WO2004067730A1

WO2004067730A1 - Bioremediation using fungal isolates of order polyporales

Info

Publication number: WO2004067730A1
Application number: PCT/NZ2004/000018
Authority: WO
Inventors: Roberta Farrell; Richard Lamar; Richard White
Original assignee: The University Of Waikato
Priority date: 2003-01-31
Filing date: 2004-02-02
Publication date: 2004-08-12

Abstract

The invention provides a method of degrading environmentally persistent and toxic chemicals in contaminated materials, such as soil. The method includes the use of fungi with selected characteristics to degrade the contaminants. The selected characteristics include colony morphology, growth rates, tolerance of boron, cellulose activity, proteinase activity. Specific isolates nominally identified as B101 and B102 with the selected characteristics and their use in the method are described.

Description

BIOREMEDIATION USING FUNGAL ISOLATES OF ORDER POLYPORALES

FIELD OF INVENTION

The invention relates to fungal isolates and the use of these fungal isolates in methods for the bioremediation of hydrocarbon contaminated materials. Particularly, although not exclusively, the invention relates to fungal isolates and their use in a method for the bioremediation of contaminated soil.

BACKGROUND

Environmentally persistent chemicals are resistant to degradation in the natural environment and may also • bioaccumulate.

Environmentally persistent chemicals include aromatics halogenated aromatic compounds (HACs) such as pentachlorophenol (PCP) and pentachloranisole (PCA), polynuclear aromatic hydrocarbon components of coal tar and creosote (PAHs), polychlorinated biphenyls (PCBs), such as DDT, and polychlorinated dibenzo dioxins (PCDDs) and dibenzo furans (PCDFs)

These chemicals present risks to both human and environmental health. The medical literature is replete with data on adverse health effects due to exposure to these chemicals.

Contamination of soils and ground water with these chemicals is a serious, ongoing problem.

The scope of the problem can be appreciated when one considers that there are thousands of toxic waste sites where large quantities of these chemicals have been used historically and have either been spilt, deliberately dumped, or have leaked out of storage tanks. It has been estimated that of the one million tons of PCBs made, up to a third is in the environment.

The disposal of contaminated materials, e.g., storage containers, manufacturing equipment, and materials treated with the chemicals such as lumber, presents an additional problem.

PCP has been used extensively as a wood preservative. DDT has been widely used as an insecticide! Others HACs have been used in agriculture and manufacturing. The PCPs and PCBs used in agriculture and manufacturing also contain dioxins such as PCDDs and PCDFs.

Whereas PCPs and PCBs cause toxic effects at levels of parts per million (ppm) dioxins are toxic at levels of parts per trillion (ppt).

In order to reduce the substantial health risks presented by these chemicals they should be destroyed, contained or degraded to non-toxic or at least reduced toxicity products. Treatment strategies that have been suggested or tried include:

1. Excavation, removal and isolation of the contaminated material; 2. Incineration of the contaminated material; and

3. Degradation of the chemical in the contaminated material.

Incineration is extremely expensive due to the energy requirements and the necessity to excavate and transport the contaminated material to remote locations. This method is also impractical due to the large quantities of waste that need to be processed.

Excavation, removal and isolation of the contaminated material is also expensive and does nothing to effect a long term solution.

The scientific literature is replete with laboratory data on methods for the biodegradation of HACs. The limitation of many of these methods is that while they may be effective under the controlled conditions achievable in a laboratory or small scale field trial, the methods often lack effectiveness or practicality under the less well controlled conditions of large scale trials.

Furthermore, published methods are not always effective when applied to "field-contaminated" materials. The term "field-contaminated" is meant to refer to a solid material, i.e. soil, sludge, or iignocellulosic (e.g. wood) debris, that has been contaminated through use or accident as compared to samples that are artificially contaminated under controlled conditions specifically for use in a trial and therefore of a more homogenous nature.

In the case of wood products, "field-contaminated" includes wood that has been contaminated or treated for use in commerce, or wood that is a contaminated by-product of industrial treatment. An example of wood that has been intentionally treated for use in commerce is wood which has been treated with a preservative.

Biodegradation of HACs by bacteria has proven less than ideal due to the bacteria's specificity for particular chemicals, inability to degrade the chemicals to non-toxic products, sensitivity of the bacteria to the toxic chemicals themselves, and variable environmental conditions.

Other biodegradation methods that have been the subject of experimental research in the past decade include the use of a class of wood degrading fungi, known as "lignin-degrading fungi", to degrade HACs.

In the early 1980's, it was reported that the fungus Phanerochaete chrysosporium (P. chrysosporium) could degrade chlorinated organics in the effluent of a Kraft pulp mill (Eaton et al., TAPPI, vol. 65 (1982) pp. 89- 92; Huynh et a/., TAPPI, vol. 68 (1985) pp. 98-102). At about this same time, other researchers isolated and characterized the enzymes responsible for the lignin degrading ability of these fungi. These isolated enzymes, termed "lignin peroxidases" or "ligninases", were found to oxidize a wide variety of compounds in addition to lignin. Such compounds include PAHs (Hammel et al., J. Biol. Chem., vol. 261 (1986) pp. 16948-16952; Sanglard et al., Enzyme and Microbial Tech., vol. 8 (1986) pp. 209-212), dibenzo(p)dioxins (Hammel et al., J. Biol. Chem. , vol. 261 (1986) pp. 16948-16952); Haemmerli et al., J. Biol. Chem., vol. 261 (1986) pp. 6900-6903), and PCPs (Hammel and Tardone, Biochem., vol. 27 (1988) pp. 6563-6568).

It has since been reported that under controlled laboratory conditions, cultures of P. chrysosporium can also effectively degrade HACs.

In particular, Bumpus et a/, have extensively studied the degradation of the PAHs. They have shown that small cultures of P. chrysosporium conditioned by growth in the laboratory in a low nitrogen-containing growth medium at approximately 37°C will degrade DDT, 2,4,5,2',4',5'-hexachloro-biphenyl-2,3,7,8-tetra- chlorodibenzo-p-dioxin, and lindane. (See, Bumpus et al., Science, vol. 228 (1985) pp. 1434-1436; Bumpus and Aust, Applied and Environmental Microbiology, .vol. 53 (1987) pp. 2001-2007; Aust and Bumpus, U.S. Pat. No. 4,891 ,320).

Similar studies have analyzed the ability of P. chrysosporium to degrade PCP in controlled small cultures (Mileski et al., Applied and Environmental Microbiology, vol. 54 (1988) pp. 2885-2889).

Fewer studies have attempted to reproduce the conditions that are present at contaminated field sites, or investigate the relative abilities of other species of the lignin-degrading class of fungi at degrading different classes of HACs.

Several studies have reported that P. chrysosporium can degrade 2,4,5-trichlorophenoxyacetic acid (Ryan et al., Appl. Microbiol, Biotechnol., vol. 31 (1989) pp. 302-307), fluorene (George and Neufeld, Biotechnol. Bioeng., vol. 33 (1989) pp. 1306-1310), and PCP (Lamar et a/., Soil Biol. Biochem., vol. 22 (1990) pp. 433- 440) in chemically spiked sterile soil in laboratory culture.

This study of Lamar et al. also reported that the ability of P. chrysosporium to degrade the PCP varied depending on the soil type. The fungi exhibited the highest rate of transformation of the PCP in Marshan soil (sandy loam) and the lowest in Batavia soil (silty clay loam).

Lamar et al., Appl. Environ. Microbiol., vol. 56 (1990) pp.3519-3526, also tested seven species of lignin- degrading fungi in laboratory cultures. They reported significant differences between the species in both the rate and extent of degradation of the PCP.

Most recently, in a preliminary field study in 1 m² plots of soil contaminated exclusively with PCP, the PCP degrading abilities of P. chrysosporium and Phane ochaete sordida (P. sordida) were compared (Lamar et al., Appl. Environ. Microbiol., vol. 56 (1990) pp. 3093-3100). The results showed that in the very alkaline soil (pH approximately 9.6) that had been tilled prior to initiation of the study to allow evaporation of mineral spirits, sterilized, and supplemented with peat moss, both fungi efficiently degraded the PCP over a 45 day period.

Although these laboratory and preliminary field study data are useful in defining a promising technology for the degradation of HACs, such a technology is of little utility if it will not work effectively on the large scale needed to clean up the vast quantities of soils and materials contaminated with HACs.

As noted, many technologies that were thought to hold great promise for solving the problem of environmentally persistent chemicals have not proven effective when put to a large scale test.

One such example was the technology of degradation of PCBs with certain genetically engineered or adapted bacteria. Results showed that under controlled laboratory conditions, successful degradation was effected. However, when field trials were conducted, the bacteria were found to be too sensitive to the varying environmental conditions for effective use (R. Unterman, "Bacterial Treatment of PCB- Contaminated Soils," Hazardous Waste Treatment by Genetically Engineered or Adapted Organisms, p. 17, Nov. 30-Dec. 2, 1988, Washington, D.C.)

In large scale use the parameters controllable in the laboratory are uncontrollable.

In the laboratory, parameters such as the temperature, humidity, concentration and distribution of the contaminating chemical, purity of the chemical, oxygen (0₂) content, pH, presence of toxins, and organics/nutrient content of the cultures can be controlled.

In large scale use, the temperature and humidity will constantly fluctuate. The concentration and distribution of the contaminating chemical will vary in different areas of the contaminated site. The site will be contaminated with other chemicals, including chemicals that are toxic to micro-organisms. The 0₂ content will depend on such parameters as water content, the particle size of the soil or contaminated material, and how tightly it is packed. The pH and organics/nutrient content will depend in part on the geology and nature of the contaminated site.

Compounding these problems are unknown factors, such as the effect the total environment will have on fungal physiology. The environment as a whole, including the parameters listed above, affects not only the growth rate of fungi, but also the stimulation/repression of their enzymatic systems.

