CA2101057A1 - Solid state culture of white rot fungi - Google Patents

Abstract

White-rot fungi are grown on a sugar beet pulp substrate.
By-products of fungal growth, such as lignin-degrading enzymes, can be recovered from the culture. The culture or enzymes recovered from the culture can be used to degrade aromatic compounds in bioremediation procedures.

Description

W ~ /13960 2 ~ O ~ ~ ~ 7 PCT/US92/00871 ~Solid Sta~e Cul~ure of White Rot Fungir Backgro~nd o~_~h~ L~yç~ n Enzymes for degrading aromatic compounds have 05 potential commsrcial application in the pulp and paper indu~try, the production of fuels and chemicals from .:.
lignocellulos~, the enhancemsnt of live~ock f2eds, and the bior~ed~ation of arom~ti~ ha~ar~ou~ wa~tes.
~ignin i8 a comples polym~r of phenyl propanoid 10 units with a vari~ty of interunit linkagas forming a nonlinear, rsndom structure. Lignin compri~es 10-356 . .
of the dry wei~ht of lignocellulose-rich materials . ::
such a~ wood, straw, and corn stover. Lignin is resistant to biologica} de~truction, although it is 15 enzymatically ~egraded by various higher order fungi. :`~
In nature, tha :~U ~L~ ~D ~ that cause white-rot wood decay aro m~or ~egrad~r~ of lignocellulo~e.
White-ro~ fungi osid$ze ligni~ completely to carbon dioside. E~tracellular enzyme compleses ~ecreted by 20 these fungi catalize o~idative reactions of the lignin -: .
structure. White-rot fungi have al~o been shown to osidi2e and d~gr~e ~ wi~e r~nge:o~ other aromatic : structures inc~uding a Yariety of:man-made, tosic aromatic compounds. The term:~white-rot fungi~:as : 25 used herein i~ intended to include fungi having enzymes capable o 02idizing and thereby degrading aromatic compounds. ;

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WO92/13~0 ~ PCT/US92/0087 There are an estimated 1700 species of white-rot fungi. However, research on enzymatic lignin degradation has concentrated on one organism:
Phanerochaete chr~sosporium. Lignin-degrading enzymes 05 from this organism have been purified and characterized. A large volume of research literature describes processes for growing P. chrvsosporium in liquid media for lignin degradation or production of lignin-degradiny enzymes. The conventional production 10 of lignin-degrading enzymes in liquid media occurs during secondary metabolism and is initiated by nitrogen or glucose starvation. For instance, in U.S.
Patent 4,554,075, Chang e~ al. describe a process for ~rowing white-rot fungi by carrying growth into 15 secondary metabolism wherein nitrogen starvation occurs. See also Ming Tien in an article.in Ç~
CritiGal Revie~s in Micr~bioloQy, titled UProperties of ~ignina~ From Phansrochaete Chrysosporium and Their Possible Applications~, Volume lS, Issue 2 (1987) at p. 143 and U.S. Patent 4,891,230 to Aust et al.
The slow growth rates and low cell mass -~
production a~sociated with starvod cultur~ results in long growth times and low yields thus~ making this 25 impractical for commercially producing enzymes for pretreating wood pulp in paper;making processes, for in ~i~ treatment of to~ic waste, or for enhancing lignocellulose for livestoc~ feed. Tien notes on page 144 in the same article llsted above that scalo-up 30 from liquid culture grown in flasks has proven difficult.

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2 ~ 7 PCT/US~2/00871 To overcome the low cell mass production, the art has suggested growing several species of white-rot fungi using solid culture media in solid state reactors. In these instances, the fungus grows on a 05 substrate of moist solid lignocellulose-containing materials. Straw, several types of wood, and milled corn cob have been disclosed as ~ubstrates in the literature. These materials have b~en selected as culture su~s~rates primarily because they are l0 typical of the materials degraded by the white-rot fungi in nature. They have a relatively high lignin content of l0-35%, low nitrogsn levels, and limited access to cellulose ~s a carbon source. White-rot fungi can be grown in ~uch ~olid-state cultures, but 15 obtaining lignin-degrading enzymes in cell and solids free estracts of such cultures has proved an elusive task as the enzyme activity remains bound to the substrate.
Several patents as well as other lit~rature 20 disclose processes for preparing ligninsse in solid cultures includi~g U.S. Patent 4,71},787 to Odakra, ^
which describes using okra as a su~strate for the production of livestock feed. Rolz, ~ aL,, in an article in 25 titled, nWhite-Rot Fungal Growth on Sugarcane Lignocel-lulosic Residue~,~Volume 25 (1987) pp.
535-541, report us~ing sugarcane residue as a substrate. In U.S. Patent 4,891,320, Aust et al. list as typical materials used to grow white-rot fungi for 30 us in degradation of aromatic compounds shredded paper, wood shavings, sawdust, corn cobs, and humus.
None of these references discloses the production of `

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w092/13~60 2 ~ O ~ 0 5 7 PCT/US92/00~7 ~

enzymes during the primary metabolic growth phase or the production of cell-free extracts of the culture containing lignin-degrading enzymes.
It is believed that the reason why extracting 05 cell-free enzymes is difficult in conventional solid state proce~ses for producing enzymes is th~t the enzymes are absorbed into the lignocellulosic substrate materials. Thus, whsn using substrates of the type norm~lly associated in nature with white-rot 10 fungi, lignin-degràding enzymes are difficult to e~tract or purify in ~ctive form. These substrates typically have a high lignin content and low pro~ein content. On the other hand, small amounts of cell-~rae enzymes are prasent in liquid cultures, 15 presuma~ly because ~h~re are no surfaces for enzyme absorption.
~ oth liguid and solid substrate cultures of white-rot fungi have been ~he subject of at least 15 year6 of intensive ressarch in numerous laboratories, ~ -20 as evidenced by the volume of research literature and patents granted in this field. However, the problems of producing enzymes during the primary metabolic growth phase, of producing cell-free enzymes from solid culturs and of producing lignin degrading enzyme 25 preparations with comm~rcially useful enzyme concentrations remajn unsolved.

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WO92/13960 _5_ ICT/US92/0087l Summary of Iavention This invention pertains to a novel composition of matter comprising a solid state culture of white-rot fungus in a mixture with a substrate comprising as an 05 important ingredient sugar beet pulp. This invention also pertain~ to the process for growing white-rot fungus in solid state culture u~ing sugar beet pulp and the use of tha fungal culture to degrade aromatic compounds ~uch as lignin or other aromatic organic 10 pollutants. Tha culture also can be used for production of by-products of fungal growth ~uch as lignin-degrading enzymes. The culture advantageously permits the production of lignin-degrading enzymes by $
tha white-rot fungi during the primary metabolic 15 growth phase o~ the f u~gu~ rather than-during secondary metabolism. Furthermore, the ligni~-degrading enzymes can be separated easily from the substrate material for the production of cell-free enzymes preparations.
The culture is prepared by growing white-rot fungus under growth-supportive conditions on a substrate comprising sugar beet pulp. An inoculum culture of white-rot fungus is prepared for inoculating th~ substrate. Water and nutrients are 25 added. A substrate of ~ugar beet pulp is prepared typically by sterilizing the substrate as by autoclaving and then cooling the substrate. The substrate is inoculated with the prepared inoculum.
The inoculated substrate is then placed in a solid 30 state reactor for growing fungi, and the mixture is aerated to enhance growth. Nonlimiting e~amples of white-rot fungi that can be grown in the substrate include species from the genera Phane~ochaete, Phle~ia, I~¢~haQ, ~5~ , and Bierkandera.

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, WO92/13960 ~ O ~ 7 PCT/US92/00871 At the conclusion of the growing period, the culture can be used without further processing. For e~ample, the culture can be used in bioremediation processes to degrade aromatic organic pollutants (e.g.
05 polynuclear aromatic hydrocarbons and chlorinated aromatic compounds) in a soil or water mass.
Alternati~ely, e~tracts rich in lignin-degrading enzymes may be separated from the substrate.
For production of by-product o~ ~ungal growth, 10 one can isolate ~y-products from the culture after an appropriate growth period. For e~ample, the substrate can be washed with water to bring aqueous-soluble enzymes such as ligninases into solution. The lignln-degrading enzymes can be recovered separate 15 from the subs~rate using ~his process. The enzyme-rich solution can be centrifuged and filtered to provide a cell free liquid enzyme preparation containing lignin-degrading enzymes that have been removed from the substrate.
The growth of white-rot fungi on sugar beet pulp substrate results in the ability to produce lignin-degrading enzymes during the primary metabolic ' , growth phase of the fungus when an abundance o9 nutrients are available and growth rate i8 optimal 25 rather than~in ~eco~dary metabolism with limited , ',, nitrogen or carbon. The ability to produce ' ' lignin-degrading enzymes commerc,ially during the primary metabolic growth phase and to produce aell free lignin-degrading enzymes is an advantage of this ' invention over conventional solid state or liquid culture process used to produce these enzymes using white-rot fungi.
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WO92tl3~60 PCT/US92/00871 2~ 0~

~rief ~es~rip~ion of thç. Figures . .
Figures lA and lB are gas chromatograms of polychlorinated biphenyl compounds in control and .
fungus-treated samples of soil.
05 Figures 2A and 2B are the same for a different experiment.

