, it is important that fungal mycelia rather than fungal spores be used. According to the process of the present invention, fungal mycelia of a white rot fungus is initially grown in a nutrient medium for about 5 days. Once it is grown, it is then added to the contaminated site where it can reduce the toxic chemicals. It must be noted that any of the nutrient media well known in the art can be used to grow the fungal mycelia. Some examples are sawdust, straw, and corn cobs. See, e.g.. Fernando, et al., Chemosphere. 1989, Vol. 19, pp. 1987-1398.

The amount of fungal mycelium to be added to the environmental site will depend upon the buffering capacity of the fungus and the pH of the environmental site. The pH of the site (or other medium in which the process is taking place) can be determined by known methods of titration. If the pH is high, more of the nutrient should be added to the site. One of ordinary skill in the art would be able to judge how much nutrient should be added to the site to enable the fungus to adjust to the pH. Only simple experimentation would be required.

Once the reduction has occurred, further degradation, consisting of oxidation, can then take place. At this point, manganese dependent peroxidases produced by the white rot fungus begin to oxidize the reduced chemicals. When the fungus produces lignin peroxidases, mineralization of the chemicals occurs.

It must be noted that in prior art systems, although chemicals have been able to be reduced by fungus, only very little mineralization occurred afterwards. No more than 2% mineralization was observed. In prior art systems, fungal spores rather than fungal mycelium were used to try to degrade the environmental pollutants. Unfortunately, as is now recognized in light of the present invention, the fungal spores were not able to reduce and detoxify the pollutants to any high degree. In contrast, by the process of the present invention, mineralization of the chemicals is realized in a significantly higher degree. At least 20% mineralization over a 30 day period has been observed, and greater amounts of mineralization can be achieved by reaction for a longer period of time, by further additions of fungal mycelium, and by other methods of desired design of the process. A "significant" amount of mineralization will be considered to be at least about 20% mineralizat-ion.

Additionally, the rate of reaction is much faster in the present invention, and can be adjusted by adjusting the amounts of materials used. For example, it has been found that the rate of reduction can be increased by increasing the pH of the environment in which the process occurs.

Successful application of the present invention depends upon several factors. These factors, as above- described, include the chemical to be degraded, the concentration of chemical, the initial amount of fungus, any nutrient used, the initial pH, and the buffering capacity of the matrix, soil or water, in which the process occurs. Initially, sufficient nutrients must be available for active growth of the fungi to occur. Additionally, sufficient initial fungal mycelia must be present to counteract any toxicity of the chemical to the fungus. The amount of fungal mycelia used in the reaction, then, is related not only to the pH of the site where the process occurs, but also to the toxicity of the chemical to be degraded and potential rate of detoxification. Again, simple experimentation by one skilled in the art could determine the appropriate amount. The more toxic and recalcitrant the chemical, either the lower the concentration of chemical or the larger amount of fungus must be used.

The natural buffering ability of fungi can also be a factor in stimulating reduction by the plasma membrane redox system. Maximum reduction occurs at high pH. However, when the pH is too high, the fungi will try to lower the pH of their environment to a more suitable level. (Typically, a suitable pH level will be between about 2.5 to about 6, and preferably around 4.5.) The fungi do this by excreting protons through its plasma membrane. The rate of this proton excretion is directly dependent on the pH. The higher the pH, the faster the rate of proton excretion. Additionally, since excretion of protons and electrons are coupled, electron excretion will occur. The electrons excreted can be used for reductions.

The buffering capacity of the soil or liquid matrix also becomes important when mineralization of chemicals is desired for the same reason. Although maximal rates of reduction occur at high pH, the optimal rate of mineralization of chemicals by _j_ chrysosporium occurs at lower pH. Therefore, the fungus will excrete protons in order to regulate the pH. The proper amount of fungus must be used to appropriately regulate the pH. Examples of successful systems are shown in the example below. Other successful combinations can be discovered by one skilled in the art without undue experimentation.