By way of illustration of this latter point, in the laboratory, the lignin peroxidases of P. chrysosporium are induced under conditions of low nitrogen and repressed under conditions of high nitrogen (Fenn and Kirk, Arch. Microbiol., vol. 130 (1981) pp. 59-65), but the lignolytic enzymes of other studied species of fungi are not regulated in this manner.

Furthermore, the ability of lignin-degrading fungi to degrade HACs may not depend entirely on enzymes of the lignolytic system (Kohler et al., Appl. Microbiol. Biotech., vol. 29 (1988) pp. 618-620), and under identical laboratory conditions, the lignolytic enzymes expressed by P. chrysosporium and P. sordida differ (R. Lamar, unpublished data).

As these data indicate, little is known or can be predicted about how particular environmental and contaminated site conditions influence the enzymatic pathways and/or growth of individual fungi.

Little is known or can be predicted about how effectively or ineffectively the enzyme systems of different fungal species will function in degrading HACs in uncontrolled field conditions.

As a consequence the outcome of large scale field trials cannot be predicted reliably.

What is required are fungal isolates that are tolerant of the range of conditions present in a contaminated site, including the presence of toxins, whilst retaining the ability to degrade the chemical contaminants to non-toxic components, or at least components of reduced toxicity. .

The object of this invention is to provide fungal isolates and methods for the large scale bioremediation of HAC contaminated material using these isolates, or at least to provide the public with a useful choice.

STATEMENT OF INVENTION

Accordingly, in a first aspect, the invention consists in an isolated culture of a fungal isolate belonging to the class Basidiomycetes having at least one of the identifying characteristics of one of the isolates nominally identified as B101 (AGAL accession no. NM03/33520) and B102 (AGAL accession no. NM03/33521) the isolate being effective to degrade hydrocarbon contaminants in contaminated material.

Preferably, the isolated culture is a biologically pure culture.

Preferably one of the identifying characteristics is tolerance of boron. More preferably the tolerance of boron is when the boron is present in the contaminated material at a concentration of at least 2000 ppm. Even more preferably the tolerance of boron is when the boron is present in the contaminated material at a concentration of at least 6,300 ppm. Most preferably the tolerance of boron is when the boron is present in the contaminated material at a concentration between 2,000 ppm and 20,000 ppm.

Preferably one of the identifying characteristics is tolerance of benlate. More preferably the tolerance of benlate is when the benlate is present in the contaminated material at a concentration of at least 0.06 g/L.

In one embodiment the invention provides an isolated culture of the isolate nominally identified as B101 (AGAL accession no. NM03/33520). In another embodiment the invention provides an isolated culture of the isolate nominally identified as B102 (AGAL accession no. NM03/33521).

Preferably the isolated culture is a biologically pure culture.

Preferably the isolate is an isolate belonging to the order Polyporales. More preferably the isolate is an isolate belonging to the family Polyporaceae.

Preferably the contaminated material is solid material. More preferably the material is soil, sludge, sediment, or debris from the processing of wood such as chips, shavings and sawdust, or a mixture.

Preferably the hydrocarbon contaminants are one or more polycyclic hydrocarbons, aromatic hydrocarbons or halogenated hydrocarbons. More preferably the contaminants are halogenated aromatic hydrocarbons (HACs). Most preferably the contaminants are one or more of polychlorophenols, such as pentachlorophenol (PCP), pentachloroanisole (PCA), polychlorinated biphenyls (PCBs), such as DDT, polybrominated biphenyls, and dioxins such as PCDDs, PCDFs, OCDD, HpCDD, and HpCDF.

Preferably the isolated culture is effective to degrade the hydrocarbon contaminants in the contaminated material without excavation of the material or the removal of the material from the locality where contamination has occurred.

Preferably the isolated culture is effective to degrade the hydrocarbon contaminants in contaminated material to non-toxic components, or at least components of reduced toxicity, when the culture is applied to the material. More preferably the isolated culture is effective to degrade both PCP and pentachloranisole (PCA) in contaminated material when the culture is applied to the material. Most preferably the culture is effective to degrade PCP, PCA and dioxins when the culture is applied to the material.

Preferably the isolated culture is effective to degrade dioxin in contaminated material when the culture is applied to the material and the initial concentration of dioxin in the material is less than 60 ppb, most preferably less than 20 ppb. In yet another embodiment the invention provides an isolated culture of an isolate of Basidiomycete having all the identifying characteristics of one of the isolates nominally identified as B101 (AGAL accession no. NM03/33520) and B102 (AGAL accession no. NM03/33521), the isolated culture being effective to degrade the hydrocarbon contaminants in contaminated material when applied to the material.

In a second aspect, the invention consists in a method for the degradation of hydrocarbon contaminants in contaminated material including the step of:

• applying an inoculum containing at least one culture of the first aspect of the invention to the contaminated material in an amount being effective to degrade hydrocarbon contaminants in the contaminated material. Preferably the method includes the step of:

• applying a lignocellulosic substrate to the contaminated material in an amount sufficient to promote degradation of the hydrocarbon contaminants.

Preferably the method includes the step of:

• aerating and/or hydrating the contaminated material to promote degradation of the hydrocarbon contaminants.

More preferably the method includes the step of: • applying an inoculum containing at least one culture of the first aspect of the invention to a lignocellulosic substrate;

• incubating the inoculated lignocellulosic substrate for a period to allow growth of the culture; and

• applying the inoculated lignocellulosic substrate to the contaminated material.

Preferably the contaminated material contains boron. More preferably the boron is present in the contaminated material at a concentration of at least 2000 ppm. Even more preferably the boron is present in the contaminated material at a concentration of at least 6,300 ppm. Most preferably the boron is present in the contaminated material at a concentration between 2,000 ppm and 20,000 ppm.

Preferably the contaminated material is solid material. More preferably the material is soil, sludge, sediment, or debris from the processing of timber or lumber such as wood chips, shavings and sawdust. Most preferably the material is soil.

Preferably the inoculum is not conditioned prior to its application to the solid material.

Preferably the percentage decrease in concentration of one or more of the hydrocarbon contaminants is greater than 50% of the initial concentration of the hydrocarbon contaminant, more preferably greater than 60%, more preferably greater than 70%, more preferably greater than 80%, most preferably greater than 90%.

It will be understood that an isolated culture containing an isolate derived from an isolated culture of the first aspect of the invention and effective to degrade hydrocarbon contaminants in contaminated material when applied to the material, is intended to be within the scope of the invention. In this context "derived" will be understood to include mutagenised, genetically engineered and hybridised (e.g. by protoplast fusion) derivatives. The invention will now be described by way of example with reference to the Figures of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Growth rates of isolate B101 ;

FIG. 2 Growth rates of isolate B102;

FIG.3 Lipase activity of B101 and B102 isolat

FIG.4 Proteinase activity of B101 and B102 isolates;

FIG.5 Cellulase activity of B101 and B102 isolates;

FIG. 6 Graph of pentachloroanisole (PCA) concentration versus time in the Completely Random Design study of fungal action (Comparative Example);

FIG. 7 Graph of PCA concentration versus time in the Balanced Incomplete Block study of fungal action (Comparative Example);

FIG. 8A Graph of the percent decrease in PCP concentration versus time for Phanerochaete chrysosporium fungal action on various woods (Comparative Example);

FIG. 8B Graph of the percent decrease in PCP concentration versus time for Phanerochaete sordida fungal action on various woods (Comparative Example);

FIG. 9A Graph of the accumulation of PCA versus time for Phanerochaete chrysosporium fungal action in various woods (Comparative Example);

FIG. 9B Graph of the accumulation of PCA versus time for Phanerochaete sordida action in various woods (Comparative Example);

FIG. 10 Bar graph showing the percentage PCP decrease after 3 weeks in PCP-contaminated softwood chips supplemented with different sources of nitrogen and/or carbon and inoculated with P. chrysosporium (Comparative Example);

FIG.11 PCP bioremediation in Kinleith soils by isolates B101 and B102; FIG.12 PCP bioremediation in Brookside soils by isolates B101 and B102;

FIG.13 Fate of PCA during bioremediation of Kinleith and Brookside soils by isolates B101 and B102;

FIG.14 Bioremediation of OCDD by isolates B101 and B102;

FIG.15 Bioremediation of HpCDD in Kinleith and Brookside soils by isolates B101 and B102; and

FIG.16 Bioremediation of HpCDF in Kinleith and Brookside soils by isolates B101 and B102.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides fungal isolates and their use in a method for the biodegradation of hydrocarbon contaminants in contaminated material. The invention is particularly applicable to the biodegradation of HACs in soil.

The invention can be used most advantageously without the need to remove the soil from the locality of the contaminated site prior to the degradation. The removal of large amounts of contaminated material from a contaminated site, such as a wood treatment yard or waste disposal site, is neither economical nor practical. Many tonnes of contaminated material may be required to be decontaminated or isolated at a remote location.

The invention permits large amounts of contaminated material to be treated locally. It is recognised that excavation and local containment of the contaminated material is desirable at some contaminated sites to manage the risk to human and environmental health associated with exposure to the contaminated material.

In order to practice the invention fungal isolates are first obtained by the methods described.

Laboratory based studies are used to evaluate the potential of the fungal isolates to be effective in degrading HACs in contaminated soil on a large scale. Such methods are described in the Comparative Examples.

The identifying characteristics of selected isolates are determined. These identifying characteristics include:

Morphology and growth characteristics; Wood degradation; Cellulose plate growth; Growth rates;

Lipase activity; Proteinase activity; and Cellulase activity.

The characterised isolates are evaluated for their ability to biodegrade PCP, dioxin and furan containing soils from several former wood-treating facilities.

The invention may most advantageously be applied to soils, including, clay soil, sandy soil, acidic soil, or sandy solid having a pH below 4, contaminated by HACs such as pentachlorophenol. The concentration of HAC may be between 1500 μ/g to 5000 μ/g of soil. The invention is most advantageously applied to soil containing inhibitory factors such as boron and/or benlate, copper, chromium or arsenic.

The "identifying characteristics" of the isolates of the invention are those shared with the isolates nominally identified as B101 (AGAL accession no. NM03/33520) and/or B102 (AGAL accession no. NM03/33521). These characteristics are described with reference to the isolates B101 and B102.