Detailed~ iPtion of the Inv~n~i~n Sugar b~et pulp is u~ed as the substrate material for fungal growth ln accordance with this invention.
10 Sugar beet pulp i~ produ~ad in large ~mount~ and is readily available for high-volume, commercial applicationfi for growing white-rot fungi.
Sugar beet pulp has not been reported as a natural substrate or white-rot fungi. I.t has a . .
15 relatively low lignin ~ontent o~ l~ to 3%. White-rot fungi occurs naturally as decay organisms on woody material~ with high ligni~ content ~uch as okra, sugarcane, shredded paper, wood shavings, Rawdust, corn cobs and humus. These materials have been used 20 in~conventional production of lignin-degrading enzymes.
&ugar beat pulp co~tains~8-lO~ protein and up to 5% rasidual suorose a~d i~ not a~carbon and nitrogen limited su~strate. Yet, white-rot fungi produce gnin degrading enzymes when grown on sugar beet pulp 25-during the primary metabolic growth phase. ~:
Lignin-degrading enzymes are produced by white-rot. .
fungi when grown on sugar beet pulp supplemented with glucose and the additional nitrogen sources peptone (a soluble protein hydrolysate) and yeast e~tract. This 30 result is une~pected because production of these ~ . enzymes uslng conventiona~l processes typically occurs : ~.
;: only with nitrogen or~carbon~starvation during ~ secondary metabolism.

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W092~l396~ - . . . PCT/US92/00871 5 ~ ~' Sugar beet pulp is a byproduct of the processing of sugar beets for sugar (sucrose). In a typical process, sugar beets are sliced and extracted with hot water to recover the sùgar. Sugar beet pulp is the 05 residue of sugar beets remaining after the e~traction process. In most ~ugar beet processing plants, the sugar beet pulp is dried and sold as cattle feed.
Sugar beet pulp is composed o the following constituents with the typical proportions shown as a 10 percentage on a dry weight basis.
Mea~ ~h*mi~l_g~ iLi~n g raw æuqar bee~ ~ulP
~omponen~s ._ Raw Pulp Dry mstter . 91 5 Total Nitrogen (~ 6.25). 10 8 15 Protein ~itrogen ~s 6.25~ 9.0 Ashes 4-3 Organic Matter , 95.7 ADFa 23.3 NDFb ~ 51 9 ~0 Lignin 1 0 Cellulose (ADF-Lignin)22.3 Hemicellulose ~NDF-ADF)2B.6 Gross Energy ~ -. (k~ k~ dry matter) 4217 25 a This is acid:detergent fi~er.
b This is neutral detergent fiber. ~ :

: ~ A. Duranl and~:D. Cherau (198:8); ~A New Pilot Reactor for Solid ~tate Fsrmentation: Application to the Protein Enrichm~nt~of Sugar ~eet Pulp~; ~iotech~olo , Vo1.~ 31, pp 476-48~.
~ Particles of sugar;beet pulp are typically 0.5 to 1 cm in the largest dimension and irregularly shaped.
Sugar beet pulp can be prepared for use as a solid culture substrate as follows. Dry sugar beet 3s pulp is moistened with one of a number of standard : ~ . , ~ nutrient solutions:supportive of ~ungal growth and :

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wos~13960 PCT/US92/0087~
~ 2 ~ 7 g--then sterilized by autoclaving, e.g., at 125C, 15 psi for 20 minutes. Other generally accepted methods for sterilization can be used involving different temperatures, pressures, and durations as long as the 05 sugar beet pulp is sterilized before inoculation. The sugar beet pulp is then cooled to between 20-40C.
An inoculum of white-rot fungi is then aseptically and thoroughly mi~ed with the cooled sugar beet substrate. The inoculum can be prepared in any 10 conventional manner such as by first selecting a pure culture of a white-rot fungus and maintaining this fungus on nutrient agar slants. Next, the culture on the agar slants is transferred to either a liquid or solid media and grown at 20-40C. The media selected 15 varies somewhat depending upon which organism is selected for growth. If a liquid media is selected for ~rowing the inoculum, the liquid inoculum media should contain glucose, a nitrogen source, and nutrient salts. Liqui~ cultures can be held 20 stationary or agitated during the culture growth phase. If a solid media is selected for growing the inoculum, either sterilized sugar beet pulp, prepared ~-~
as described above, or other known materials can be used as a substrate. G~n~rally, sufficient inoculum 25 culture is grown to provide approximately 1-20% by volum~ of the mass of substrate to be inoculated.
According to the present invention, the inoculated~sugar beet pulp comprises a so:id state culture characterized by a solid phase of particles of 30 sugar beet pulp, an aqueous phase sorbed into the particles o~ the pulp and a gas phase in the interparticle spaces. Moisture content of the sugar beet pulp is 40 to 80%, typically 66% by weight.

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wo s2/l3s6n PCI'/US92/00871 5 7 ~, Optionally, 2-10% sterilized straw can also be added to the sugar beet pulp. Straw may be added before or, more typically, after the beet pulp is wetted. The straw improves the physical characteristics of the 05 solid culture by increa~ing the Yolume and maintaining integrity of interparticle spaces resulting in improved aeration, temperature control, and moisture control.
The fungus grows on the surface of, and 10 penetrates into, the particles of sugar beet pulp.
The inocul~ted substrate is placed in a vessel designed as a solid culture reactor or in a trench or pile. The shape and dimensions of the vessel used as the solid culture reactor may be varied widely~ In 15 one currently developed embodiment, the inoculated substrate is placed in cylindrical or rectangular vessel in a bed appro~imately 70 cm deep. The vessel is designed 80 that air at controlled temperature and humidity can be circulated through the bed and 20 appropriate means are provided for this.
In a solid state reactor, the temperature, nutrients, aeration rate, and growing period can be varied to regulate the meta~olic rate of the fungus.
Metabolic condi~ions also can d~termine the specific 25 types of lignin-degrading enzymes produced by the : fungus. Typically, the temperature of the substrate is maintained between 20-40C depending on the organism and en~yme preparation bei ng produced. A
nutrient solution may be added to the substrate as ~ ;
30 necessary to maintain primary metabolic growth phase.
Sufficient conventional nutrient solution is provided during the growing period to prevent nitrogen or carbon starva~ion or secondary metabolism.

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WO 92/13960 PCl`f US92/0087 1 ~ 2 1 ~

An atmosphere of air, or an artificially created atmosphere having an oxygen concentration of 7-100%, is circulated through the substrate during the growing period. An aeration rate of between .05 to 20 unit :
05 volumes of air per minuts per unit volume of substrate may be used. The aeration atmo~phere preferably i~
maintained between 70-99% relative humidity. The relative humidity typically is varied to maintain the absorbed water content of the substrate between ahout 10 40-80% initially, and then between about 60-80% at the end of the growing period, with 66-72% being typical.
The growing period of the culture is ~aried from 4 to 30 days, depending on the identity of the organism and ~he type of enzyme to be produced.
At the completion of the growing period, the culture comprises a fungal cell mass, unutilized culture substrate, and extracellular enz~mes. For some applications, particularly in situ degradation of tosic waste~, the-whole wet culture may be used 20 without further processing by merely turning the culture into the soil.
The method of this invention can be used to degrade polyaromatic hydrocarbons and polyhalogenated aromatic compounds such as~polyhalogenated bipbenyl 25 compounds in a variety of materials. The method can be used in the bioremediation of so1ls, aquatic sediments, gravels or other solid materials contaminated with polyhalogenated biphenyl compounds.
For bioremediation of soils, whole wet culture is 30 spread on the soil surface and mi~ed to thoroughly disperse the particles of white-rot fungus, sugar beet pulp culture through~the soil. In laboratory experiments mixing can be accomplished by stirring.
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w092/13960 2 1 ~ 1 0 ~ 7 PCT/US92/00871 ~

In many contaminatsd sites, contaminants have spilled on the surface and contamination is confined to the top ~5-50 cm of soil. In these cases the fungus, sugar beet.pulp culture is spread on the soil surface 05 and mi~ed using tilling equipment such as a rototiller, tractor and plow, etc. The methods and implement~ to accomplish mising may vary if uniform dispersion of white-rot fungus culture through the soil can be achieYed. Where contamination e~tends too lO deep for efective mising or is not accessible to direct mixing a6 in ~he case of underwater seaiments, the mat~rial to be treated may be e~cavated and mi~ed with the white-rot fungus, sugar beet pulp culture. -~
The misture can thsn be ~prsad in windrows or lifts on 15 a sur~ace or placed in a container such as a lined trench or tank.
The ~olume of white-ro~ fungus, sugar beet ~ulp culture aaded to a given volum~ of soil varies with soil characteristics (such as pH and de~ity) 20 concentration of polyhalogenated biphenyls and treatment time. For low concentrations of contaminant generally lO0 ppm or les~, one application of a volume of fungu~ culture ~qual to 25~ of the volume of soil may be sufficient to achieve the desired level of 25 remediation. With high concentrations of contaminant or for more rapid degradation, up to 150% volume fungus culture to volume of soil may be necessary.
Alternatively, several additions of 25% fungus culture volume at lO to 20 day intervals may be the most 30 effective.

