?,EPA
                                    United States
                                    Environmental Protection
                                    Agency
                 EPA/540/MR-93/514
                 June 1993
                                    SUPERFUND  INNOVATIVE
                                    TECHNOLOGY EVALUATION
                                    Demonstration  Bulletin

                                       Fungal Treatment Bulletin

                                           USEPA-RREL/USDA-FPL
Technology Description:  Fungal Treatment is a bioremedia-
tion 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,
Wl, 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 (POP) and select poly-
nuclear 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
POP and PAHs). The fungal treatment process involves inocula-
tion of the contaminated soil with selected fungal strain(s) fol-
lowed 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 mix-
ing 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-inoculurn  mix must then  be
spread over the ground. Land farming procedures such as irriga-
tion, aeration and nutrient addition are then implemented periodi-
cally to sustain the fungal activity within the soil  matrix  As a
result of the fungal activity, the hazardous compounds are trans-
formed 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  contami-
nant reductions.

Two species of white-rot fungi,  Phanerochaete chrysosporium
and Phanerochaete sordida, have exhibited the best growth po-
tential 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 poly-
nuclear aromatic hydrocarbons (PAHs) found in creosote. Warm
temperatures (greater than 80°F) 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 con-
taminants.

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:
                                /-v-y
                               U%) Printed on Recycled Paper

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  • Levels of POP and the target PAHs found in the underlying sand   For Further Information:
    layer and the leachate from each of the plots were insignificant,
    indicating low leachability and loss of these contaminants due to   EPA Project Manager:
    periodic irrigation of the soil and heavy rainfall.                 Kim Lisa Kreiton
                                                             U.S. EPA
  • Levels of POP, the target PAHs, and dioxins in the active air   Risk Reduction Engineering Laboratory
    samples collected during the soil tilling events were insignifi-   26 West Martin Luther King Drive
    cant, indicating a very  low potential of air-borne contaminant   Cincinnati, OH 45268
    transport as a result of  Fungal Treatment activities.             (513) 569-7328

An  Applications Analysis Report  and a Technology  Evaluation   Developer:
Report describing the complete Fungal Treatment SITE demon-   EPA/Biosystems- John Glaser
stration will be available in Summer of 1993.                      (513) 569-7568
_.   .    ,        „                                           USDA/FPL: Richard Lamar
The developers collected data independently and will  produce   (608) 231 -9200
results via other publications.

•U.S. Government Printing Office: 1983 — 750-071/80011
   United States
   Environmental Protection Agency
   Center for Environmental Research Information
   Cincinnati, OH 45268

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