EXAMPLES

The following examples are illustrative of the process of the present invention, but are not intended to limit the scope of the present invention.

Example 1

In this example, TNT was reduced using cultures of

Phanerochaete chrysosporium. The reduction of the TNT occurred through the plasma membrane redox system of the

P. chrysosporiu . TNT was obtained from Chem Service, Inc. (West

Chester, PA) . Other chemicals, such as the dyes and inhibitors used in the examples, were obtained from Sigma Chemical Company (St. Louis, MO) .

P. chrysosporium (BKMF-1767) was obtained from the United States Department of Agriculture, Forest Products Laboratory (Madison, WI) . The microorganism was maintained on malt agar slant cultures at room temperature and routinely subcultured every 30 to 60 days.

Nutrient nitrogen sufficient and deficient cultures of P. chrysosporium were grown in 150 ml erlenmeyer flasks containing 150 ml medium consisting of 56 mM glucose, 1.2 mM ammonium tartrate, mineral salts, thiamine (lmg/1) , and Tween 80 (0.05%) in 10 mM sodium acetate buffer, pH 4.5. The cultures were inoculated with a spore suspension (10% of the total volume at OD₆₅₀ = 0.35) and grown under an atmosphere of air at 39C. The agitation speed was 200rpm.

TNT was then placed into contact with the cultures. After reduction of the TNT by the plasma membrane redox system, the concentration of the TNT was measured. The concentration of TNT, 4-amino-2,6-dinitrotoluene (4-AmDNT) , and 2-amino-4,6-dinitrotoluene (2-AmDNT) were determined by extracting 50 ul samples with 500 ul of ethyl acetate and analyzing the extracts using a gas chromatograph (Varian, Model 3700, Sunnyvale, CA) equipped with a Durabond™ DB-5 megabore capillary column (Alltech, San Jose CA) , an electron capture detector and a digital integrator (Hewlett-Packard Co., Model 3390, Palo Atlo, CA) . The injector temperature was 220C, the detector temperature was 330C, and the column was held at 185C for 2 minutes then increased to 250C at 7C/minute. Nitrogen was used as the carrier gas. Triplicate injections of triplicate samples were analyzed. Samples were taken at 0, 0.5, 1, 2, and 4 hours and the data were used to determine the initial rate of TNT reduction based on TNT disappearance. The concentration of ferricyanide and ferrocyanide were determined spectrophotometrically. Reduction of TNT was studied using nonligninolytic (nutrient nitrogen sufficient, 12 mM ammonium tartrate) cultures of P____ chrysosporium to minimize oxidation of the reduced products and to eliminate any reductive reactions of the lignin peroxidases.

The results obtained in this example indicate that TNT is reduced by the plasma membrane redox system of P. chrysosporium. An initial reduction of 0.4 nmol/ml/min. was found in five day old cultures. (Table 1) .

Table 1. TNT reduction by Pj_ chrysosporium.

TNT Reduction Rate (nmol/ml/min)

A. Aerobic

Mycelium culture 0.4 (± 0.03)

Washed mycelium 1.1 (± 0.2)

Extracellular fluid < 0.03

Lysed mycelia (a,b) < 0.03

Intracellular fraction (c) < 0.03

Killed mycelia (d,e) < 0.03

B. Anaerobic (f)

Mycelium culture 0.08 (± 0.06)

Washed mycelium 0.6 (± 0.5)

Extracellular fluid < 0.03

Lysed mycelia (a,b) < 0.03

Intracellular fraction (c) < 0.03

Killed mycelia (d,e) < 0.03

Whole cultures or various culture fractions were analyzed for TNT reduction as earlier described. The extracellular fluid was collected by filtration. The intracellular fractions were prepared as described by Hughes, Methods of Microbiology. Vol. 5B, p. 1-58, 1971. The extracellular fluids, lysed mycelia, intracellular fractions, and killed mycelia were all assayed alone and with 1.0 mM NADH, NADPH or ATP, both aerobically and anaerobically. The lysed mycelia and intracellular fluids were in 100 mM potassium phosphate buffer, pH 7.6, containing 1 mM EDTA. a. Ground at 4C for 3-30 second periods with glass beads. b. Frozen in liquid nitrogen and thawed. c. Supernatants from a or b centrifuged at 10,000 rpm for 10 minutes. d. 10% formaldehyde added. e. Autoclaved for 10 minutes. f. Reactions performed under argon.