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wo g2/139~0 2 1 0 1 0 ~ 7 PCT/US92/00871 . ~
~13-Moisture content of the mi~ture of soil and fungus culture is typically maintained at 40-60%, though this may vary depending on water capacity of the soil and volume of fungus culture used.
05 Temperature for treatment must be within a range supportive of growth and metabolism of the species of white-rot fungus being introduced. Generally this is in the range of 10 to 40C. Time required to achieve a specific level o~ degradation will vary with 10 contaminant, its concentration, soil characteristic, volume of culture, temperature and moisture.
Significant degradation of polyhalogenated biphenyls may be achieved in a few days up to se~eral months.
In addition to th~ use of whole, wet culture for 15 remediation, cultures may be processed by forming a slurry that can be pumped and mi~ed more easily in some types of materials. Cultures may al~o be dried for improved storage and transportation and rehydrated immediately prior to application.
To produce a cell-free liquid enzyme preparation containing lignin-degradin~ enzymes, one can e~tract the culture by mixing it with water. Alternatively, water together with conventional, biologically compatible detergents, such as TWEEN 80, may be used 25 as an extractant. A cell-free solution containing lignin-degrading enzymes can be produced by mixing the culture with the extractant, then centrifuging and filtering to remove all cells and solids 'with, for example a 0.8 micron filter).
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W0~2/13960 ~ 0 ~ 7 PCT/US92tO0871 The sugar beet pulp substrate is capable of sustaining growth of a variety of white-rot fungi to induce production of at least four types of enzymes, namely, pero~idases, manganese peroxidases, oxidases 05 and laccases. To determine the nature of the enzymes present in various e~tracts, conventional ~ssa~
procedures such as those based on enz~matic osidation of compounds such as phenol red, veratryl alcohol, vanillylacetone and ani~ alcohol with and without the 10 presence of hydrogen pero~da or o~ygen or manganese are used.
~ 8~ay8 of pero~idase are based on oxidation of phenol red or Yeratryl alcohol in the presence of hydrogen peroside. See 2.g., Tien, ~. (1987? Criti~al 15 ~eoi~Li~L~ oh~ s~ 2):144; Farrell, R., U.S.
Patent No. 4,687,741; Kuwahare, M. ~ ~1~ (lg84) E~
Ic~J~ls~ lh2(2):247-250; Walder, R. 8~ al. ~198B) Applied Mi~rQbiolo~Y and BiotechnoloqY ~:400~407.
Assays for manganese p~ro~idase measure o~idation of 20 phenol red, veratryl alcohol or vanillacetone with the presence of both hydrogen pero~ide and~manganese. See Kuwahare, M. et al.~and Walder, R. ~ 21., supra;
~onnarme, P. and ~efferieR, T.W. ~1990) Appl~d~ ~nd Environmental ~ Qki~l~gy ~fi(1):210-217. Assays for 25 o~idase are based on oæida~ion of veratryl alcohol or anis alcohol with the pr~sence of o~ygen. See Muheim, Ao et al. Er~3~ and Mic~Q~ Technoloqy; Walder, R.
et al., supra. Assays of laccase activity is based on oxidation of phenol red or 2,6-dimethoxy phenol in the 30 a~sence of hydrogen peroxide and manganese. See Kuwahare, M. Q~ al. and Walder, R. et al~ E~
Haars, A. and Huttermann, A. (1980) Archive~ of Microbiolo~y 125:233-237. ~ - ;;~ ;

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W09~t139S0 21 O 10 ~ 7 PCT/US92/00871 As illustrated in the examples below, culture extracts grown by the processes of this invention have been assayed using each of these procedures. The presence or absence of hydrogen pero~ide, manganese, 05 and o~ygen in the en~yme reactio~ provides a basis for distinguishing the di~ferent ~ypes of activities.
It is an important feature of the invention that all of these different types of enzymes can be produced. Different commercial applications may 10 require specific types or combinations of these types of enzyme activitie~. Furthermore, the different t~pes of enzyme~ produced by various white-rot fungi grown by this process, dif~er in substrate specificity, pH optima, buffer re~uirements and 15 stability. These diferences may confer relative advantages of one organism and or one type of enzyme in specific commercial applications.
The invention i8 illustrated further by the following eYamples. All percentages are ~y weight and 20 all inoculum mi~ture proportions are by volume unless otherwise noted.
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Production of Mn~Perosidase using P . ch FysQ spo r iur~

P. Ghryso~p~ m obtained from the USDA FQrest Products Laboratory (strain BKM) was grown without agitation for 10 days at 25C in a high-nitrogen, stationary-liquid medium composed of 10 g/l glucose, 5 g/l peptone and 3~g/1 yeast extract (Difco). This 30 liquid culture was used~ as an inoculum culture for the .
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solid culture medium. The solid culture medium consisted of dried sugar beet pulp wetted to 66%
moisture with a nutrient solution disclosed in Table 1:

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Table 1 05 TYPICAL ~UTRIENT SOLUTION USED

SUb~t~n~ç-- 9~1 __ Su~stanc~e _ g~l Glucose 10.0 cacl2.2H2o NH4H2Po4 ,05 Trace Elements 5 ml stock solution 10 KH2PO4 1.0 Veratryl Alcohol 0 or .14 MgSO~.7H20 1.0 Peptone .05 Yea~t eYtract .05 The wetted sugar beet pulp was autoclaved at 120C, 15 psi, ~or 20 minutes, cooled, and inoculated 15 at the rate of 10 ml ~noculum cultures per 100 ml of sugar beet pulp subs~rate. The solid culture was incubated for 5 day~ at 2~C with an air f}ow of .2 volume of air per volume of culture per minute with the air at 90% relative humidity. At 5 days, the 20 culture was extracted by adding 3 volumes of water per one part wet weight of whole culture, blended for one minute, centrifuged, and passed through a 0.8 micron ~-~
filter to produce a cell and solids-~ree, liquid enzyme preparation. The e~tracted enzyme preparation 25 was assayed using the phenol red and vanillylacetone assays. In the presence of both hydrogen peroxide and manganese, activity was 80 Phenol Red Units per ml as ~ .
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~0 g'/13960 2 1 ~ 1 0 ~ 7 PCT/~S92/00871 . ~ .

assessed by the phenol red assay and .92 International units per ml by vanillylacetone assay. Mn peroxidase was the only activity detected in this preparation.
~Phenol Red Units" may be defined as a 0.1 absorbance 05 change in the optical density of a standardized assay in 30 minutes. An ~International UnitW may be defined as the production o~ 1 ~mole of reaction product per minute using conventional as~ay techniques such as ~hose ~xploiting veratryl alcohol, anis alcohol, and 10 vanillylacetone.

Production of Mn peroxidase and laccase using P. sh~Y~UEalLiUm P. ~hrYsosporium was grown under the conditions 15 described in Example 1 t except that the inoculum volume was 5%, and the dry sugar beet pulp was wetted to 66% moisture with a nutrient solution including 10 9/l glucose, 5 q~l peptone, and 3 g/l yeast extract. Cultures were grown for 14 days and 20 extracted with two volumes of water per 1 volume wet weight culture.
E~tracts which were assayed with phenol red contained 6~ Phenol Red Units per ml of Mn pero~idase activity and 27 Phenol Red Units per ml of laccase 25 activity.
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~ ,' WO92/13960 2 1 0 1 ~ 5 7 PCT/US92/00871 ~$ ' ' E~mple 3 Pilot scale production of Mn pero~idase Cultures were grown under conditions described in Example 1 except that 5% by weight (dry basis) milled 05 straw was added to the sugar beet pulp preparation.
Cultures were grown in a 20 litor vessel with a substrate bed depth of 70 cm, aerated with 1 volume air per volume of culture per minute at 27-30C.
Extracts of culture6 harv~sted at 10 days showed Mn 10 peroxidase activity at ~6 Phenol Red Units. ~

