Demonstration Bulletin

Fungal Treatment Bulletin

USEPA-RREL/USDA-FPL

Technology Description: Fungal Treatment is a bioremediation process utilizing white-rot fungi as a means of destroying

organic contaminants in soils. The Fungal Treatment technology

was developed jointly by the Forest Products Laboratory (FPL) of

the U.S. Department of Agriculture’s Forest Services in Madison,

WI, and the U.S. EPA Risk Reduction Engineering Laboratory’s

Biosystems Branch. The Fungal Treatment demonstration was

conducted on the site of a former wood treating facility, the

Brookhaven Wood Preserving (BWP) in Brookhaven, MS. The

goal of this demonstration was to determine the use of Fungal

Treatment to destroy pentachlorophenol (PCP) and select polynuclear aromatic hydrocarbons (PAHs: creosote constituents) in

contaminated soil.

White-rot fungi are the major degraders of lignin, a polymeric

component of wood which is resistant to biodegradation. Interest

in these fungi for biotechnological applications has increased

during the past decade, spurred by the ability of these organisms

to degrade a wide variety of hazardous compounds (including

PCP and PAHs). The fungal treatment process involves inoculation of the contaminated soil with selected fungal strain(s) followed by addition of nutrients (if needed), irrigation, and aeration

of the soil through tilling/cultivating to provide optimal fungal

growth conditions. Inoculation is accomplished by physically mixing the soil and the inoculum. Mixing can be performed in solids

mixing equipment, or in-situ by placing the inoculum on the

contaminated soil and tilling until the two are thoroughly mixed. In

the case of ex-situ mixing, the soil-inoculum mix must then be

spread over the ground. Land farming procedures such as irrigation, aeration and nutrient addition are then implemented periodically to sustain the fungal activity within the soil matrix. As a

result of the fungal activity, the hazardous compounds are transformed and become irreversibly bound to soil organic matter, in

which state they are not biologically active and thus do not

present toxicity problems. The fungal treatment can take several

weeks to several months to achieve the desired level of contaminant reductions.

Two species of white-rot fungi, Phanerochaete chrysosporium

and Phanerochaete sordida, have exhibited the best growth potential in soil contaminated with wood preserving wastes. Studies

conducted by the developer have indicated that lignin-degrading

enzymes generated by these fungal species are capable of

oxidizing and detoxifying some normally recalcitrant PAHs. In

addition, these fungal species have been known to detoxify

phenolic compounds (PCP is one of them) by methylation of the

phenolic group. The methylated product is susceptible to further

transformation by the fungi.

Prior to treatment, a treatability study either in the field or in a

laboratory is performed using representative contaminated soil.

Based on the treatability data, the developer will determine: (1)

the applicable fungal species, (2) the inoculum loading level, (3)

length of treatment time, and (4) the need for nutrients or other

soil supplements.

Waste Applicability: This Fungal Treatment has been tested

for treatment of soils contaminated with organic wood preserving

compounds such as pentachlorophenol (PCP) and select polynuclear aromatic hydrocarbons (PAHs) found in creosote. Warm

temperatures (greater than 80oF) and sufficient moisture (greater

than 30%) in the target matrix are desirable for the optimal

growth of the fungus and, thus, for the degradation of the contaminants.

Demonstration Results: Over a 20-wk period, from June

1992 through November 1992, the Fungal Treatment technology

was tested under actual field conditions. The test matrix was

produced from excavated wood preserving wastewater treatment

sludges from unlined onsite surface impoundments. This soil-like

material was screened to remove solid debris larger than three

inches in size, and further mixed/diluted with clean soil to bring

the PCP concentration down to an acceptable level for fungal

growth and viability. This test soil was then used in the test and

the two control plots as described below.

On the BWP site a Test Plot and two Control Plots, A and B,

were constructed. In Control Plot B, the test soil mix only was

homogenized and placed as a 10-in. bed on top of a sand layer.

In Control Plot A, the test soil mix was homogenized with the

sterile (i.e., non-inoculated or non-fungal) spawn material at a

10:1 w/w ratio and placed as a 10-in. bed on a sand layer. In the

Test Plot, the test soil mix was homogenized with the fungal

inoculum at a 10:1 w/w ratio and placed as a 10-in. bed on a

sand layer. The Test and the two Control Plots were rototilled/

cultivated about once a week over the duration of the study. Soil

temperatures were recorded on a daily basis. Moisture content of

the soil from each of the plots was determined on a weekly basis.

If the moisture content in a plot dropped below the target level, it

was irrigated with leachate collected from the same plot and/or

with the municipal tap water. Soil samples from each of the plots

were collected at the start of the study, and then at the end of the

5th, 9th, and 20th weeks of the study.

Data from the Demonstration are undergoing final review. Some

key initial findings are as follows:

EPA/540/MR-93/514

June 1993

United States

Environmental Protection

Agency
• Levels of PCP and the target PAHs found in the underlying sand

layer and the leachate from each of the plots were insignificant,

indicating low leachability and loss of these contaminants due to

periodic irrigation of the soil and heavy rainfall.

• Levels of PCP, the target PAHs, and dioxins in the active air

samples collected during the soil tilling events were insignificant, indicating a very low potential of air-borne contaminant

transport as a result of Fungal Treatment activities.

An Applications Analysis Report and a Technology Evaluation

Report describing the complete Fungal Treatment SITE demonstration will be available in Summer of 1993.

The developers collected data independently and will produce

results via other publications.

For Further Information:

EPA Project Manager:

Kim Lisa Kreiton

U.S. EPA

Risk Reduction Engineering Laboratory

26 West Martin Luther King Drive

Cincinnati, OH 45268

(513) 569-7328

Developer:

EPA/Biosystems: John Glaser

(513) 569-7568

USDA/FPL: Richard Lamar

(608) 231-9200

BULK RATE

POSTAGE & FEES PAID

EPA

PERMIT No. G-35

United States

Environmental Protection Agency

Center for Environmental Research Information

Cincinnati, OH 45268

Official Business

Penalty for Private Use

$300

EPA/540/MR-93/514