Example 2-6

The experiment of example 1 is repeated with similar results except that the white rot fungus for in the experiments of examples 2-6, respectively, are Pleurotus ostreotus. Phellinus weirri. Trametes versicolor. Fomus fomentarius. and Phlebia radiata.

Example 7 The experiment of example 1 was repeated except that only fungal mycelia was used. Five day old cultures were filtered through cheesecloth and washed with distilled water.

Greater reduction was observed aerobically. The results illustrate that washed mycelium is more active than the entire culture.

Example 8 The experiment of example 1 was repeated, except that extracellular fluid or intracellular fractions of the cultures were placed into contact with the TNT.

No reduction was observed by the extracellular fluid or intracellular fractions of the cultures even when reducing equivalents (NADH or NADPH) or ATP were added either aerobically or anaerobically. This experiment illustrates that reduction does not take place when the plasma membrane redox system of the fungus is not present. Examples 9-12 Ferricyanide and membrane impermeable dyes have been widely used as indicators of membrane redox systems. Reduction of the dyes results in insoluble formazan salts which precipitate on the mycelial hyphae. Although redox potentials of the dyes are unknown, their relative ease of reduction are known. The inhibition of TNT reduction by the most easily reduced dyes and ferricyanide suggests either direct or indirect competition for membrane reducing equivalents. The following examples illustrate the effects of ferricyanide and membrane impermeable dyes on the system of the present invention.

Example 9 The example of example 1 was repeated, except that washed mycelia (5.0g) in sodium tartrate buffer (10.0ml, 0.22M, pH 4.5) was incubated with TNT (130uM) in the presence of potassium ferricyanide (l.OmM). The concentration of the ferricyanide was determined spectrophotometrically. As can be seen in Figure 1, addition of ferricyanide inhibited TNT reduction.

Example 10 This experiment was used to show reduction of potassium ferricyanide to ferrocyanide by the process of the present invention. Washed mycelia (5.0g) in sodium tartrate buffer (10.0ml, 0.22M, pH 4.5) was incubated with potassium ferricyanide (l.OmM). The concentration of ferricyanide and ferrocyanide were determined spectrophotometrically and can be seen in Figure 2. The concentration of ferricyanide decreased while the concentration of ferrocyanide increased, thereby illustrating reduction of the ferricyanide. Example 11 This experiment was used to show the effect of membrane impermeable dyes on TNT reduction. Washed mycelia (5.0g) in sodium tartrate buffer (10.0ml, 0.22M, pH 4.5) was incubated with TNT (130 μM) in the absence or presence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) , tetranitroblue tetrazolium (TNBT) , nitroblue tetrazolium (NBT) , 2,3,5-triphenyl tetrazolium (TTC) , or blue tetrazolium (BT) , (l.OmM). The results of the experiment can be seen in Figure 3. MTT and TNBT both inhibited TNT reduction and were themselves reduced. The impermeable dyes were found to be reduced by the fungal mycelium.

Example 12 This experiment was used to show the effect of 2,4- dinitrophenol, FCCP and sodium azide on TNT reduction. Washed mycelia (5.0g) in sodium tartrate buffer (10.0 ml, 0.22M, pH 4.5) was preincubated with 2,4-dinitrophenol, FCCP or sodium azide for 15 minutes, then incubated with TNT. The results can be seen in Figure 4.