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Production of peroxidase, Mn peroxidase and laccase/oxidas~ using ~ ~51Si~QiQ~
,. ~ , An inoculum culture of Trametes Y~ash3aLQ~ (ATCC
15 48424) was grown in stationary culture in~the salts solution of Esample 1 at 27C for 7 days. The inoculum culture was used to inoculate (5~ v/v) a series of identical solid cultures composed of sugar beet pulp wetted to 66~ moisture with the high 20 nitrogen solution of Example 2. Each of the cultures were incubated at 27C with an air flow of .2 vol/vol culture per minute at 90%RH. These identical cultures were extracted in 4 volumes of water at different time intervals and assayed for enzyme activity using phenol 25 red. Results are shown below:
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W092/l3960 2 1 0 1 ~ ~ 7 PCT/VS92/00871 ~, . . .
:- --1 9--Culture Phenol Red Phenol Red Phenol Red Time in Units of Mn Units of Units . of Days P~ro~idase l~o3~LilQi~ accas~Qxi~a~ç

05 17 25 . 17 44 haccase/o~idase activity is o~idation of phenol red without hydrogen pero~ide or manganese. Assay technique~ use~ in th$s example do not dist~nguish 10 betw~en laccase an~ osida~e type activitie~.
An additional type of enzyme activity may bP
produced by growing T~am~ ~sl~bkDLL~ according to the method of ~his eYampl~. This is an activity that o~idize~ phenol r~d in the presence of mangane6e but 15 without hydrogen peroside. This activity i8 present in 10 day cultures at 12 Phenol Red Units per ml extract and in 17 day cultures with 47 Phenol Red Units per ml.
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E~ample 5 Production of Xn perosidase and perosida~e using ~ ve~si~olD~
.~" ' Cultures were grown and extracted under the conditions described in Example 4 except that the inoculum nutrient solution was 10 g/l glucose, 5 g/1 25 peptone and 3 g/l yeast e~tracts instead of the salts solution. At 10 days culture the extracts contained 22 Phenol Red Units of Mn pero~idase activity and 33 : Phenol Red Units:of pero~idase activity per ml.
Extracts showed no laccase or oxidase activitiesO -, : .

~1010 ~ rl ExamPlç 6 Pilot scale production of Mn pero~idase and peroxidase using T. versi~Qlor : -Culturss were grown under conditions described in 05 Example 4 e~cept that 3~ by weight (dry basis) milled straw was added to the ~ugar beet pulp preparation.
Cultures were grown in a 20 liter v~sel with ; : .
æub~trate b~d dopth o~ 70 cm, aerate~ w~th l volume of air per volume of culture per minute. Temperature was lO maintained at 27-30C. ~tract~ of ~ultures were made at lO days with 2 volumes of water per volume wet weight of culture. E~tracts contained 37 Phenol Red Units per ml Mn pero~idase, 72 Phenol Red Units per ml -perosidase, and 27 Phenol ~ed Units per ml lS laccase/o~idase activity by phenol red assay. . . .
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Inoculum cultur~s of Phl:~ki~ tr~me~ su5 were grown at 27C:for l~ d~ays in unagitated high nitrogen liquid media. Sugar beet pulp was wetted to 57%
:moisture with the nutr1ent solution shown below:
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W~ 92/13960 PCr/US9~/00871 ~" 21~10~7 . --2 1--qrams,~l~iters NH4H2Po4 . 2 KH2P04 2 . 72 Mg S04 . 7H20 . 5 05 CaC12 .1 Yeast Extract . 05 Thi ami ne . 0 01 Veratryl Alcohol .10 Tr~ce Elements 5 . Oml Glucose 10 g~l Thr~e cultures were grown in this e~psriment.
The first with the nutrient ~olution, the second with the nutrient s~lution supplemented with an addi~ional 20 g/l glucose, and the third supplemented with an 15 additional 20 9~1 glucose plus 5 g~l pepto~e and 3 g/l yeast ~tr~ct. Cultures were grown for 12 day~, at 27OC, with 0.2 volume~:o~ 90% RH air per volume of culture per minute. Culture~ were estracted with 2 volumes of water per volume wet weight culture.
20 Extracts of all three cultures contained high levels of Mn perosidase activity in phenol red assay as shown : below: :
,:
Pheno l Red Uni t s Cultu~g Medium of Mn PQroxid~ase : .
Salts 10 Salts plus glucose 25 Salts plus glucose, : 78 peptone and yeast e~tract ~
~ ~ 30 Mn pero~idase~was produced regardless of glucose - ~ or nit~ogen concentration a~nd wa~s the only activity detected.

~VO 92/13960 PCr/US92/00871 ~1010~7 E~ample ~

Production of peroxidase and Mn peroxidase using ~

Inoculum cultures of Bierk~de~a ~ (C~S
05 595.78~ were grown for four days at 28C in an agitated nutrient solution comprising 10 9/1 glucose, 5 g/l peptone and 3 g/l yeast e~tract. Sugar beet pulp was w~tted to 70% moisture with the same high nitrogen media and inoculated at 10% v/v with the inoculum culture. Inoculated ~ugar beet pulp was incubated for 10 days at 27C with an air flow of 2 ~olumes of air per volume of culture per minute with ~ -the air a~ appro~imately 90% rslative humidity.
After 10 days, e~trac~s were made with the addition of two ~olumes of water per volume wet weight culture by the method o~ E~ample 1. Estracts w2re assayed for p~ro~idase, ~n perosidase and oxidase using phenol red. The estracts contained 47 Phenol Red Units per ml Mn perosidase and 45 Phenol Red Units 20 per ml perosidase. Extracts showed no oxidase or laccase activity.

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Production of Mn pero~idase using B. adust2 B. adusta was grown, e~tracted, and assayed as 25 described in Example 8, excep~ cultures were grown at 20OC. Extracts were made at 14 days culture time.
Assays showed 101 Phenol Red Units per ml Mn peroxidase. Estracts~a}so showed manganese pero~idase .~ - .
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, WO 9~t13960 2 1 0 1 0 ~ 7 PCT/US92/00~71 activity as assessed by veratryl alcohol assay at .93 International Units/ml. Extracts showed no o~idase or - -laccase activity.

05 Production of pero~idase using ~

was grown and extracted as described in ~xample 8 e~cept that ~stracts were made at 12 days culture time. E~trac~8 contained 9~ Phenol Ræd Units per ml perosidase aCtivity by phenol red a8say.
10 E~tracts ~howed no Mn pero~idase, oxidase or laccase activity.

E~ample }1 . ' Production of aryl alcohol osidase u8ing j~ ad~a . . . .
~i53~ L~ ~gy~ was grown under the same conditions as Ezample 8, e~cept that the sugar beet pulp preparation was w~tted~ with;water and tih~ culture grown for 14 day~ at 300C. Aqueou~ e~tracts con~ained aryl alcohol osidase as demonstrated by assay using 2Q anis alcohol ~nd veratry}:alcohol.
E~tracts 8howed no manganese or hydrogen peroxide dependent activity in these assays. Oxidase activity . was .667 International Units per ml of e~tract by anis alcohol assay and .30 International Units per ml.by 25 veratryl alcohoI assay. ~ ~

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WO92/13960 ~ 1 0 1 ~ ~ 7 P~T/~S92/0087l :~:x~m Production of pero~idase using B, adu~

~ ierkand~ra adu~ta was grown under the same conditions as E~ample B e~cept that 5% milled barley 05 straw was added to the sugar beet pulp and the aulture was grown in a ~0 liter vessel aerated with 1 volurne of air per volume of culture p~r minut~ in a 70 cm deep ~ubstrate bed~ Estracts of cultures at 10 days showed paro~idase activity assayed using phenol red.
10 Activity was 56.5 Phenol Red Units per ml.
. .

Degradation of chlorinated herbicides using cultures of B. ~ grown on sugar beet pulp .. .. . .
Soil contaminated with chlorinated herbicides 15 2,4-dichlorophenosyacetic acid ~2,4-D) and ~-2,~,5-Trichlorophenosyacetic acid (2,4,5-T) was decontaminated using a culture of ~. adusta grown on .
sugar beet pulp. The contaminated site is in Joliet, Montana. Contaminated ~oil is under the raised wooden 20 floor of a buildi~g used to store herbicides. The : .
building and the floor prevented any photodegradation of the chlorinated compounds from taking place.
- Inoculum cultures of B. adusta were produced as described in example 8 and used to inoculate 5 liter 25 volumes of sugar beet pulp substrate prepared as in .
e~ample 8. Inoculated substrate was placed in 10 '....
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3~60 2 1 0 1 0 ~ 7 PCT/US92/00871 , ., -~5-liter vessels in a 10 cm deep bed and incubated for 10 days at 22-25OC with a flow of 1 volume of air per volume per volume of culture per minute at approximately 10% RH.
05 After 10 days, three separate cultures were pooled, tran~ported to the ~ite and mi~ed with soil.
A volume of culture equal to 18% of the volume of soil was used in Plot 1 whilQ a volume o~ culture equal to

4% of the 80i 1 was u6ed in Plot 2. Each plot was 10 appro2imately one meter s~uare with contamînation e~tending down one meter. The conce~tration of contaminants was differ~nt in the two plots. Soil was treated to a depth of approsimately 13 cm through rototill~ng. Treated 80il wa~ sprayed lightly with 15 water as necessary to maintain soil moisture. A third plot was used as a control plot. No fungus was applied to thi~ plot.
Samples of contamipated 80il were removed ~rom the two treatment plots prior to addition of the 20 fungus. A soi} sample was also taken from the control plot at this time. Final soil samples were taken 74 days later. Soil sample~ were analyzed~for chlorinated herbicides by~an EPA approved laboratory using standard EPA method 8150. Laboratory results 25 are shown in the table below:

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WO 9~/13960 ~ 1 0 ~ ) 7 PCr/US92/0087l ~1 :

CONCENTRATION IN PPM

Plot ID Contaminant InitialFinal Conc. Conc.
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Plot 1 2,4-D 1,100.00 680.0 05 Plot 2 2,~-D 6~0.00 4.4 Control 2, 4-D 320 . 00 370 . 0 Plot 1 2,4,5-T 12.0 13.0 Plot 2 2,4,5-T .1 1.3 Control 2,4,5-T 370.0 390.0 .