In the description the term "tolerance" is used to mean the ability of an isolate to grow in the presence of a concentration of an inhibitory factor such as an antibiotic (e.g. benlate), or toxin (e.g. boron), at growth rates greater than about 50% of the growth rate observed in the absence of the inhibitory factor. Ideally growth in the present of an inhibitory factor is at growth rates greater than 66% of the growth rate observed in the absence of the inhibitory factor.

ISOLATION

Methods for the isolation of the isolates of the invention are described with reference to the isolation of the isolates nominally identified as B101 and B102.

ISOLATION OF B101 AND B102

A sample of soil is placed directly on the surface of a solidified, agar-containing selective medium. The selective medium is prepared by autoclaving water containing:

15 grams per litre malt extract; 2 grams per litre yeast extract;

200 mg/ml chloramphenical; 0.06 grams per litre benlate;and 15 grams per litre agar.

After autoclaving the medium is allowed to cool and 2 ml of lactic acid and 100 mg/ml streptomycin added while the medium is still molten. The medium is then poured to provide plates of selective medium. Plates containing samples of soil are incubated at 30°C for at least 2 to 3 weeks. As colonies appear samples of the growth are removed aseptically and used to inoculate fresh plates of the selective medium.

IDENTIFYING CHARACTERISTICS

The identifying characteristics of the isolates are determined.

MORPHOLOGY AND GROWTH CHARACTERISTICS OF B101 AND B102

Isolates B101 and B102 grow as white mycelia displaying clamp junctions.

Mycelial growth is rather flat in appearance and somewhat translucent.

WOOD DEGRADATION

The ability of the isolates B101 and B102 to degrade wood in the form of woodchips was measured. Woodchips were inoculated with the isolate and incubated at 27°C or 35°C.

The initial fresh weights and final dry weights of uninoculated woodchips (control) incubated at 27°C and 35°C were used to calculate the average dry content of the woodchips (% dry weight).

The weight loss for woodchips inoculated with an isolate (% weight loss) was calculated using the fresh weight of the wood chips in the bag (initial fresh weight) and the final dry weight after incubation (final dry weight) according to the formula:

% weight loss = [(initial fresh weight x % dry weiαhϋ-final dry weight] x 100 (initial fresh weight x % dry weight)

The percentage weight loss of three replicated samples was used to calculate a mean and standard deviation shown in Table 1. When the standard deviation was larger than one half of the mean, the outlier was removed and the mean and the standard deviation were calculated for the remaining two replicated samples. Weight loss after fungal treatments

Fungal isolate 27°C 35°C

B101 4.5% 2.9%

B102 7.9% 1.7%

Table 1

CELLULOSE PLATE GROWTH TEST

Plugs of fresh culture of an isolate were used to inoculate an agarose plate containing carboxymethyl cellulose as the carbon source.

The plates were incubated at 35°C for one week. The plates were then overlaid with a solution of 0.1% Congo Red. The overlaid plates were allowed to stand for 30 minutes. The Congo Red solution was then aspirated or tipped out. The plates were then overlaid with a solution of 1 M sodium chloride. The plates were allowed to stand until a zone of clearing was visible. The diameter of growth and zone of clearing were then measured. The measurements are shown in Table 2.

ORGANISM Growth Diameter (cm) Zone Clearing (cm)

B102 5.2 0.4

5 0.43

B101 5.5 0.6

5.8 0.8

Table 2

GROWTH RATES

Plates of medium 4 and medium 7 were inoculated with an isolate. Colony diameters were measured on a daily basis with incubation at either 20°C, 27°C, 35°C and 40°C.

Medium 4 is prepared as follows:

Yeast extract 2g/L

Malt extract 15g/L

Agar 18g/L

Chloramphenicol 200mg/L

Streptomycin 100mg/L Chloramphenicol is added directly to media, before autoclaving. Streptomycin is dissolved in sterile water and aseptically added to the autoclaved media via filter sterilization.

Medium 7 is prepared as follows:

grams/litre of distilled water

Malt extract 15g/L Yeast extract 2g/L Agar 15g/L Benlate 0.06g/L

After autoclaving, add streptomycin 0.01 g/L, and lactic acid 2ml/ L

Growth rate measurements are provided in Table 3 and represented graphically in Figure 1 and Figure 2.

time (hrs) 20 deg rees 27 degrees 35 degrees 40 degrees

B101 media 4 media 7 media 4 media 7 media 4 media 7 media 4 media 7

48 0 . 0 0 0 0 0 0 0

120 0 0 1 0.8 1.5 1.2 0 0

168 0.4 1 1.3 1.6 1.8 1.8 0 0

216 2.1 1 2.2 2.5 2.2 3 0 0

288 3.5 2.5 3.4 2.6 2.5 2.6 0 0

408 4.1 2.5 4 2.7 2.8 2.9 0 0

480 4.6 2.8 5 3.6 3 3 0 0

576 5 5 5 4.5 4.7 3.6 0 0

720 5 5 5 . 5 5 5 0 0

time (hrs) 20 deg rees 27 degrees 35 degrees 40 deg rees

B102 media 4 media 7 media 4 media 7 media 4 media 7 media 4 media 7

48 0 0 0 0 0 0 0 0

120 0 0 . 0 0.7 0.7 1 0 0

168 1.2 0.3 1.1 1.1 2.4 2.4 0 0

216 3 2.8 2.5 1.8 3.7 1.6 0 0

288 3 2.8 3.2 4.3 4.1 2.8 0 0

408 4.1 2.8 3.6 4.6 4.3 3 0 0

480 4 3 4 4.8 4.5 3.8 0 0

576 5 4.6 4.4 5 5 4 0 0

720 5 5 5 5 5 5 0 0

Table 3 LIPASE ASSAY

Cultures grown in specific media containing gelysate peptone as carbon source.

Buffer:

MOPS 10OmM + 7.5mM CaCI₂ (pH 7.5 @ 20°C )

Substrate:

1 mg/ml p-Nitrophenol palmitate (pNP) in ethanol.

Per assay:

MOPS - 800μl pNP substrate - 100ml

Incubation Temperature: 37 °C for 10 mins.

Stop Reagent:

0.1 M Na₂C0₃ 10% Triton

Procedure:

1. 900 μl of buffer/ substrate mix incubated with 10Oμl of enzyme @ 370C for 10 mins.

2. Controls treated similarly except enzyme added after incubation. 3. 500 μl of Stop Reagent added after incubation period, enzyme added to control tubes.

4. Cooled on ice, centrifuged and optical density read @ 400nm.

Lipase Enzyme activity calculated as follow:

Δ A ⁴⁰⁰ / 0.011 (slope on std curve)X 10 )(dil.factor) / Time(incubation)

Specific Activity = Activity/ Dry weight = mU/ml/g

Measurements of lipase activity are provided in Table 4a and b. Strain No: B101

Enzy. Activity Biomass(g) Highest Specific Activity ve\

(mU/ml) (after 9 days) (mU/ml/g)/ 9 days

0

1

0 (cultures grown in triplicates) 0

3.04

3 5.06 4.96 20.90

5 23.36 22.75

24.31

7 21.11 23.89

9 9.72 0.23 105.69

7.04 0.21 111.23

10.04 0.26 91.86

Table 4b

Strain No: B102

Enzy. Activity Biomass(g) Highest Specific Activity

Timp fDA V_ \

(mU/ml) (after 9 days) (mU/ml/g)/ 9 days

1

0 (cultures grown in triplicates) 0

0

3 5.87 4.97 5.42

5 20.54 22.48 18.03

7 23.96 26.94 25.31

9 10.04 0.23 104.17

7.90 0.27 99.77

18.63 0.24 105.45

Table 4b PROTEINASE ASSAY

Unit Definition:

1 UNIT = ΔA₄₂₀ of 1.0 per hour

Substrate:

0.2% Azocasein in 50mM HEPES containing 5 mM CaCI₂, Ph 7.0 @ R.T.

Stop Reagent: 15% Trichloroacetic acid

Procedure:

1. Cultures grown in specific media containing peptone and starch as the carbon source.

2. Sample tubes: 100 μl of supernatent incubated with 900μl of substrate at 37°C for 15 minutes. 3. Control tubes: 900μl of substrate, culture supernatant added after incubation, incubated at above.

4. Reactions stopped by adding 500μl of 15% TCA, supernatant added to control tubes.

5. Mixed well, and cooled for at least 15 mins at room temperature to ensure complete precipitation of unhydrolysed substrate.

6. Centrifuged for 5 mins at full speed, Absorbance read at 420nm against a water reference.

Change in Absorbance determined:

ΔA420 = ΔA420 (sample) " ΔA₄2θ(coπtrol)

Activity: 1AU/hr/mg = ΔA x incubation time x dilution factor.

Measurements of proteinase activity are provided in Table 5a and b..

Strain No : B101

Enzy. Activity Biomass(g) Highest Specific Activity

Time (DAYS)

(AU/hr/mg) (after 9 days) (AU/hr/mg)/ 9 days

1 0 0 0 0

3 0

0

0.02

5 0.01 0.06 0.09

7 0.07 0.11

0.10 0.33 0.208

9 0.05 0.29 0.344

0.10 0.23 0.478

Table 5a

Strain No : 102

Enzy. Activity Biomass(g) Highest Specific Activity

Time (DAYS)

(AU/hr/mg) (after 9 days) (AU/hr/mg)/ 9 days

1 0 0 0

3 0 0 0

5 0 0.01 0.03

7 0.10 0.08 0.12

9 0.11 0.29 0.344

0.12 0.34 0.352

0.10 0.25 0.480

Table 5b CELLULASE ASSAY

PAHBAH solution: 0.5 M Na₃ Citrate.2H₂0

M Na₂S04.7H₂0 0.2 M CaCI₂.2H₂0 5.0 M NaOH 10mls of each of the above stock sol. mixed well, 1.52g of PAHBAH( p-hydroxybenzoic acid hydrazide) added, total vol. Made up to 100ml.