FCCP and 2,4-dinitrophenol are compounds that disrupt membrane potential gradients. Both compounds act to equalize the concentration of protons across membranes. (Without a proton gradient there would be no driving force for reduction.) It was found that when the membrane potential was disrupted, TNT reduction was inhibited.

Dinitrophenol was found to be a stronger inhibitor than FCCP probably because the nitro groups may be reduced, therefore acting as a competitor for the reducing equivalents as well as disrupting the proton gradient of the membrane.

Example 13

This experiment was performed to show the effect of pH on proton excretion and TNT reduction rates. Washed mycelia (5g) in potassium phosphate buffer (10m, 1.0M, pH

4, 5, 6, 7, 8,) was incubated with TNT (130uM) . Concentration of protons generated was calculated from the pH change. TNT reduction rate was calculated as described in example 1. The results can be seen in Figure 5. As the pH was raised from pH 4 to pH 8, the rate of reduction of TNT increased.

Example 14-16 The present invention has also been found to reduce halobenzoates. An experiment was conducted wherein various halobenzoates (5mM) (3-Chlorobenzoate, 4-Chlorobenzoate, and 4-Bromobenzoate) were incubated with 1 gram wet weight of £i chrysosporium washed mycelium. Incubations were conducted in 200 mM phosphate buffer, pH 8.0, with 1 mM glucose with rotary shaking (200rpm) at 37C. The halobenzoates remaining after 90 minutes of incubation were extracted after acidification to pH 2 (using HCL) with 2 consecutive 2 ml extractions of diethyl ether. The ether was removed and the samples redissolved in methanol and analyzed by HPLC using a C18 column with absorbance monitored at 254 nm.

The results are as shown in Table 2. TABLE 2

Halobenzoate Amount Dehalogenated mM/hr 3-Chlorobenzoate 1.4 4-Chlorobenzoate 1.5 4-Bromobenzoate 1.5

Example 17

P. chrysosporium is grown on rye in order to prepare a fungal inoculum. The cultures are prepared under sterile conditions and grown and 37C. The inoculum is mixed (20%) with a locally available, economic nutrient.

Approximately 5 days after inoculating the substrate (i.e.. after good primary fungal growth is achieved, , the inoculated nutrient is mixed with 2.5 kg (dry weight) of sieved (2mm) contaminated soil (at ratio of 10. 20. and 30 percent by dry weight) ♦ The soil is moistened to 50 percent of water holding capacity and trays are covered with gas permeable plastic to retain moisture but allow gas exchange. Incubation for the treatability test proceeds at 37C.

Samples (approximately lQg each) are taken from each of four travs (control and three inoculated ratios) at day zero and after 10. 20. and 30 days of incubation. The samples are air dried. sieved (2mm) to remove - the substrate, and analyzed to determine the concentration of contaminants. Reduction and mineralization of TNT is observed. The extent to which TNT disappears will be dependent upon the initial concentration of TNT, the amount of starting fungus, and other factors which affect the rate of TNT disappearance such as pH. buffering capacity, etc. Thus, with either high initial levels of TNT or slow rates of disappearance, toxicity may be observed.

Example 18 The experiment of example 17 is performed except that inoculated nutrient is added a second time about 30 days after initial inoculation. Further mineralization is observed.

Discussion of the Experiments

It has been found that conditions that killed or disposed of the fungus or disrupted the membrane, destroyed the reductase activity. Reduction, observed during both primary and secondary growth phases of nutrient sufficient cultures of __?____ chrysosporiu . was inhibited.

Compounds were added to the cultures to determine whether TNT reduction could be inhibited. Addition of ferricyanide resulted in inhibition of TNT reduction while the ferricyanide was at the same time reduced by the washed mycelium. Washed mycelia was also capable of reducing five membrane impermeable dyes — MTT, TNBT, NBT, TTC, and BT. The most easily reduced of these dyes (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl (MTT) & tetranitroblue tetrazolium (TNBT)) inhibited TNT reduction.