Ea~ample 14 Degradation of chlorinated herbicides using cultures : .
of ~ chryso~porium grown on sugar beet pulp Soil contaminated with chlorinated herbicides .
2,4-dichlorophenosyacetic acid (2,4-D) and 15 2,4,5-Trichlorophenoxyacetic acid ~2,4,5-T) was .
decontaminated u~ing a culturs of P. ~h~xsQ~Qrium grown on sugar beet pulp. Chlorinated dio~ins were also present in the soil and most likely were a by-product of the 2,4,5-T manufacture. The 20 contaminated site is in Joliet, Montana. Contaminated .:
soil is under the raised wooden floor of a building used to store herbicides. The building and the floor prevented any photodegradation of the chlorinated compounds from taking place.
: .
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WO92/13960 PCT/US~2/00871 Inoculu~ cultures of ~1 hrysosp~Fi~m were produced as described in example 2 and used to inoculate S liter volumes of sugar beet pulp substrate prepared as in example l. Inoculated substrate was 05 placed in lO li~er vessels in a lO cm deep bed and incubated for 6 days at 22-25C with a flow of l volume of air per volume per volume of culture per minute at appro~imately 10% RH.
After 6 days, two separate cultures were pooled, transported to the ~it~ 3nd mi~ed with soil. A volume of culture e~ual to 18% of the volume of ~oil was used in Plot 3. Th~ plot was approsima~ely one meter square with contamination estending down one meter.
Soil was treated to a d~pth of approximat~ly 13 cm through rototilling. Treated 80~1 was spr~yed lightly wit~ water a~ necessary to main~ain soil moisture. An untreated pIot was u~ed as a control plot.
Samples of contaminated soil were removed ~rom tha treated plot prior to addition of the fungus. A
soil sample was also taken from the control plot at this time. Final soil samples wera takan 74 days : . .
later. Soil samples:were analyzed for~chlorina~ed ~
herbicides and diosin~ uæing EPA approved laboratories : ~:
using standar~ EPA ~ethods. Herbicides were analyzed for using Methoa 8150 ~hile dioxins were~analyzed for using an EPA approved method incorporating Low ~esolution Mass Spectrometry. Laboratory results are shown in the following tablPs: . :
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W~92/13960 ~ 1 0 ~ PCT/~S92/00871 CHLORINATED HERBICIDES . .
Concentration in ppm Plot ID Contaminant Initial Final Conc. Conc.
. . ~
05 Plot 3 2,4-D l,l00 17 Control 2,4-D 320 340 Plot 3 2,4,5-T 12 .b.26 Control 2,4,5-T 370 390 .. . .

Site Demonstration - Dio~in Results ' ': , ' ....
l0 Dio~in ~artin~ Conc. Final COnC. Detection Compound Limit .
..... . ._ .... __ .
TCDD (total) O .16 ppb N.D. . 090 PeCDD cO.10 N.D. .090 : -HxCDD <0.13 :~ N.D. .012 :
15 HpCDD 0.88 0.079 .021 `.
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WO92/13960 PCT/USg2/00871 2 ~ 0 ~ 7 _~9_ ~mP.l~

Degradation of polynuclear aromatic hydrocarbons (PAH) in creosote contaminated soils using cultures of P. chry~o~po~ium grown on sugar beet pulp 05 Cultures of ~ ch~y~osDorium grown on sugar beet pulp were prepared as ~scribed in Example l. At the time the cultures were mi~e~ with the contaminated soil, the cultures con~ain~d 30.7 unit~ per gram wet weight oE Mn Perosidase activity assayed u~ing phenol 10 red.
The soil was obtained from a site contaminated with creosote. 50g soil samples were placed in one liter bottl~s. Fungal cultures were mi~ed in with the soil samples at 25, 50, and 75% volume of fungus to 15 volume of soil. The ~oil ~amples were incubated for -~
either 30 or 45 days at room temperature. After either 30 or ~S days, depending on the sample, the entire sample o soil and fungal culture was e~tracted and analyzed. EPA method 8100 for analysis of PAH was 20~used. Concentrations of the four principal PA~
compounds are shown in the following table:

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WO92/13960 2 ~ O 1 0 5 I PCT/~S92/00871 -30- ~

-Constituent Untreated 25% 50% 75% Time Naphthalene*~500 ppm50 ppm 50 ppm 50 ppm 30 d.
Acetnaphthene6S000 29000 20000 20000 30 Fluorene 42000 26000 16000 10000 30 05 Anthracene14500 600 550 700 30 Naphthalene~2500 ~0 50 55 45 d.
Acetnaphthene65000 14000 9000 10000- 45 Fluorene 42000 12000 6500 6500 45 .
Anthracene14500 150 175 160 45 10 ~When fungal growth substrate i~ e~tracted prior to. ,~
fungal growth and run on the G.C. using the PAH
program, this peak occurs at the ~ame time and magnitude as NaphthaleneO FlorosiI does not totally remove it. All PAH analy~is of soi Vsolid fungal 15 inoculum mixtures indicate naphthalene at approYimately 50 ppm. ~owever it is unlikely that it is naphthalene in the soil. Additional analysis will be required to determine what this compound is.
Gas chromatography o~ the untreated control and :.
20 of the 25 and 50~ volume treatments after 45 days incuba~ion was per~ormed. Treated samples showed significan~ reductions in PAH concentration as ; :
indicated by the reduc~d number and area of the ::
chromatographic peak. .

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wos2/13960 ~ PC~/US92/00871 ExamPle 16 Degradation of polynuclear aromatic hydrocarbons (PAH) in water using cell-fres extracts of Phanerochaete chrYsosporium, sugar beet pulp cultures 05 Cultur2s of P. chrY~o~Porium grown on sugar beet pulp were prepared as described in E~ample 1.
Cultures were e~tracted by adding 2 volumes of water per one part weight of culture. The culture and water were blended ~or one minute, centrifuged, and filtered 10 through a 0.8 micron filter. The cell-free, solids-free, filtrate contained 30.7 units per ml of Mn. Peroxidase activity as determined by phenol red assay. 20 ml samples of creosote contaminated water were dispensed to reaction vials. 0.5g, 2.0g, or 3.0g 15 of culture e~tract was added to duplicate samples and the vials sealed. Three contaminated water samples were not mixed with culture estract. These samples were the controls. After 12 hours of incubation at room temperature, the controls and treated water 20 samples were e~tracted and analyzed for PAH
concentration using EPA method 610.

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WO92tl396~ ~ 1 0 1 0 ~ ~ PCT/US~2/00871 concentrations of PAH in untreated and treated samples are shown below:

20 gram water samples; white-rot fungi - liquid enzyme extracts :
05 12 hour treatment tim~
GC Analysis: EPA Method 610 Fungus Strain - ~1 sbuy~ Ei~m .
Liquid enzyme dose :.
o 0.5g. 2.0g. 3.0g.

10 compound concentration in micrograms/liter Acenaphthene 70 53.7 37.6 15.4 Fluorene 45 27 23.1 12.1 Phenanthrene 23 11.8 13.9 4.3 :;
.

` E~am~le 17 : ~
....
: 15 Degradation of polynuc}ear aromatic hydrocarbons .
PAH) in water using cell-free e~tracts of ~ierkandera adust~, sugar beet pulp cultures Cultures of B. ~g~ grown on sugar beet pulp were prepared as described in Example 8. Cultures 20 were extracted by adding 2 volumes of water per one : part weight of culture. The culture and water were blended for one minute, centrifuged, and filtered through a 0.8 micron filter. The cell-free, sollds-free, flltra~te contained 95.1 units per ml of : 25 Mn. Pero~idase activity::as determined by phenol red , .