Substrate:

1.0% Carboxymethyl cellulose in 100mM MOPS buffer, pH 7.0 @20°C

Procedure:

1) Assay tubes prepared:

Sample tubes: 300μl of substrate

100μl of sample 100μl RO water

Control tubes: 300μl substrate

10Oμl substrate Culture supernatent added after incubation.

2) Incubated at 37°C for 30 mins, cooled on ice.

3) 1 ml of PAHBAH reagent added to each tube, enzyme added to controls.

4) Reagent mixture boiled for 6 mins, cooled on ice, centrifuged. Abs. measured at 420nm against air ref.

Activity :

ΔA₄₂₀/min/ml = (mean of A ₂₀(_Sam_Pie) - Aβ. (bla_nk)) / incubation time/sample vol./ dilution

= Cellulose units (micromoles of cellulose realeased / min.) = (ΔA₄₂o/min/ml) x slope on std curve (80) /1000 = CU/ml Measurements of cellulase activity are provided in Table 6a and b.

Strain No : 101

Enzy. Activity Biomass(g) Highest Specific Activity

Time (DAYS)

(CU/ml) (after 9 days) (CU/ml)/ 9 days

1 0 0 0

3 0.003

0.001

0

5 ^' 0.006 0.01 0.01

7 0.16 0.15 0.12

9 0.18 0.19 0.947

0.15 0.17 0.882

0.17 0.18 0.944

Table 6a

Strain No: 102

Enzy. Activity Biomass(g) Highest Specific Activity

Time (DAYS)

(CU/ml) (after 9 days) (CU/ml)/ 9 days

1 0 0 0

3 0 0 0

5 0.02 0.01 0.05

7 0.10 0.13 0.16

9 0.17 0.19 0.894

0.13 0.21 0.619

0.12 0.16 1.0

Table 6b METHODS OF ANALYSIS AND BIODEGRADATION OF HYDROCARBON CONTAMINANTS

Methods for the analysis and biodegradation of hydrocarbon contaminants are described.

The following section of the description (methods of analysis and biodegradation of hydrocarbon contaminants) is not intended to indicate that the isolates and methods referred to in the Comparative Examples are within the scope of the invention.

The described methods are known methods used to evaluate isolates for their ability to degrade hydrocarbon contaminants.

In the following examples, organic solvent extracts of soil and chip samples were prepared by known methods and analyzed by gas chromatography. Pentachlorophenol was analyzed as the trimethylsilyl derivative using Sylon BTZ (Supelco Inc., Bellefonte, Pa.) as the derivatizing agent and quantitated using authentic derivatized standards. Pentachloroanisole was quantified with authentic standards.

Analyses of extracts were performed on a Hewlett Packard Model 5890A or 589011 gas chromatograph equipped with ⁶³ Ni electron capture detectors, Model 7673A autosamplers, and split-splitless capillary column injection ports. Operating temperatures were: injector 220°C. and detector 300°C, carrier gas, He; and make-up gas, N₂. The columns were 30 m by 0.321 mm DB-5 fused silica capillary columns, film thickness 0.25 .μm (J & W Scientific, Folsom, Calif.).

For analysis of data, an analysis of variance (ANOVA) was used at each sample time. If by ANOVA either CRD or BIB (as defined below) treatments were shown to be significantly different, a Tukey multiple comparison test (α.=0.05) was performed to determine which treatments were different from others.

SOIL STUDIES

COMPARATIVE EXAMPLE 1 Fungi and Inocula Preparation

The fungi P. chrysosporium, P. sordida, Ceriporiopsis subvermispora (C. subvermispora), and Trametes hirsuta (T. hirsuta) were obtained from the culture collection of the Center for Forest Mycology Research (Forest Products Laboratory, Madison, Wis.). The fungi were grown and maintained on yeast malt peptone glucose (YMPG) agar slants (10 g/L glucose, 10 g/L malt extract, 2 g/L Bacto-Peptone, 2 g/L yeast extract, 1 g/L asparagine, 2 g/L monobasic potassium phosphate (KH P0₄), 1 g/L magnesium sulfate (MgS0₄7H₂ O), 1 g/L thiamine, and 23.g/L Bacto Agar). Fungal inocula described herein were prepared either by inoculating a sterile solid growth substrate of primarily lignocellulosic material (L. F. Lambert Spawn Co.) with the pure agar slant cultures ( Comparative Examples 3 and 4 described below) or by aseptically transferring pieces of fungal mycelium from the YMPG slants to 2% malt agar plates (100 mm by 20 mm) (Comparative Examples 5-9 described below). The fungi were incubated until colony growth completely covered the plates. Fungal inoculum treatments are indicated by inoculum density in percentages (Comparative Examples 3 and 4), i.e., the percentage dry weight of the inoculum to the dry weight of the treated soil.

COMPARATIVE EXAMPLE 2 Soil Characteristics

Systematic sampling of an 18 m by 18 m level section of a waste sludge pile (former Brookhaven Wood Preserving facility, Brookhaven, Miss.) was conducted. Samples were taken to a depth of 30 cm along five rows that were 18 m long and 4.5 m apart. Ten samples were taken from each row at 1.8 m intervals. The soil samples were analyzed in duplicate for PCP, soil chemical characteristics, and volatile and semi-volatile organics.

The PCP concentration in the waste sludge pile ranged from 15 to 342 μ/g of soil. The soil chemical characteristics were: pH=3.8, cation exchange capacity 8.87 milliequivalents/100 g of soil, base saturation 54.8%, total nitrogen 0.04% and total carbon 2.17%. The soil was also found to contain small concentrations of volatile compounds and significant concentrations (approximately 2500 ppm total) of polyaromatic hydrocarbon components of creosote as indicated in Table 5 below.

COMPARATIVE EXAMPLE 3

Fungi Decontamination in Completely Random Design (CRD) Plots

To analyze the ability of lignin-degrading fungi to degrade PCP in the soil of the waste sludge pile, an experimental plot section was constructed.

Soil was excavated from the upper 30 cm of the section of the waste sludge pile from which samples were initially taken for characterization. The excavated soil was sieved to exclude materials greater than 1.9 cm in diameter and replaced in plots as follows. An 18 m by 24 m area with an approximate 3% slope along the short side was constructed using a sandy clay material. Three 3 m wide drainage swales with 10% slopes from outer edge to nadir were cut into the incline. Eleven 3 m by 3 m plot borders with 0.70 m high vertical side walls were constructed of #14 galvanized sheet metal. The plot borders were placed over the drainage swales and sunk into the sandy clay material so that the tops of the side walls were level. Each plot was lined with 4 mil polyethylene sheeting and drained using a perforated polyvinylchloride pipe placed on the liner in the middle of the V and connected to a drainage hose through a hole in the front wall of the plot. The drainage pipe was completely covered with gravel which was further covered with sand leaving approximately 45 cm to the top of the plot border. Each plot was then filled to a depth of 25 cm with sieved soil from the sludge pile. Concentrations ^a(mg/kg) of Volatile and Semi-Volatile Organic Compounds in Soil from the Waste Sludge Pile

Compound Concentration (mg/kg)

Volatiles

1,1,1-Trichloroethane 0.057

Toluene 0.100

Total xylenes 0.950

Semi-volatiles

Pentachlorophenol 51

Napthalene 250

2-Methylnapthalene 95

Acenapthylene 6.4

Acenapthene 210

Dibenzofuran 110

Fluorene 170

Phenanthrene 470

Anthracene 110

Fluoranthene 290

Pyrene 270

Benzo(a)anthracene 55

Chrysene 59

Benzo(b)fluoranthene 43

Benzo(k)fluoranthene 43

Benzo(a)pyrene 21

National Environmental Testing Method 8270 Table 7

Six treatments were evaluated in a completely random design (CRD). Six of the eleven 3 m by 3 m square plots were randomly assigned treatment with either P. chrysospoάum (5%), P. chrysosporium (10%), P. sordida (10%), combination of P. chrysosporium (5%) and T. hirsuta (5%), lignocellulosic substrate (10%), or no amendments. On the day prior to application of the fungal inocula or standard substrate, the five plots receiving these treatments were amended with sterile aspen wood chips at a rate of 2.5% (w/w). Chips were sterilized three days before application and mixed into the soil to a depth of 20 cm with a rototiller. One day after chip application, the lignocellulosic substrate or fungal inocula was mixed into the soil to a depth of 20 cm with a rototiller. During treatment application, the soil in each of the CRD plots was physically divided into four 1.5 m by 1.5 m sections. These sections were then treated as replications during maintenance and sampling. Soil water content was determined daily and maintained at a minimum of 20% by the application of tap water when necessary. Additionally, the soil in each plot was mixed once a week by tilling and manual shoveling to provide aeration.

Soil samples were taken with a core sampler to a depth of 20 cm. The soil samples were collected in triplicate from each section 1 , 7, 14, 28, and 56 days after application of the fungal inocula or standard substrate. Samples were analyzed for PCP and pentachloroanisole (PCA) concentration. The results of the study are shown in Table 8.

Effect of CRD Treatments on the Percentage of PCP Remaining in Soil Over Time³

^a If the ANOVA showed a significant difference among treatment means Tukey's multiple determine treatment differences. ^b Treatment means within a column followed by the same letter are no significantly different (α= 0.05). ^c Soils in all treatments, except the no amendment treatment, were amended with wood chips at a rate of 2.5%. ^d P equals the probability of a larger F value for difference among treatment means.