Inhibition of TNT reduction was also found by the proton uncouplers 2,4-dinitrophenol (I₅₀, about 70uM) and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) (I₅₀, about 150 μM) , and by sodium azide (I₅₀, about 200 μM) . Incubating the mycelium in buffers of higher pH stimulated proton flow across the plasma membrane - and increased TNT reduction.

The results from these experiments strongly implicate a plasma membrane redox system of P_j_ chrysosporium in the reduction of TNT. No TNT was reduced by the extracellular or intracellular fraction of the fungus, and the addition of reducing equivalents had no effect on the reduction ability of the fluids, either aerobically or anaerobically. Conditions that disrupted the integrity of the membrane, such as grinding with glass beads or one cycle of freezing in liquid nitrogen and thawing, destroyed the reductase activity.

P. chrysosporium is known to maintain its external environment at a physiological pH of approximately 4.5. Presumably it does this by producing organic acids or excreting protons to lower the pH as needed. The major organic acid produced by P^. chrysosporium appears to be oxalate. However, it was found that oxalate was not produced until between days 2 and 3 of culture. Thus, when the fungus was placed in a media well above the physiological pH, the fungus was forced to increase the rate of proton pumping through the plasma membrane. As proton excretion is tightly coupled to electron transport through the membrane, an increased rate of proton excretion allowed more reducing equivalents to be generated, and thus a faster TNT reduction rate was observed. B. DETOXIFICATION OF TNT BY P. CHRYSOSPORIUM

It has been suggested in the prior art that P. chrysosporium is not a good candidate for bioremediation of TNT-contaminated sites containing high concentrations of explosives because little fungal growth from a spore inoculum was observed in the present of 20 ppm TNT. However, by the present invention, wherein use of fungal mycelia rather than fungal spores are used, degradation of TNT by Pj. chrysosporium at 100 ppm in water and 10,000 ppm in soil has been observed. Reduction was found to play a role in the detoxification of TNT and other explosives-.

A biodegradation system can be designed to degrade high concentrations of TNT. Initial conditions to optimize fungal growth and TNT reduction need to be employed. Once the toxic effects of TNT have been alleviated, conditions that promote the expression of the lignin degrading system can be employed to promote mineralization of TNT.

The following examples illustrate the role of reduction in the detoxification of TNT.

Example 19 P. chrysosporium (BKMF-1767) was obtained from the United Stated Department of Agriculture, Forest Products Laboratory (Madison, WI) . The microorganism was maintained on malt agar slants at room temperature and routinely subcultured every 30 to 60 days.

TNT was obtained from Chem Service, Inc. (West Chester, PA) and C-TNT (ring labeled; specific activity, 21.58 mCi/mmol) was purchased from Chemsyn Science Laboratories (Lenexa, KS) . Radioactive glucose (uniformly ^uC-labeled, specific activity, 268 mCi/mmol) was purchased from Sigma Chemical Company (St. Louis, MO) .

Culture Conditions: Nutrient nitrogen sufficient and deficient cultures of P_j_ chrysosporium were grown in 250 ml

Wheaton bottles or 500 ml Erlenmeyer flasks as described in

Fernando et al., APPI. Environ. Microbiol.. 56, 1666-1671, 1990. For experiments requiring only mycelia, five day old nutrient nitrogen sufficient cultures grown in 500 ml Erlenmeyer flasks were filtered through cheesecloth and washed with distilled water.

Mineralization and Metabolism of TNT: TNT (44 μM) and ¹⁴C- TNT (0.4 μM) were added to nutrient nitrogen sufficient and deficient (24 mM, 2.4 mM) media and £^ chrysosporium inoculum. Evolution of volatile radioactivity and C0₂ were measured every three days as described in the Fernando et al. reference cited above. The concentration of TNT, 4- AmDNT, and 2-AmDNT were determined by gas chromatography.