WO g2/13960 2 1 0 1 ~ ~ 7 P~T/~S92/00871 assay. 20 ml samples of creosote contaminated water were dispensed to reaction vials. 2.0g or 5.0g of culture extract was added to duplicate samples and the vials sealed. Three contaminated water samples were 05 not mi~ed with culture extractO These samples were the controls. After 12 hours of incubation at room temperature, the controls and treated water samples were e~tracted and analyzed for PAH concentration using EPA method 610.
Concentrations of PAH in untreated and treated samples are shown below:

20 gram water samples; white-rot fungi - liquid enzyme e~tracts 12 hour treatment time 15 GC Analysis: EPA Method 610 ,'..
Fungus Strain - ~. ~dusta Liquid enzyme dose ; 0 2.0g. 5.Qg.

compound concentration in micrograms~liter 20 Acenaphthene 70 70 Fluorene 45 31.6 31.6 Phenanthrene 23 25 26 , : : :

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- :-WO92/13960 ~ 5 7 PCT/US92/00871 ~.
-34- t::~

ExamPle 1~

Degradation of PCB's Using Cultures of ~
Bierkandera adusta Grown on Sugar Beet Pulp Polychlorinated biphenyls (PCs~s) in soil were 05 degraded by treatment with cultures of B. a~ust~ grown on sugar beet pulp. PCB contaminated soil was obtained from an e}ectric utility maintenance yard.
The PC~'~ were a commercial mi~ture designated as Aroclor 1~60. PCB type and concentration in soil was 10 determined by e~traction and ga~ chromatograph according to Environmental Protection Agency (EPA), method 8080. PCB analysis was performed by Mycotech Corporation (Butte, MT~ and by independent, EP~
certified laboratories.
15Inoculum cultures of B. adusta CBS 595.78 were grown for 4 days at 28C in an agitated flask in a nutrient solution of 10 9/1 glucose, 5 g/l peptone and 3 g/l yeast e~tract. Sugar beet pulp was wetted to 70% moisture content with the same high nitrogen medium sterilized, cooled and inoculated at 10% volume with the inoculum culture. Inoculated sugar beet pulp was incubated for 10 days at 27C with an airflow of .
0.2 volumes air per v~}ume of culture per minute with the air at appro~ima~ely 90% relative humidity. At 10 25 days a sample of the culture was extracted by adding 3 volumes of water per volume of culture and homogenizing with a hand held blender for 20 seconds, centrifuging and filtering through a filter with a 0.8 micron pore size. The cell-free filtrate was assayed 30 for the presence of pero~idase and manganese .
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WO92/13960 2 1 0 1 ~ ~ 7 PCT/US92/00871 peroxidase using phenol red and for o~idase using anis alcohol by standard procedures. E~tracts contained 18.3 units per ml pero~idase and 99.5 units per ml manganese peroxidase and no oxidase activity at the 05 time of application to soil.
Whole culture with a moisture content of 78% was mixed at 25% by volume with 50 grams of contaminated soil containing 45 ppm total PC~ and the mi~ture placed in a covered glass bottle and incubated at room 10 temperature for 30 days with periodic addition of wa~er. Controls were prepared by treating contaminated soil with fungus culture that had been destroyed by autoclaving at 121C for 20 minutes prior to addition to soil. After 30 days, treated and 15 control soil samples were e~tracted and assayed for PCB concentration. Controls showed 45 ppm total PCB
and treated samples 5 ppm total PCB. Gas chromatograph analysis showed degradation o~ all PCB
congeners in the sample. Figures lA and lB are 20 chromatographs of the con~rol samples and treated samples showing uniform degradation of the PCB mi~ture.

E~ample 1~

Degradation of~PCB's Using Cultures of B, adusta Grown on Sugar Beet Pulp ~ PCB's in contaminated soil were degraded by treatment with cultures of adusta grown on sugar beet pulp. Cultures were grown and soil treated as described in Example 1 escept that soil contamination ~ ~ was 330 ppm total~ PCB and equal~volumes of whole wet ;~ 30 culture and soi~l were~used. After 30 days;incubation . -:: .

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WO~/13960 21 U i U 5 7 PCT/US92/00~71 PCB concentration in the treated soil was 15 ppm with uniform reduction of all congeners in the PCB
mixture. Figures 2A and 2B are chromatographs of .-extracts of control and treated soil samples.

05 ~xamDle 2Q

Degradation of PCB's in a Time Course Using a Slurry of B. ~ , Sugar Beet Pulp Cultures B, adus~a sugar beet pulp cultures were prepared as described in Example 1. After 10 days culture time, a slurry o~ the culture was prepared by adding 3 volumes of watèr per volume of we~ culture. The mixture was homogenized in a blender. The resulting slurry contained 6.7~ solids by weight. The slurry can be pumped or poured as a liquid for addition to soil or water. This slurry was stored in the refrigerator and used as the base stock for repeated addition of slurry.
The slurry as prepared contained 7.1 units per ml pero~idase activity and 76.4 units per ml Mn 20 pero~idase activity by phenol red assay.
This experiment was designed as a time course using repeated applications of slurry to eight 50 gram dupIicate soil samples. One of the 50il samples was e tracted without any slurry being added. This sample 25 established the starting concentration. The other 7 soil samples had 50 ~rams o~ slurry added to them.
~fter 7 days, all of these samples had appro~imately 50 grams of slurry added to them. Seven days later, another soil and slurry sample was extracted and 30 analyzed for PCB~s. The remaining 5 samples had . . .
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wos2/1396o 2 1 ~ 7 Pcr/usg2/oo87 l ~;~ 37_ :

approximately 50 grams of slurry added. This process ;
was repeated until 35 days had elapsed. No slurry was .
added to the remaining samples at 35 or 45 days. The results of the time course are sum~arized in the 05 following table:

TIME COURSE
Slurry Application - B. adusta PC~ contaminated soil _ . . , Weight Elapsed Concentration l0Inoculum Time ppm 0 g. 0 days 325 l00 1~ 122 ;- :
140 l9 66 .::~
275 26 35 ~ :
315 35 20 ~:

335 55 less than l0 -- - .

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W092/139fiO 2 1 0 1 0 ~ 7 PCT/US92/00~7~ .

Example 21 The Use of B. adusta, Sugar Beet Pulp Culture Slurries to Degrade PCB~s in a Field Demonstration ~ adus~a sugar beet cultures were prepared as 05 described in E~ample l. After 10 days culture time, a slurry of the culture was prepared by adaing 3 volumes of water to one volume of culture~ This preparation was homogenized in a blender for one minute.
The slurry as prepared contained 10.3 units per lO ml perosidase activity and 72.7 units per ml Mn pero~idase activity by phenol red assay.
Three soil plots approsimately 46 cm in diameter with contamination e~tending to a depth of 15.5 cm were used for the field demonstration. These plots 15 contained approximately 0.049 cubic meters of so~il or 49 liters of soil. Eight liters of slurry were added to two of the plots. Seven days later, slurry was added to the third plot. Samples were taken before ~: s}urry addition, at 7 and 14 days. The results are :
20 shown in the following table:

Results of Field Demonstration .~ B, ~ , Sugar~ Beet Pulp Culture Slurry i Initial Conc.7 days 14 days ppm Elapsed Time Elapsad Time 25 Plot l 410 370 ppm 330 ppm Plot 2 260 230 ppm 210 ppm :
Plot 3 260 ~230 ppm ~ : .
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WO92/13960 2 1 0 1 a 5 7 Pcr/usg2/oo87l . . ..