Table 8

Compared to the PCP concentrations on day 1 , which ranged from 500-1000 .μ.g/g, all of the fungal inoculum treatments resulted in substantial decreases in the PCP levels by 7 days post amendment. These decreases ranged from a high of 63% in the plots treated with P. chrysosporium (5%) and T. hirsuta (5%) to a low of 42% in the plots treated with P. sordida (10%). The PCP level then remained fairly stable through 28 days in all of the fungal treatments. By 56 days however, the PCP concentration in the plots treated with either P. chrysosporium (10%) or P. sordida (10%) had decreased substantially further giving 67% and 89% depletion, respectively, at the end of the test period. It is noted that the two other fungal treatments (P. chrysosporium (5%) and P. chrysosporium (5%)/T. hirsuta (5%)) had final PCP concentrations higher than those at days 7, 14, and 28. This was presumed to be due to incorporation of fresh PCP from nontreated soil, introduced into the upper 20 cm layer during the periodic tilling, and that the biomass of the fungal inoculum was not sufficient to metabolize. In comparison, the two control plots, one of which was nonamended and the other amended with lignocellulosic substrate alone, showed no significant decrease in PCP concentrations over the 56-day period. The accumulation of PCA, a less toxic methylated derivative of PCP (Ruckdeschel and Renner, Appl. Environ. Microbiol., vol. 53 (1986) pp. 2689-2692), in soils inoculated with P. chrysosporium or P. sordida has been observed, therefore, soil samples from the plots treated with fungi were also analyzed for PCA. PCA was not detected in initial soil samples or at any time in samples taken from the nonamended plot. However, PCA accumulated in all fungal treated plots. As shown in Figure 6, the increases were maximal by 14 to 28 days after which they either stabilized or decreased. The largest increase was seen in the plot treated with P. chrysosporium (10%); the smallest were in the plots treated with P. chrysosporium (5%) alone or in combination with T. hirsuta (5%) (Figure 6). In the case of the plots treated with P. sordida (10%), although PCA did accumulate to levels almost as high as that seen in the plots treated with P. chrysosponum (10%), unlike P. chrysosponum, it was apparently able to metabolize the PCA further such that the final level of PCA was comparable to that seen in the plot treated with P. chrysosporium (5%)' (Figure 6).

COMPARATIVE EXAMPLE 4

Balanced Incomplete Block (BIB) Design Plots

Five treatments were evaluated in a balanced incomplete block design (BIB). The remaining five 3 m by 3 m plots constructed as described in Comparative Example 3 (hereinafter referred to as blocks), were divided into four 1.5 m by 1.5 m squares. Each treatment was randomly assigned to four one square sections in the five blocks. Thus, each treatment was replicated four times. The treatments were either P. cri/ysospor/um (10%), P. chrysosporium (13%), P. chrysosporium (10%, day 0 with 3% added at day 14), T. hirsuta (10%), or chips alone (2.5%).

Application of the treatments, sampling of the soil, and care and maintenance of the plots were as described in Comparative Example 3. The results of the study are shown in Table 9.

All of the treatments with P. chrysosporium resulted in decreases in the concentration of PCP by 7 days, and although there was some variability, the levels of PCP then gradually declined further through the 56 days. The final percent of PCP decreases for treatment with P. chrysosporium (10%), P. chrysosporium (13%), and P.jchrysosponum (10%, day 0; 3% day 14) were 72%, 52%, and 55%, respectively.

Treatment with T. hirsuta (10%) also caused decreases in the PCP concentration, however, there was a lag time. No decrease was observed at 7 or 14 days, but by 26 days PCP levels had been decreased 14%, and by 56 days the decrease had increased to 55%. In contrast, no decrease in PCP levels was obtained in the squares amended with chips alone. These results, along with those of Comparative Example 3, clearly show the ability of lignin-degrading fungi to degrade PCP under conditions of high contaminant concentrations and mixed contaminant conditions. Effect of BIB Treatments on the Percentage of PCP Remaining in soil Over Time³

Treatment⁰ PCP remaining (%) at various days after treatment"

7 14 28^d 56

P. chrysosporium (10%) 58a 45a 37a 28a

P. chrysosporium (13%) 87a 72a 92ab 48a

P. chrysosporium 81a 103a 57a 45a

(10%, day 0)

(3%, day 14)

T. hirsuta (10%) 101a 102a 86ab 45a

Chips only (2.5%) 103a 102a 136b 114b pβ 0.3017 0.1547 0.0604 0.0051 ^a If the ANOVA showed a significant difference among treatment means Tukey's multiple comparison test was used to determine treatment differences. ^b Treatment means within a column followed by the same letter are not significantly different (α= 0.05).

⁰ Soils in all treatments were amended with wood chips at a rate of 2.5%. ^d The Ftest generated by the ANOVA for sample 6 (day 28) had a significance level of 0.0604. Therefore, Tukey's test was performed unprotected.

⁸ P equals the probability of a larger F value for difference among treatment means.

Table 9

Analysis of the PCA level in these squares in shown in Figure 7. All of the squares treated with P. chrysosporium showed increases in PCA which maximized at 14 to 28 days and then only decreased slightly. This agrees with the data in Comparative Example 3 where it appeared that P. chrysosporium .as not able to metabolize PCA at any appreciable rate under these conditions. In contrast, the squares treated with T. hirsuta showed very little accumulation of PCA (Figure 7).

WOOD STUDIES

COMPARATIVE EXAMPLE 5 Contaminated Wood Preparation

To analyze the ability of lignin-degrading fungi to degrade PCP in contaminated wood, six PCP-treated ammunition boxes were obtained for study. The boxes were variously constructed of lodgepole (Pinus contorta Dougl. ex Loud), ponderosa pine (Pinus ponderosa Dougl. ex Loud), yellow poplar (Liriodendron tulipifera L.), all softwoods, and the hardwoods blackgum (Nyssa sylvatica Marsh.) and sweetgum (Liquidambar styraciflua L). The boxes were disassembled and the hardwood and softwood materials separated. The materials were chipped using a hammer to pass a 3.8 cm screen. Sterile chips were prepared by adjusting the moisture content of the chips to 60% with distilled water and autoclaving at 121 °C. for 30 min. on 3 successive days. COMPARATIVE EXAMPLE 6

Effects of wood type, chip sterilization, and fungal species (P. chrysosporium and P. sordida) on

PCP decontamination

In this experiment, the effects of wood type, chip sterilization and fungal species on concentrations of PCP in wood chips were evaluated. Chip cultures were prepared by aseptically placing approximately 10 g of either sterile or nonsterile softwood or hardwood chips in a sterile aluminium foil-covered Erlenmeyer flask. Approximately half of the agar from an inoculum plate of P. chrysosporium or P. sordida (see, Comparative Example 1) was then aseptically added to the flask along with 5000 ppm glutamine. Noninoculated control cultures were also set up. There were five cultures per treatment. Cultures were incubated at 30°C. Initial concentrations of PCP and PCA were determined on 10 replicate samples from each batch of sterile or nonsterile wood chips. Concentrations of PCP and PCA were determined at 1 , 2, 4, and 6 weeks post inoculation by gas chromatography of organic solvent extracts. Analyses were performed in duplicate at each sample time and the results are given in Table 10 below.

Initial Concentrations of PCP in Autoclaved and Nonautoclaved Hardwood and Softwood Chips

Table

As shown in Table 10, the initial concentrations of PCP in the chips was affected by autoclaving; the concentration was higher in nonautoclaved than in autoclaved chips in both wood types.

As best seen in Figures 8A and 8B, inoculation of sterile and nonsterile, softwood or hardwood chips with either P. chrysosporium or P. sordida resulted in decreases in the PCP concentration in the chips after 6 weeks. As shown in Figure 8A, in chip cultures inoculated with P. chrysosporium, decreases in the PCP concentration in sterile and nonsterile hardwood and sterile softwood were rapid and extensive between days 7 and 14 post inoculation and reached a maximum measured decrease of 63-72% by 42 days. In nonsterile softwood, P. chrysosponum did not give as large or as rapid an effect, with the decrease in PCP rising slowly to reach 30% after 42 days. As shown in Figure 8B, in chip cultures inoculated with P. sordida, decreases in the PCP concentrations in sterile and nonsterile softwood and sterile hardwood also mostly occurred during days 7-14, with the final decreases reaching from 50-66%. In nonsterile hardwood, significant decreases in the PCP concentration did not occur until the period between days 28-42 by the end of which PCP concentrations were decreased 45%. No decreases in PCP concentrations were observed in either sterile or nonsterile noninoculated chips, indicating that the observed PCP decreases were due to the activities of the inoculated fungi. As seen in Figures 9A and 9B, the depletion of PCP was always accompanied by formation of PCA. However, no accumulation of PCA was observed in noninoculated cultures, indicating that accumulation in inoculated chips was due to the activity of the fungi. Accumulation of PCA in sterile cultures was greater than in nonsterile cultures of both fungi, but particularly those inoculated with P. sordida, as shown in Figures 9B. However, it should be noted that the lowest PCP decreases were also associated with P. sordida inoculation of nonsterile hardwood and softwood. Also, in nonsterile hardwood and softwood chips inoculated with P. chrysosporium, 65% and 72%, respectively, of the PCP decrease was due to conversion of PCP to PCA. In sterile chips inoculated with either fungus, virtually all of the PCP decrease was due to conversion to PCA.

COMPARATIVE EXAMPLE 7

Effect of carbon and nitrogen supplementation on PCP decontamination using P. chrysosporium

A study was conducted to ascertain the effects of different carbon and nitrogen source supplementation on the concentrations of PCP and PCA in softwood chips inoculated with P. chrysosporium. Chip cultures were set up as described in Comparative Example 6 except each culture was supplemented with either glucose (5.1 g/g of chips), glycerin (5.3 g/g), ammonium chloride (NH₄ Cl) (3.7 g/g), glutamine (5 g/g), or potassium nitrate (KN0₃) (2.5 g/g). Control cultures with no supplementation were also set up. Three inoculated and two noninoculated cultures were prepared for each treatment. Concentrations of PCP and PCA were determined on duplicate samples from each culture after 3 weeks.

As seen in Figure 10, there was a decrease in the PCP concentration in inoculated chips regardless of supplement treatment. The largest decreases were seen with glucose, glutamine and no supplement, averaging around 70%. Again, these decreases were always accompanied by increases in PCA concentration. However, the percentage of the total decrease in the PCP concentrations as a result of PCA formation varied greatly among the treatments. When chips were supplemented with glycerin, virtually all of the PCP decrease was due to conversion to PCA. In chips receiving inorganic sources of nitrogen, the majority of the PCP loss (77-89%) was due to conversion of PCP to PCA. Finally, in chips supplemented with glucose or glutamine and in chips receiving no supplement, slightly less than two-thirds (61-63%) of the PCP decrease was due to conversion to PCA.