Peroxidase Assays: Extracellular fluid (1.0 ml) from quadruplicate cultures were collected each day and analyzed for veratryl alcohol oxidase activity essentially as described by Tien and Kirk, Proc. Natl. Acad. Sci. USA. 81,

2280-2284, 1984. The reaction mixtures contained 500 μl extracellular fluid and 1.5 mM veratryl alcohol in 100 mM sodium tartrate buffer, pH 2.5. Addition of 500 μM H₂0₂ initiated the reaction. Manganese dependent peroxidase activity was determined from the oxidation of guaiacol.

The reaction mixtures contained 500 μl extracellular fluid,

100 μM MnS0₄, 1.0 mM guaiacol in 100 mM sodium tartrate buffer, pH 4.5, and initiated with the addition of 100 μM

H₂0₂. One unit of activities produce 1 μmol of product formed in one minute at 25C.

Toxicity of TNT: Toxicity to the fungus was estimated by quantitating glucose-dependent respiration. Cultures were grown in 250 ml Wheaton bottles as described in Fernando et al. cited earlier. To measure the toxicity of TNT to cultures started with spores, TNT and [^UC] glucose (0.05 μCi) were added to fungal spores in nutrient nitrogen limited culture media. The head spaces were flushed with oxygen (99%) every three days (for 12 days) and the ^UC0₂ was collected in a solution containing ethanolamine:methanol:safety solve scintillation cocktail (1:4:5). The amount of ¹⁴C0₂ trapped was determined by scintillation spectrometry (Beckman, LS 5801) . TNT toxicity under other conditions was measured similarly except the TNT was added at different times. The toxicity of TNT to ligninolytic and nonligninolytic cultures of P. chrysosporium was measured under nutrient nitrogen limiting and nonlimiting conditions, respectively. The head spaces were flushed with oxygen (99%) every three days (for 18 days) and the ¹⁴C0₂ was collected. TNT was added immediately after flushing on day 6.

The toxicity of TNT to various amounts of mycelia was measured by incubating TNT, glucose (1 mM) , [^UC]-glucose (0.05 μCi) and mycelia for one day in 250 ml Wheaton bottles equipped with gas-exchange manifolds. The amount of ¹⁴C0₂ evolved was collected and analyzed as described above.

Results

TNT disappearance and 4-AmDNT and 2-AmDNT appearance were observed during the first two days of culture and were essentially identical under both nutrient nitrogen limiting (Figure 6) and nonlimiting conditions (Figure 7) . Both 4-AmDNT and 2-AmDNT were identified by GC-MS (data not shown) . Under nutrient nitrogen limiting conditions, manganese peroxidase activity was first observed on day three and reached a maximum activity on day four (Figure 8) . The concentrations of both 4-AmDNT and 2-AmDNT rapidly decrease during this time (Figure 6) . In nutrient nitrogen sufficient cultures, the 4-AmDNT and 2-AmDNT concentrations decreased much more slowly (Figure 7) and 2,4-DAT and 2,6-DAT were observed (data not shown).

Mineralization of TNT began between days three and six in nitrogen deficient cultures (Figure 6) , the same time as lignin peroxidases were detected (Figure 8) . In nitrogen sufficient cultures, little mineralization of TNT or peroxidase activities were observed. Further, the added radioactivity was accounted for in the extracellular fluid throughout the experiment under the nitrogen nonlimiting conditions (data not shown) . Under nutrient nitrogen limiting conditions, 40% of the TNT metabolites were water soluble, 40% evolved as ¹⁴C0₂, and 23% was collected in the mycelia by day 30.

Low concentrations of TNT were toxic to cultures started with spores (Figure 9) . Respiration was inhibited about 50% by 44 μM TNT and 96% by 200 μM TNT. The amount of TNT required for 50% inhibition of glucose respiration was increased to just above 660 μM TNT when the TNT was added to two day old cultures (Figure 10) . Toxicity was identical in both ligninolytic and nonligninolytic cultures (Figure 11) . The amount of mycelia (dry weight) under both culture conditions were nearly identical (data not shown) . Also, the TNT was completely gone by day 21 in the cultures which exhibited no TNT toxicity.