COMPARATIVE EXAMPLE 8

Ability of T. hirsuta and C. subvermispora to degrade PCP

The ability of T hirsuta or C. subvermispora to deplete PCP in sterile softwood chips was evaluated. Chip cultures were set up as described in Comparative Example 6 except that the fungi used for inoculation were T. hirsuta or C. subvermispora. Six cultures were set up for each treatment. Initial concentrations of PCP and PCA were determined on 10 replicate samples for each batch of chips. Concentrations of PCP and PCA were determined after 2 and 4 weeks of incubation on replicate samples and the results are given in Tables 11 and 12 below. Concentrations of PCP and PCA in Sterile Softwood Chips Inoculated With Trametes hirsuta or Left Noninoculated.³

Means followed by the

0.05). Table 11

Concentrations of PCP and PCA in terile Softwood Chips Inoculated With Ceriposiopsis subvermispora or Left Noninoculated.³

PCP (μg g^'1) PCA (μg g-¹)

Day 0 14 28 0 14 28

C. subvermispora 448.0a 300.0b 266.1c 5.1a 4.4a 6.2a

Noninoculated 448.0a 408.9a 418.9a 5.1a 4.1a 3.6a ^a Means within compound followed by the same letter are not significantly different according to Scheffe's test (α= 0.05). Table 12

As seen from the results in Table 11 , inoculation of the sterile PCP-contaminated softwood chips with T. hirsuta resulted in the PCP concentration decreasing from 382 μg/g to 145 μg/g by the end of the four week incubation period. This represented a 62% decrease in the amount of PCP which is similar in magnitude to that removed by P. chrysosponum and P. sordida in Comparative Example 5. However, the decrease affected by T. hirsuta was not due to transformation to and accumulation of PCA as it was in chips inoculated with P. chrysosporium or P. sordida.

As seen in Table 12, inoculation of PCP-contaminated softwood chips with C. subvermispora resulted in a decrease in the concentration of PCP from 448 .μ.g/g to 266 .μ.g/g after 4 weeks. This 37% decrease was the least of any of the fungi evaluated. However, as was observed with T. hirsuta, this decrease was not due to accumulation of PCA.

COMPARATIVE EXAMPLE 9

Ability of the four fungal strains to effect dry weight loss of wood

The ability of the different fungal strains to effect dry weight loss was also evaluated. Chip cultures were set up as described in Comparative Example 6 using sterile softwood chips inoculated with one of the four fungal strains. After 4 or 9 weeks, dry weight loss was determined by removing mycelium from the chip surfaces, drying the chips at 105°C. for 24 hours, and comparing the weight to the preincubation dry weight. The results are given in Table 13 below. Percentage Dry Weight Loss of Sterile Softwood Chips 4 or 9 Weeks After Inoculation With P. chrysosporium, P. sordida, T. hirsuta or C. subvermispora

FUNGUS TIME WEIGHT LOSS (%)

P. chrysosporium (9 weeks) 18

P. sordida (9 weeks) 14.9

Noninoculated (9 weeks) 0.1

T. hirsuta (4 weeks) 24.5

C. subvermispora (4 weeks) 17.4

Noninoculated (4 weeks) 1.6

Table 13

Results in Table 13 show that inoculation of softwood chips with T. hirsuta resulted in a 25% weight, loss after 4 weeks. This weight loss was much greater than the 18% and 15% decreases obtained from inoculation with P. chrysosporium or P. sordida, respectively, after 9 weeks. After 4 weeks, C. subvermispora decreased the dry weight of the PCP-contaminated softwood chips by 17%. This loss was greater than those obtained form inoculation with P. chrysosporium or P. sordida but less than that obtained with T. hirsuta. No weight loss was observed in noninoculated chips.

EVALUATION OF ISOLATES B101 AND B102 - STUDY 1

The isolates B101 and B102 were evaluated for their ability to degrade PCP in contaminated soils obtained from two sites. The methods described and illustrated in the Comparative Examples were employed and adapted as appropriate.

The two sites were Brookside and Kinleith. Analysis of soil samples from each of these two sites demonstrated contamination with PCP and dioxins, as well as high levels of boron (Table 14).

The isolates were also evaluated for their ability to biodegrade dioxins (OCDD, HpCDD, HpCDF). The biodegradation of dioxins is illustrated in Figures 14 to 16. Notably, a reduction in dioxin content of about 90% was achievable.

The isolates were found to be effective to degrade hydrocarbon contaminants in contaminated soil in the presence of boron at concentrations up to about 20,000 ppm.

The ability of the isolates B101 and B102 to degrade the hydrocarbon contaminants PCP and dioxins in these soils was evaluated. PCP bioremediation for the Brookside and Kinleath soils by the isolates is shown in Figures 11 and 12. Notably, PCP bioremediation was accompanied by a reduction in the levels of PCA. The concomitant bioremediation of PCP and PCA is desirable. Arsenic

PCP Boron Copper Chromium

Site Ref (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg)

Brookside Stockpile high 1950 2900

Brookside UW1 high 4060 4100

Brookside UW2 high 3370 3990

Brookside UW3 high 2900 2820

Brookside WM1 high 3100 2980

Brookside WM2 high 3250 5320

Brookside WM3 high 2530 2390

Brookside ERL1 high 3300 3850

Brookside E L2 high 3410 3520

Brookside ERL3 high 2840 2990

Brookside Stockpile high 3650 1570

Brookside UW1 low 2840 2600

Brookside UW2 low 2110 1630

Brookside UW3 low 4660 3580

Brookside WM1 low 4270 2170

Brookside WM2 low 3610 2440

Brookside WM3 low 4420 2820

Brookside ERL1 low 3080 2030

Brookside ERL2 low 1780 1660

Brookside ERL3 low 3960 3170

Kinleith store UW kinleith #1 2390 17700 82 37 72

Kinleith store UW kinleith #2 2100 17700 77 36 55

Kinleith store WM kinleith #1 1570 18400 74 36 62

Kinleith store WM kinleith #2 1820 17600 192 36 57

Kinleith store ERL kinleith #1 1840 17700 65 37 49

Kinleith store ERL kinleith #2 1560 18200 75 34 53

Table 14

EVALUATION OF ISOLATES B101 AND B102 - STUDY 2

Outline

The ability of species of lignin-degrading or "white rot fungi" (WRF) including the isolates B101 and B102, grown on either radiata pine or eucalyptus pulpwood chips, to decrease the concentrations of HACs in contaminated soils from three former wood-treating facilities were evaluated.

A preliminary analysis of the soils revealed that the following PCDD/PCDF congeners were present in the highest concentrations:

1 ,2,3,4,6,7,8-heptachlorodiebenzo furan (HpCDF); 1 ,2, 3,4,6, 7,8-heptachlorodibenzo dioxin (HpCDD); and octachlorodibenzo dioxin (OCDD).

The following factors were evaluated in a study on soil obtained from a Whakatane site:

fungal species; inoculum application rate; and surfactant addition.

The following factors were evaluated in a study on soil obtained from Brookside and Kinleith sites:

fungal species; and inoculum application rate.

Inoculum substrates that are readily available in New Zealand (i.e. radiata pine and eucalytpus pulpwood chips) were evaluated.

Materials and Methods

Solid inoculum was prepared by inoculating sterile substrates with mycelial slurries of the selected fungi. The slurries were prepared by homogenizing liquid fungal cultures.

Soil samples were obtained from the Whakatane, Brookside and Kinleith sites. The soils were air-dried, sieved to pass a 2-mm screen and thoroughly mixed. Soils were then stored dry, in sealed containers, until use.

The concentrations of target chemicals were determined on soil subsamples using appropriate extraction and analytical techniques.

Prior to the study, a moisture content to be used for each soil was determined. This was done by gradually adding water to soil and mixing until a good working consistency is reached. The moisture content was then determined gravimetrically.

Because PCDDs and PCDFs are extremely hydrophobic, the effect of amending the Whakatene soil with a surfactant to enhance fungal degradation of PCDD/PCDF congeners was evaluated. The surfactant evaluated was emulsified vegetable oil (EVO) which was applied at a rate of 3% (weight of oil to dry weight of soil). The EVO was mixed with the water that was used to adjust the moisture content of the soil to provide homogeneous distribution of the surfactant.

Five WRF species were evaluated, including B101 and B102. The three other isolates were of the species Phanerochaete gigantea, Resinicium bicolor, and Pleurotus ostreatus. Fungal inoculum was prepared by cultivating pure cultures of each of the fungal isolates on sterilized radiata pine and/or eucalyptus wood chips. The moisture contents of the chips were adjusted to 60% (wet weight basis) and then sterilized by autoclaving at 15 psi and 121 °C for 1 hour on two successive days.

The chips were inoculated with mycelial slurry inocula produced from liquid cultures (2% glucose and 2% malt extract) of each fungal isolate. The inoculated chips were then incubated at 30°C until they were thoroughly colonized by the fungi (about 2 weeks).

Soil treatments for both studies were conducted in 272 ml canning jars with lids modified to allow adequate air exchange. Each jar contained approximately 30 g of the test soil (i.e. wet weight) and the appropriate amount of fungal inoculum and amendments.

Three replicates were prepared for each treatment for each sample time-except for day 0. For day 0, samples were prepared on the side for each treatment from which 2 sub-samples were taken for analyses.

The cultures were incubated at 30°C under high relative humidity to prevent moisture loss. Soil moisture contents were maintained as needed.

Contaminant concentrations were evaluated on the following days: 0, 14, 28 and 56.

Soil and soil inoculum mixtures from each experimental unit were air dried in plastic weigh boats and then ground to a fine powder using a commercial coffee grinder. The ground samples were stored dry in sealed glass containers.

To determine the concentrations of PCP, HpCDD, HpCDF, and OCDD 3 g subsamples from each sample were extracted with a 50:50 mixture of hexane and acetone with a Dionex Accelerated Solvent Extractor. Subsamples of the extracts were then analyzed using GC/ECD methods to determine extract concentrations of the analytes.

PCP was analyzed as the trimethylsilyl derivative. PCP in extract subamples was derivitized using Sylon BTZ (Supelco Co.). GC/ECD analyses of derivatized extracts were performed on a Hewlett-Packard model 5890 gas chromatograph equipped with a 63Ni electron capture detector, a model 7673A autosampler, and a split-splitless capillary column injection port. Gas flows were: column flow 2 ml min^"1; total flow 60 ml min^"1. Operating temperatures were: 220°C (injector) and 300°C (detector); the carrier and makeup gas was nitrogen. The column was a DB-5 fused silica capillary column (30 m by 0.321mm; film thickness 0.25 urn). The temperature program was as follows: initial 60°C; hold for 1 min; split off for 0.5 min; ramp A, 10°C min^"1 for 9 min (60 to 150°C); ramp B, 2°C min^"1 for 20 min (150 to 190°C); hold at 190°C for 5 min.

GC/ECD analysis of extracts for HpCDD, HpCDF and OCDD were performed on the same instrument using the following conditions: Gas flows were: column flow 2 ml min^"1; total flow 30 ml min^"1. Operating temperatures were: 280°C (injector) and 300°C (detector); the carrier and makeup gas was nitrogen. The column was a DB-5 fused silica capillary column (30 m by 0.321mm; film thickness 0.25 urn). The temperature program was as follows: initial 185°C; hold for 2 min; split off for 0.5 min; ramp A, 8°C min^"1 for 8 min (85 to 285°C); hold at 285°C for 8 min.

Analyses of variance (ANOVA), using Δ = 0.05, were performed on the percent difference between concentrations of the analytes on day 0 and day 56. The main effects included in the ANOVA were fungal treatment, inoculum application rate and surfactant addition.

Results

Whakatane soil

Initial concentrations after treatment applications are given in Table 15. There was significant variation in initial analyte concentrations among the treatments for all four analytes. This was an indication of the heterogeneity of the soil with respect to contaminant concentrations.

Initial concentrations of PCP (mg/kg), HpCDF (μg/kg), HpCDD (μg/kg), and OCDD (μg/kg) immediately after treatment application.

Treatment PCP HpCDF HpCDD OCDD (mg/kg) (μg/kg)

Control 83 313 135 472

P. ostreatus 92 262 189 508

B101 182 340 351 743

B102 115 378 331 1045

R. bicoiour 154 323 307 644

P. gigantea 136 356 380 792

Table 15

Fungal inoculation had a significant effect on the mean percent decreases of all four analytes among fungal inoculation treatments (Table 16). In all cases inoculation with any of the tested fungi resulted in a significantly greater decrease than no inoculation (i.e. control).

Among the tested fungi, the greatest percent PCP decrease occurred in soils inoculated with B101.

There were no significant differences among the fungal treatments in the degradation of HpCDF and HpCDD. Average percent decrease of these compounds was greater than 90% in all fungal treatments.

Degradation of OCDD was greatest in soils inoculated with P. gigantea (Table 2). The percent OCDD decrease in all other fungal inoculated soils was less, significantly so, in soils inoculated with R. bicolor. Effect of fungal inoculum and control treatments on mean¹ percent decrease of PCP, HpCDF, HpCDD, and OCDD after 56 days of treatment.

Treatment PCP HpCDF HpCDD . OCDD

%

Control 15.6c 5.4b (33.3)b (22.4)c

P. ostreatus 75.2b 98.5a 97.8a 82.1ab

B101 90.3a 95.7a 95.9a 69.3ab

B102 75.7b 97.0a 92.4a 81.0ab

R. bicolour 83.5ab 95.0a 91.6a 68.2b

P.gigantea 76.6ab 93.7a 91.5a 86.2a

Table 16

¹ Means within columns followed by the same letter are not significantly different.

Mean concentrations of all four analytes after 56 days of treatment were significantly less in fungal inoculated treatments compared to control treatments (Table 17).

The lowest residual PCP concentration occurred in soils inoculated with B101.

There were no significant differences among the fungal treatments in residual concentrations of HpCDF and OCDD. The lowest residual concentration of HpCDD occurred in soil inoculated with P. ostreatus. However, as with HpCDF and OCDD all the fungal treatments resulted in very extensive decreases in the concetration of HpCDD.

Mean¹ fungal inoculum treatment concentrations of PCP, HpCDF, HpCDD, OCDD after 56 days of treatment.

Treatment PCP HpCDF HpCDD OCDD (mg/kg) (μg/kg)

Control 70c 263b 264c 557b

P. ostreatus 28b 4a 3a 98a

B101 13a 12a 12ab 210a

B102 28b 15a 30b 196a

R. bicolour 22ab 14a 24b 188a

P.gigantea 32b 21a 30b 95a

¹ Means followed by the same letter are not significantly different. Table 17

The rate of fungal inoculation did not have a significant effect on the average percent decrease of any of the four analytes (Table 18). Application of EVO had no effect on the mean inoculum application rate percent decrease of PCP, but significantly decreased the percent degradation of HpCDF, HpCDD and OCDD (Table 19). Effect of inoculum application rate on mean treatment percent decrease for inocuolum application rate of PCP, HpCDF, HpCDD, and OCDD after 56 days of treatment.

Inoculum application rate

PCP HpCDF HpCDD OCDD (wt inoc/wt soil) (% decrease)

10% 83.7 96.1 92.6 78.9

20% 77.1 95.9 95 75.8

Table 18

Effect of EVO application rate on mean percent decrease of PCP, HpCDF, HpCDD, and OCDD after 56 days of surfactant treatment.

EVO addition rate PCP HpCDF HpCDD OCDD (% decrease)

0 71.8a 91.3a 82.9a 79.0a

3 76.8a 84.2b 81.6b 57.6b

Table 19

The treatment combination that resulted in the greatest overall total percent decrease for the four analytes was inoculation with P. ostreatus (which is a US strain included for comparative purposes only) using an inoculum application rate of 10% and augmentation of the soil with 3% EVO. The second most effective was inoculation with the B101 at an inoculum application rate of 10% in the presence of 3% EVO (Table 20).

Based on the degradation of PCDD/PCDFs only, the most effective treatments were inoculation with P. ostreatus at a rate of 10% with or without EVO and inoculation with the B102 isolate at a rate of 20% with or without EVO.

Brookside and Kinleith soils

The relative abilites of B101 and B102 to decrease the concentrations of PCP HpCDF, HpCDD and OCDD in the two soils were evaluated. Initial (Day 0) concentrations are given in Table 20.

After 56 days treatment of the Brookside soil the concentration of PCP decreased 98.7% and 98% in soils inoculated with B101 or B102, respectively.

After 56 days treatment of the Kinleith soils the PCP concentration in the soils inoculated with B101 and B102 decreassed by 99% and 98.9%, respectively.

Fungal inoculation in both soils, using either fungus resulted in rapid and extensive decreases in the concentration of PCP. Initial concentrations of PCP, HpCDF, HpCDD and OCDD in the Brookside and Kinleith inoculated with either B101 orB102 or noninoculated.

PCP HpCDF HpCDD OCDD

Treatment (μg kg^"1)

Kinleith soil

Noninoculated 710 1.3 16.1 51.4

B101 682 na na na

B102 738 na na na

Brookside soil

Noninoculated 473 1.3 9.6 18.3

B101 481 na na na

B102 464 na na na

Na = not assessed Table 20

Percentage decreases of OCDD, HpCDD and HpCDF in the Brookside and Kinleith soils inoculated with either B101 or B 102 or noninoculated, after 56 days.

HpCDF HpCDD OCDD

Treatment (%)

Kinleith soil

Noninoculated 10.0 3.0 (2.0)

B101 63.6 94.7 93.4

B102 53.3 93.5 91.6

Brookside soil

Noninoculated 11.3 9.1 (2.9)

B101 74.3 82.3 78.3

B102 68.0 80.3 75.15

Table 21

There were only slight decreases in the concentrations sof HpCDF, HpCDD and OCDD in noninoculated Kinleith and Brookside soils after the 56-day treatment period (Table 21).

Inoculation with either B101 or B102, however, resulted in large decreases of all three analytes in both soils. Decreases were similar for both fungi.

Decreases in the concentrations of HpCDD and OCDD were greater in the Kinleith soil than in the Brookside soil, whereas decreases in the concentration of HpCDF were greater in the Brookside than those observed in the Kinleith soil. Summary

Inoculation of PCP/PCDD/PCDF-contaminated soils with the selected isolates B101 or B102 grown on locally available radiata pine or eucalyptus pulpwood chips resulted in rapid and extensive decreases in the concentrations of the contaminants.

Minor decreases in the concentrations of the contaminants indicated that the decreases in the fungal inoculated soils were due to the pollutant-degrading. activity of B101 and B102.

Treatment with either of these two fungal isolates effectively decreased the concentrations of the PCP, HpCDF, HpCDD, and OCDD in the HAC contaminated soil.

Where in the foregoing description reference has been made to integers or components having known equivalents then such equivalents are herein incorporated as if individually set forth.

Although the invention has been described by way of examples (excluding the comparative examples) and with reference to possible embodiments thereof it is to be appreciated that improvements and/or modification may be made thereto without departing from the scope or spirit of the invention.

REFERENCES

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1989. CABA Abstract 91 :24540. ,3. Lamar et al. "Use of Lignin-degrading fungi in the disposal of PCP treated wood." 1992. Biosis Abstract 92:409260.

4. Lamar et al. "Use of lignin-degrading fungi in the disposal of PCP-treated Wood". J. of Industrial Microbiology, vol. 9 No. 3-4 (1992) pp. 181-191.

5. Bumpus, et. al. "Oxidation of Persistent Environmental Pollutants by White Rot Fungus," Science 288, pp. 1434-1436 (1985).