The toxicity of TNT was inversely related to the amount of fungal mycelia. Respiration was only reduced 6.6% when 4.4 mM TNT was incubated with 360.9 mg of mycelium compared with 62.6% inhibition with 5.5 mg (dry weights) of mycelium. The rate of reduction on TNT was directly correlated with mycelial mass (Figure 12) and the initial TNT concentration (Figure 13) .

Discussion of the Experiments These results suggest a pathway of metabolism of TNT by £i_ chrysosporium which starts with reduction of the TNT. The AmDNT's then appear to be oxidized by the manganese peroxidases and the resulting metabolites must then be further degraded by ligninases. The fungus may then absorb some metabolites and use them for cellular metabolism under low nutrient nitrogen conditions; 23% of the TNT was eventually bound in mycelia and 40% was evolved as C0₂. In the absence of peroxidase activity, only reduced metabolites were observed. These results show the interdependency of several degradative systems for the mineralization of TNT. Any of the individual activities in the system could be rate limiting and disruption of any system may inhibit mineralization.

TNT toxicity to low nutrient nitrogen cultures of P. chrysosporium was dependent on mycelial mass. The concentration of TNT required to inhibit glucose respiration of 50% was increased from 44 μM to 600 μM TNT was added to fungal spores versus two day old cultures. Further, an inverse relationship existed between fungal mass and toxicity.

Several facts support the possibility that toxicity is influenced by the fungal plasma membrane reduction system. TNT toxicity was not affected by the presence of the lignin degrading system. TNT was only observed in cultures where glucose respiration was inhibited, and TNT reduction rates were directly correlated with both mycelial mass and TNT concentration. TNT was not found in any culture where glucose respiration was not inhibited.

All these results support the conclusion that a biodegradation system can be designed to degrade high concentrations of TNT. Initial conditions to optimize fungal growth and TNT reduction need to be employed. Once the toxic effects of TNT have been alleviated, conditions that promote the expression of the lignin degrading system should be employed to promote mineralization on TNT.

C. Summary It can be seen that for degradation of chemicals with high oxidation potentials such as TNT and other explosives, a mechanism of reduction must first take place wherein the pollutants are detoxified. By the present invention, this reduction occurs by the plasma membrane redox system of fungi which is introduced to the pollutants by introducing fungal mycelia to the pollutants. By regulating the pH of the system, rate of reduction can also be regulated. Unlike prior art reduction systems, this system has been found to work in aerobic environments. Further, with the present invention, complete mineralization and detoxification can occur. With prior art reduction systems, very little mineralization resulted.

The present invention can be used in situ in any area where the fungus can be grown, and where chemical reduction and mineralization is desired. The present invention may be embodied in other specific forms without departing from its spirit- or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

CLAIMS:

1. A process for degradation and disposal of environmental pollutants having high oxidation potentials comprising the steps of: a) combining at least one environmental pollutant with fungal mycelia of a white rot fungus so as to place the fungal mycelia in contact with the pollutant; b) allowing the plasma membrane redox system of the white rot fungus to reduce the pollutant until the pollutant is converted to less toxic degradation products; and c) allowing further degradation such that the pollutant is oxidized.