6. Bumpus and Aust, "Biodegradation of DTT by the White Rot Fungus . . . ," App. & Env. Microbio. 53, pp. 2001-2007 (1987). 7. Bumpus and Aust, "Biodegradation of Chlorinated Organic Compounds . . . ," Solving

Hazardous Waste Problems, ACS Symposium Ser., pp. 340-349 (1987). 8. Eaton et. al., "Method Obtains Fungal Reduction of the Color of Extraction-Stage Kraft Bleach Effluents," TAPPI 65(6), pp. 89-92 (1982). . 9. Fenn and Kirk, "Relationship of Nitrogen to the Onset and Suppression of . . . ," Arch. Microbiol. 130, pp. 59-65 (1981).

10. Field et. al., "Biodegradation of Polycyclic Aramatic Hydrocarbons by New Isolates of White Rot Fungi," App. & Env. Microb. 58(7), pp. 2219-2226 (1992).

I I . George and Neufeld, "Degradation of Flourene in Soil by Fungus . . . " Biotechnol. Bioeng. 33, pp. 1306-1310 (1989). 12. Haemmerli et. al., "Oxidation of Benzyl(a)pyrene by Extracellular Ligninases . . . ," J. Biol.

Chem. 261 , pp. 6900-6903 (May 1986).

13. Hammel, "Organopollutant Degradation by Lignolytic Fungi," Enz. Microb. Techonol. 11, pp. 776-777 (1989).

14. Hammel and Tardone, "The Oxicative 4-Dechlorination of Polychlorinated Phenols . . . ," Biochem, 27(17), pp. 6563-6568 (1988).

15. Huynh et. al., "Dechlorination of Chloro-Organics by a White-Rot Fungus," TAPPI 68(7), pp. 98-102 (1985).

16. Kohler et. al., "Extracellular Ligninase of Phanerochaete chrysosporium Bursdall . . . ," App. Microb. Biotech. 29, pp. 618-620 (1988). 17. Lamar et. al., "Fate of Pentachlorophenol (PCP) in Sterile Soils . . . ," Soil Biol. Biochem.

22(4), pp. 433-440 (1990). 18. Lamar et. al., "In Situ Depletion of Pentachlorophenol from Contaminated Soil . . . ," App. and Env. Microbio. 56(10), pp. 3093-3100 (1990). 19. Lamar et. al., "Sensitivity to and Degradation of Pentachlorophenol . . . ," App. and^' Env. Microbio. 56(11), pp. 3519-3526 (1991).

20. Leatham et. al., "Degradation of Phenolic Compounds and Ring Cleavage of Catechol 1 1 1," App. and Env. Microbio. 46(1), pp. 191-197 (1983). 21. Ryan, et. al., "Biodegradation of 2,4,5-Trichlorophenoxyacetic Acid in Liquid Culture . . . ,"

App. Microbio. Biotechnol. 31, pp. 302-307 (1989).

22. Mileski et. al., "Biodegradation of Pentachlorophenol by the White Rot Fungus . . . ," App. & Env. Microbio. 54(12), pp. 2885-2889 (1988).

23. Sanglard et. al., "Role of Extracellular Ligninases in Biodegradation of Benzy(a)pyrene . . . ," Enz. Microb. Technol. 8, pp. 209-212 (1986).

24. Hammel et al. "Oxidation of Polycyclic Aromatic Hydrocarbons and Dibenzo(p)dioxins by Phanerochaete chrysosporium Ligninase", The Journal of Biological Chemistry, vol. 261, No. . 36, pp. 16948-52 (1986).

25. Unterman, "Bacterial Treatment of PCB-Contaminated Soils," Proceeding of Hazardous Materials Control Research Institute Hazardous Waste Treatment by Genetically Engineered or Adapted Organisms, pp. 17-18, Nov. 30-De. 2, 1988, Washington, D.C.

26. Lamar, R. T., "Biodegradation of PCP-Treated Ammunition Boxes Using White-Rot Fungi," U.S. Army Corps of Engineers, Toxic and Hazardous Materials Agency Report No. CETHA- TE-TR-91029 Final Report (cover page through end filming page) (31 pages), Sep. 1991. 27. Lamar, R. T. and Scholze, R. J., "White-Rot Fungi Biodegradation of PCP-Treated

Ammunition Boxes," Proceedings of R&D 92 National research & development conference on the control of hazardous materials; 1992 Feb. 4-6; San Francisco, Calif. Greenbelt, Md. Hazardous Materials Control Resources Institute; 1992, pp. 89-94.

28. Joshi D. and Gold M. H. (1993) Degradation of 2,4,5-trichlorophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl. Env. Microbiol. 59:1779- 1985.

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30. Ruttimann-Johnson, C. and R. T. Lamar. 1997. Binding of pentachlorophenol to humic substances in soil by the action of white-rot fungi. Soil Biology and Biochemistry 29:1143- 1 148.

31. Valli K. and Gold M. H. (1991) Degradation of 2,4-dichlorophenol by the lignin-degrading fungus Phanerochaete chrysosporium. J.Bacteriol. 173:345-352.

Claims

1. An isolated culture of a fungal isolate belonging to the class Basidiomycetes having at least one of the identifying characteristics of one of the isolates nominally identified as B101 (AGAL accession no. NM03/33520) and B102 (AGAL accession no. NM03/33521), the isolate being effective to degrade hydrocarbon contaminants in contaminated material.

2. An isolated culture according to claim 1 where the at least one of the identifying characteristics is tolerance of boron.

3. An isolated culture according to claim 2 where the tolerance of boron is when the boron is present in the contaminated material at a concentration of at least 2000 ppm.

4. An isolated culture according to claim 3 where the tolerance of boron is when the boron is present in the contaminated material at a concentration of at least 6,300 ppm.

5. An isolated culture according to claim 4 where the tolerance of boron is when the boron is present in the contaminated material at a concentration between 2,000 ppm and 20,000 ppm.

6. An isolated culture according to claim 1 where the at least one of the identifying characteristics is tolerance of benlate.

7. An isolated culture according to claim 6 where the tolerance of benlate is when the benlate is present in the contaminated material at a concentration of at least 0.06 g/L.

8. An isolated culture of the isolate nominally identified as B101 (AGAL accession no. NM03/33520).

9. An isolated culture of the isolate nominally identified as B102 (AGAL accession no. NM03/33521).

10. An isolated culture according to any one of claims 1 to 7 where the isolate is an isolate belonging to the order Polyporales.

11. An isolated culture according to claim 10 where the isolate is an isolate belonging to the family Polyporaceae.

12. An isolated culture according to any one of claims 1 to 11 where the isolated culture is effective to degrade the hydrocarbon contaminants in contaminated material to non-toxic components, or at least components of reduced toxicity, when the culture is applied to the material.

13. An isolated culture according to claim 12 where the isolated culture is effective to degrade both PCP and pentachloranisole (PCA) in contaminated material when the culture is applied to the material.

14. An isolated culture according to claim 13 where the culture is effective to degrade PCP, PCA and dioxins in contaminated material when the culture is applied to the material.

15. An isolated culture according to any one of claims 1 to 14 where the isolated culture is a biologically pure culture.

16. A method for the degradation of hydrocarbon contaminants in contaminated material including the step of: • applying an inoculum containing at least one culture of an isolate according to anyone of claims 1 to 15 to the contaminated material in an amount being effective to degrade hydrocarbon contaminants in the contaminated material.

17. A method according to claim including the step of: • applying a lignocellulosic substrate to the contaminated material in an amount sufficient to promote degradation of the hydrocarbon contaminants.

18. A method according to claim 16 or claim 17 including the step of:

19. A method for the degradation of hydrocarbon contaminants in contaminated material including the steps of:

• applying an inoculum containing at least one culture of an isolate according to anyone of claims 1 to 15 to a lignocellulosic substrate;

• incubating the inoculated lignocellulosic substrate for a period of time and at a temperature sufficient to allow growth of the culture; and

20. A method according to any one of claims 16 to 19 where the contaminated material contains boron.

21. A method according to claim 20 where the boron is present in the contaminated material at a concentration of at least 2000 ppm.

22. A method according to claim 21 where the boron is present in the contaminated material at a concentration of at least 6,300 ppm.

23. A method according to claim 22 where the boron is present in the contaminated material at a concentration between 2,000 ppm and 20,000 ppm.

24. A method according to any one of claims 16 to 23 where the contaminated material is solid material.

25. A method according to claim 24 where the material is soil, sludge, sediment, or debris from the processing of timber or lumber such as wood chips, shavings and sawdust.

26. A method according to claim 25 where the material is soil.

27. A method according to any one of claims 16 to 26 where the hydrocarbon contaminants are one or more polycyclic hydrocarbons, aromatic hydrocarbons or halogenated hydrocarbons.

28. A method according to claim 28 where the contaminants are halogenated aromatic hydrocarbons (HACs).

29. A method according to claim where the contaminants are one or more of polychlorophenols, DDT, polychlorinated biphenyls, polybrominated biphenyls, and the like.

30. A method according to any one of claims 16 to 23 where the initial concentration of dioxin in the material is less than 60 ppb.

31. A method according to claim 30 where the initial concentration of dioxin in the material is less than 20 ppb.

32. A method according to any one of claims 16 to 31 where the inoculum is not conditioned prior to its application to the solid material.

33. A method according to any one of claims 16 to 32 where the percentage decrease in concentration of one or more of the hydrocarbon contaminants is greater than 60% of the initial concentration of the hydrocarbon contaminant.

34. A method according to any one of claims 16 to 32 where the percentage decrease in concentration of one or more of the hydrocarbon contaminants is greater than 70% of the initial concentration of the hydrocarbon contaminant.

35. A method according to any one of claims 16 to 32 where the percentage decrease in concentration of one or more of the hydrocarbon contaminants is greater than 80% of the initial concentration of the hydrocarbon contaminant.

36. A method according to any one of claims 16 to 32 where the percentage decrease in concentration of one or more of the hydrocarbon contaminants is greater than 90% of the initial concentration of the hydrocarbon contaminant.