2. A process as recited in claim 1, wherein the process occurs in an aerobic environment.

3. A process as recited in claim 1, wherein the white rot fungus comprises P_j_ chrysosporium.

4. A process as recited in claim 1 , wherein the environmental pollutant comprises a nitrocompound.

5. A process as recited in claim 4, wherein the nitrocompound comprises TNT.

6. A process as recited in claim 4, wherein the nitrocompound comprises RDX.

7. A process as recited in claim 4, wherein the nitrocompound comprises HMX.

8. A process as recited in claim 4, wherein the nitrocompound comprises PETN.

9. A process as recited in claim 4, wherein the nitrocompound comprises EGDN.

10. A process as recited in claim 1, wherein the environmental pollutant comprises an impermeable dye.

11. A process as recited in claim 10, wherein the impermeable dye comprises MTT.

12. A process as recited in claim 10, wherein the impermeable dye comprises TNBT.

13. A process as recited in claim 10, wherein the impermeable dye comprises NBT.

14. A process as recited in claim 10, wherein the impermeable dye comprises TTC.

15. A process as recited in claim 10, wherein the impermeable dye comprises BT.

16. A process as recited in claim 1, wherein the environmental pollutant comprises ferricyanide.

17. A process as recited in claim 1, wherein the environmental pollutant comprises a halobenzoate.

18. A process as recited in claim 17, wherein the halobenzoate comprises 3-Chlorobenzoate.

19. A process as recited in claim 17, wherein the halobenzoate comprises 4-Chlorobenzoate.

20. A process as recited in 17, wherein the halobenzoate comprises 4-Bromobenzoate.

21. A process as recited in claim 1, further comprising, before the step (a), the, step of growing a culture of white rot fungus with a nutrient until fungal mycelia develops.

22. A process as recited in claim 21, wherein the pH of the environment in which the white rot fungus and the pollutant are to be combined is determined by titration.

23. A process as recited in claim 1, wherein the step of combining at least one environmental pollutant with fungal mycelia comprises placing the fungal mycelia into a pollutant contaminated site and allowing degradation to proceed .in situ.

24. A process as recited in claim 1, wherein the step of combining at least one environmental pollutant with fungal mycelia comprises placing the fungal mycelia into a pollutant-containing aqueous medium.

25. A process as recited in claim 1, further comprising the step of combining a second amount of fungal mycelia to the pollutant to catalyze further degradation.

26. A process as recited in claim 1, further comprising the step of adding more nutrient to the fungal mycelium and pollutant combination.

27. A process for degradation and disposal of environmental pollutants having high oxidation potentials comprising the steps of: a) growing a culture of white rot fungus with a nutrient until fungal mycelia develops; b) combining at least one environmental pollutant with said fungal mycelia so as to place the fungal mycelia in contact with the pollutant; c) allowing the white rot fungus to reduce the environmental pollutant until the pollutant is converted to less toxic degradation products; and d) allowing further degradation such that the pollutant is oxidized. e) allowing the degradation reaction to proceed until the pollutant has been substantially mineralized to carbon dioxide.

28. A process as recited in claim 27, wherein the process occurs in an aerobic environment.

29. A process as recited in claim 27, wherein the white rot fungus comprises P_j_ chrysosporium.

30. A process as recited in claim 27, wherein- the environmental pollutant is a compound selected from the group consisting of TNT, RDX, HMX, PETN, and EGDN.

31. A process as recited in claim 27, wherein the environmental pollutant is a compound selected from the group consisting of MTT, TNBT, NBT, TTC, and BT.

32. A process as recited in claim 27, wherein the environmental pollutant comprises ferricyanide.

33. A process as recited in claim 27, wherein the environmental pollutant is a halobenzoate selected from the group consisting of 3-Chlorobenzoate, 4-Chlorobenzoate, and 4-Bromobenzoate.

34. A process for reducing environmental pollutants having high oxidation potentials comprising the steps of: a) combining in an aerobic environment at least one organic pollutant with fungal mycelium of a white rot fungus, so as to place the fungal mycelium in contact with the pollutant; b) allowing the reduction to proceed until the pollutant is reduced such that oxidation is thereafter possible.

35. A process as recited in claim 34, wherein the environmental pollutant comprises a nitrocompound.

36. A process as recited in claim 35, wherein the nitrocompound is selected from the group consisting of TNT, RDX, HMX, PETN, and EGDN.

37. A process as recited in claim 34, wherein the environmental pollutant comprises ferricyanide.

38. A process as recited in claim 34, wherein- the environmental pollutant comprises an impermeable dye.

39. A process as recited in claim 34, wherein the environmental pollutant comprises a halobenzoate.