Example 22 The Use of B. adusta, Sugar Beet Pulp Cultures to Degrade PCB's in a Field Demonstration adusta sugar beet pulp cultures were prepared 05 as described in Example 1. The wet culture contained 1~.3 units per ml perosidase activit~ and 99.5 units per ml Mn peroxidase activity by phenol red ~ssay.
Three soil plots measuriny 2 meters ~ 3 meters with contamination extending 15.5 cm in depth were 10 used for this field demonstration. Appro~imately 0.55 cubic meters of culture material were mi~ed into two of the plots. The third plot was treated 7 days later. The plots were sampled for PCB's prior to the addition of the fungus and again after 7 and 14 days 15 elaps~d ~ime. The results are shown in the following ; -table:
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Results of Field Demonstration ~-B. adusta, Sugar Beet Pulp Culture Initial Conc. 7 days 14 days ppm Elapsed Time Elapsed Time Plot 1 150 120 ppm 100 ppm Plot 2 210 180 ppm 130 ppm Plot 3 190 150 ppm ' ' , : .. ' ' :"-"

-W092/l3960 PCTlUSg2/0~871 2 1~ 1 ~7 _40_ Example 23 The Use of B. adusta, Sugar Beet Pulp Cultures to Degrade PCB's in a Field Demonstration Repeated Additions of B, adusta, Sugar Beet Pulp Cultures 05 B. ~dusta sugar beet cultures were prepared as described in Esample l. The initial wet culturecontained 33.2 units per ml pero~idase activity and 85 . 9 units per ml Mn pero~idase activity by phenol red assay. Subsequent culture~ were not assayed for 10 enzyme actiVitY~
Two soil plots appro~imately 46 cm in diameter with contamination e~tending to a depth of 15.5 cm -were used for the field demonstration. These plots contained appro~imatel~ 0.049 cubic meters of soil or 15 49 liters of soil. The whole culture was mi~ed 100%
by volume with the soil. Samples were taken prior to addition of the whole culture and again after 12 days. After the 12 day sample, whole culture was again added to the plots at approsimately 50% culture 20 per volume of dirt. The plots were sampled 22 days later. Results of the sampling are shown in the following table. All analy~es were performed by an EPA approved laboratory.
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Results of Field Demonstration i 25 B, adusta, Sugar Beet Culture Two Applications Initial Conc. 12 days 34 days ppm Elapsed Time Elapsed Time .
Plot 1 330 280 ppm 180 ppm :
30 Plot 2 210 , 180 ppm 42 ppm '' ' ,' ' wos~/13960 ~ o~a~ PCT/US92/00871 E~amPle 24 The Use of B. adusta Sugar Beet Pulp Cultures to Degrade PCB's in a Field Demonstration B, adu~ta sugar beet pulp cultures were prepared 05 as described in Esample 1. Two field soil plots at the site described in Esample 1, measuring 46 cm diameter with contamination extending 15.5 cm deep were treated~ The first plot contained a be~inning PCB concentration of 220 ppm and the second plot 130 10 ppm. Plots were treated at the rate of 66~ volume culture per volume of soil. After 34 days plots - showed no evidence of culture substrate or cell mass. ~ ~
At 34 days plots were treated a second time at 70~ :
volume with ~ a~usta sugar beet pulp cultures. Plots 15 were assayed for PCB ~concentration by an EPA approved laboratory. Assay time intervals beginning from the first addition and PCB concentrations ~ppm) are shown in the following table:

Results of Field Demonstration ~ adu~ta - Sugar 8eet Pulp Culture, Two Applications -.
Elapsed Time Days After First Application : :
0 11 23 44 76 98 :-Plot 1 220 200 180 69 52 35 ., .
Plot 2 130 110 100 95 87 12 ... . .

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- -wog2/l3s60 i i~ 10 J ~ PCT/US92/0087 E~ample 25 The Use of P. chrys~sporium, Sugar Beet Pulp Cultures to Degrade PCB's in a Field Demonstration Single Application of 05 P. chrysosPorium, ~ qar Beet Pulp Cultures Inoculum cultures of P! chrYsosporium were grown for five days at 28C in an agitated flask in a nutrient solution of 10 gfl glucose, 5 g/l peptone and 3 9~1 yeast e~tract. Sugar beet pulp wetted to 70%
10 moisture content with the same high nitrogen medium was autoclaved, cooled and inoculated at 10% volume with the inoculum culture. Inoculated sugar beet pulp was incubated for 7 days at 23C with an airflow of .2 volumes air per volume of culture per minute with the 15 air at approsimately 90% relative humidity. At 7 days, a sample was e~tracted by adding 3`volumes of water per volume of culture and homogenizing with a hand held ~lender for 20 seconds, centrifuging and filtering through a filter with a 0.8 micron pore , 20 size. The cell free filtra~e was assayed for the presence of peroxidase and manganese~pero~idase using `~
phenol red. E~tracts contained 61 units~;per ml pero~idase and 64 units per ml~manqanese pero~idase.
Whole~culture with a moist~ure of~75~ was mi~ed at 25 25% by volume into a soil plot approximately 46 cm in diameter with contamination e~tending to a depth of 15.5 cm. The plot contained approximately 49 liters of soil. The soil was contaminated with a mixture of the Aroclors 1254 and 1260 with the majority of the 30 contamination~ being Aroclor 1260. The soil pH was 8.5. Soil samples~were~taken at dlscrete lntervals : :

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~092/13960 2 ~ O 1 0 5 7 PCT/US92/00871 and sent to an EPA approved laboratory for PCB -~
analysis. The results are summarized in the following table:
Results of Field Demonstration S ~ chr~sQ~porium, Sugar Beet Pulp Elapsed Concentration Time in ppm initial 200 11 days 190 19 days lB0 50 days 170 __ - --.
E~am~le 2~

Degradation of PCB's Using Cultures of chrYsosPorium Grown on Sugar Beet Pulp , PCB's in contaminated soil were degraded by treatment with~cultures of P. chrysQs~orium grown on sugar beet pulp. Cul~ures were grown as described in E2ample 8 e~cept that the~sugar beet pulp was wetted with the salts solution shown in the table below and .
20 grown for 6~days at 28C. Duplicate 50 gram soil samples were prepared. Each sample was mised with 150% by ~olume of whole wet fungal culture. The soil - contained a mi~ture of the Aroclors 1242, 1254 and 1260 with 1254 and 1260 being the predominant types.
25 The soil pH was 4.5.

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WO92/13960 ~ 1 0 1 0 ~ 7 PCT/U~92/00~71 The whole culture was assayed for manga~ese pero~idase and peroxidase activity as described in Example ~. The culture contained 76 units per ml of manganese peroxidase activity.
05 At discrete time intervals, a soil sample was sent to an EPA approved laboratory for PCB analysis.
The results of those analyses are shown in the following table:

Degradation of PCB's Using P~ chrvsosPQrium Cultures Grown on Sugar Beet Pulp Elapsed Time Control 15 days 35 days 55 days 310 ppm 175 ppm 42 ppm 18 ppm :

Typical Nutrient Solution Used Substance g/l Substance g/l Glucose 10.0 CaC12.2H20 .03 NH4H2Po4 05 Trace Elements 5 ml stock solution KH2PO4 1.0 Veratryl Alcohol 0 or .14 20 MgSO4.7H2O 1.0 Peptone 05 : ~ Yeast e~tract .05 .', .
:

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Example 27 Degradation of PCB's Using Cultures of P. chrysosPorium Grown on Sugar Beet Pulp PCB's in contaminated soil were degraded with 05 treatments of P chrysos~orium grown on sugar b~et pulp. Cultures were grown as described in ~ample 8 e~cept that inoculum cultures were grown in a media containing .5g~1 peptone, .59/1 yeast es~ract and 5g/1 glucose. Duplicate 50 gram soil samples were 10 prepare~. The soil was contaminated with the mixture of Aroclors as described in Esample 8. Different duplicate soil samples were mi~ed with 50%, 100% and . .
150% by volume wet fungal cultures.
The whole culture was assayed for manganese 15 pero~idase and perosidase activity as described in Esample 8. The culture contained 66 per ml of manganese pero~idase activity.
The treated soil was analyzed for PCB's after 14 days. The results of those analyses are shown in the 20 following table:

Degradation of PCB's Using P. chrYsosporium : Grown on Sugar Beet PuIp PCB Concentration : Volume % After 14 days Fungus Elapsed Time -. :
: 0% (control) 310 ppm 50% 230 ppm 10~0% : 150 ppm : 150% 101 ppm '.:

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WO92/13960 . ,. PcT/uS92/oo87~
2~ol~)5746 Ex~ le 2~

Degradation of PCB's Using Varying Rates of P. chrv~osporium Sugar Beet Pulp Cu}ture P. chrvsosp~ium was grown and used to treat 50 05 gram samples of PCB soil as descri~ed in E~ample 8.
Identical soil samples were treated with different volumes of fungus culture and each treatment rate was sampled for PCB concentration at three different time intervals. Treatment rates were 25, 50, 100 and 150%
10 volume of culture per volume of soil. Results are shown in the table below:

Vol ~ Fu~gus Elapsed:Time in Days Added to Soil Control 15 35 55 0~ 310 15 25% ~oo 305 200 145 50% ~ No Value 270 190 130 100~ 305 250 130 42 150~ : No Va:lue 175 42 18 :
~OTE: PCB concentrations in ppm :
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W~9~/13960 2 1 0 1 0 ~ 7 PCT/US92/00871 E~m~~

Time Course of PCB Degradation Using P~ chrYsosporium Sugar Beet Pulp Cultures P. hrysosPQ~ium was grown as described in 05 E~ample 8 and used to treat identical 50 gram samples of the PCB contaminated soil also described in E~ample 8. Soil samples were treated with 150% volume of whole wet P. chrYso~Porium culture and incubated for 10 days. At 10 days an additional 50% volume of 10 culture was added to one half of the 50 gram samples for a total of 200~ volume treatment. Samples with 150 and 200% volume of culture we~e assayed ~or PCB ..
concentra~ion at 20, 30, 40 and 50 days elapsed time.
Results a~e shown in the table below:

15 Elapsed Total Volume PCB Conc.
Time in of in Days Solid Inoculum ppm 0 150% 332 : ~ 10 150% 224 : ....
150% 154 20~% 113 150% : 83 200% 73 .
150% 31 ~ :
200% 33 . . .
150% 13 :
200% . 8 : :
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. , WO92/13~0 2 1 0 1 0 $ 7 PCT/lJS92/00871 Exam~le 30 Degradation of Pentachlorophenol Using Cultures of P. chrysos~orium Grown on Sugar Beet Pulp Pentachlorophenol (PCP3 in soil was degraded by 05 treatment with cultures of P. Ghrysosporium grown on sugar beet pulp. P~P was widely used as a wood preservative and is considered by the United States Environmental Protection Agency (~PA) to be a hazardous waste.
Two soil samples contamina.ted with dif~erent concentrations of PCP were obtained from a commercial laboratory. Sample l contained 8050 ppm and sample 2 contained 52~6 ppm PCP.
P. chrYsosPo~L~m sugar beet pulp cultures were . 15 prepared as follows: an inoculum culture was prepared .:
by transferring ~. chrvsosPorium maintained on nutrient aqar slants to a sterile liquid medium containing lO grams/l:iter sugar:beet molasses, 2 grams~liter yeast e~tract and l gram/liter KH2 PO4 adjusted to pH 3.5 with H2SO4. The liquid inoculum ; culture wa~ cubated wit~h agitation ~or four days at 30C. ~ugar beet pulp was wetted to 65%:moisture cont~ent with water,~autocl:avea`at 12.0C, 05 psi for 20 minutes, cooled and inoculated at the:rate of lO ml -.::
. :~ 25 inocu~um culture per l00 ml volume of sugar beet puIp substrate. The inoculated sugar beet pulp was ,~ incubated for 7 days at 28C with an airflow of .2 volume air per volume of culture per minute with the .:
,q air at a relative humidity of about 90%. :~

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~92/13960 2 ~ O ~ Q ~ 7 P~T/US92/00871 Twenty-five (25) grams of contaminated soil was placed in l-liter bot~les and thoroughly mi~ed with either 25 grams or 50 grams of P. chrYsospori~m sugar beet pulp culture. Bottles with treated soil were 05 loosely covered and incubated at 25C for 21 days.
After 21 days, soil was analyzed for PCP concentration by a modification of EPA method B040. The entire contents of each treatment bottle - fungus culture and contaminated soil - was tranæferred to a so2hlet 10 apparatus and e~tracted for eight hours with he~ane.
The estract was concentrated and analyz~d by gas chromatography. Concentration was determined by comparison with standard~ of known PCP concentration.
For e~perimental controls, 25 grams of contaminated 15 soil was treated with wetted, sterile sugar beet pulp without fungu~ growth. Results of PCP assays for fungus treated and control treatments are shown in Table 1.

Table 1 Pentachlorophenol Degradation Soil #l As Measured: 8050 ppm Treatment ~onc ~ After Tr~atment % Xemaininq WRF#l Control 25g 7,040 ppm 87.5 25 ~RF#l Treated 25g 3,810 47.3 WRF#l Control 50g 5,230 65.0 WRF#l Treated 50g 1,310 16.3 WRF#l Control 25g 3,200 59.0 WRF#~ Treated 25g 2,466 ~5.~ ;
30 WRF#l Control 50g 3,801 70.1 WRF#l Treated 50g 1,456 26.8 . .
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~YO92/13960 ~ 1 0 1 0 ~ ~ PCT/US92/00871 E~ample 31 Degradation of Pentachlorophenol Using Cultures of B. adusta Grown on Sugar Beet Pulp PCP in soil was degraded by treatment with 05 cultures of B. adust~ grown on ~ugar beet pulp. Soil samples were the same as those described in E~ample 30.
~ a~usta ~ugar beet pulp cultures were prepared as described in E~ample 30, e~cept that ~ dusta was used.
Soil was treated with ~, adusta sugar beet cultures and analyzed for PCP concentration as described in E~ample 30.
Results are shown below: :

~abl~ 2 Pentachlorophenol Degradation Soil #2 A~ Measured: 5426 ppm Trea~ment Conc. After Trea~ment % Rçmaininq WRF#2 Control 25g 6,961 ppm 86.5 20 WRF#2 Trea~ed 25g 6,29S 7R . 2 WR~#2 Control 50g 7,233 89.6 : WRF~2 Treated 50g: 5,392 67.0 -:WRF#2 Control 25g 4,820 88.8 WRF#2 Treated 25g 4,016 74.0 t 25 WRF#2 Control 50g 4,603 84.8 . WRF#2 Treated 50g 4,602 84.8 .. . .

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WO92/139S0 2 1 0 1 ~ 5 7 P~T/US92/00871 E~uivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine e~perimentation, many equivalents to the specific 05 embodiments of the invention described herein. Such .
equivalents are intended to be encompassed by the following claims.

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Claims (55)

Claims

1. A method of culturing white-rot fungus, comprising combining a white-rot fungus and a sugar beet pulp substrate forming a fungal culture;
subjecting the fungal culture to growth-supportive conditions such that an aromatic compound degrading enzyme is produced by the white-rot fungus during a growing period, wherein the growing period includes both primary and secondary metabolic phases and an aromatic compound degrading enzyme is produced at least during a portion of the primary metabolic phase.

2. A method of culturing white-rot fungus comprising:
combining a white-rot fungus and a sugar beet pulp substrate forming a fungal culture;
subjecting the fungal culture to growth-supportive conditions such that an aromatic degrading enzyme is produced by the white-rot fungus without restricting the nitrogen level of the fungal culture.

3. A method of claim 1 or 2, wherein the sugar beet pulp is mixed with straw.

4. A method of claim 1 or 3, wherein the white-rot fungus is selected from the group of genera consisting of Phanerochaete, Phlebia, Trametes, Pleurotus and Bjerkandera.

5. A method of claim 1 or 2, further comprising the step of separating the at least one aromatic compound degrading enzyme from the substrate.

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6. A method of claim 1 or 2, further comprising the step of isolating a by-product of fungal growth from the fungal culture.

7. A method of claim 6, wherein the by-product is an aromatic-compound degrading enzyme.

8. A method of claim 7, wherein the aromatic-compound degrading enzyme is a lignin-degrading enzyme.

9. A method of claim 8, wherein the enzyme is selected from the group consisting of peroxidases, oxidases and laccases.

10. A method of cultivating white-rot fungus, comprising the steps of:
a) mixing an inoculum of white-rot fungus with substrate of sugar beet pulp having an absorbed water content of 40-80%;
b) growing white-rot fungus on the sugar beet pulp substrate at a temperature between 20-40°C for a growing period; and c) aerating the inoculated substrate at a rate of between .95 to 20 volumes of air per minute per volume of substrate during the growing period with air having an oxygen level above 7% and a relative humidity of 70-99%.

11. A method of claim 10 in which water is used to wet the sugar beet pulp.

12. A method of claim 11 in which a solution containing glucose and protein is used to wet the sugar beet pulp.

13. A method of claim 10, further comprising the steps of adding water to the substrate after the growing period and then centrifuging and filtering the mixture to separate a solution of cell-free enzymes from the substrate.

14 A method of claim 13, wherein the filtering step is carried out with filters having a screen mesh of no larger than 0.8 micron.

15. A method of claim 10, wherein the white-rot fungus is selected from the group consisting of Phanerochaete chrysosporium, Phlebia tremellosus, Trametes versicolor, and Bierkandera adusta.

16. A method of claim 10, wherein the growing period includes primary and secondary metabolic phases and enzyme production occurs during at least a portion of the primary metabolic growth phase.

17. A method of claim 10, wherein, prior to step A, the sugar beet pulp is sterilized by autoclaving and cooled to between 20-40°C.

18. A method of claim 10, wherein straw is added to the sugar beet pulp substrate prior to step B.

19. A method of producing lignin-degrading enzymes, comprising growing white-rot fungus on a sugar beet pulp substrate for a growing period and recovering lignin-degrading enzymes produced by the fungus in a form separate from the sugar beet pulp substrate.

20. A method of claim 19, wherein the white rot fungus is selected from the group of genera consisting of Phanerochaete, Phlebia, Trametes, Pleurotus and Bierkandera.

21. A method of claim 19, wherein growth-supportive conditions sufficient to support primary metabolic growth of the fungus are maintained substantially throughout the growing period.

22. A fungal culture comprising a ligninase-producing white-rot fungus in admixture with and grown on a solid substrate comprising sugar beet pulp.

23. The culture of claim 22, wherein the white-rot fungus is selected from the group of genera consisting of Phanerochaete, Phlebia, Trametes, Pleurotus and Bjerkandera.

24. A bioremediation method for degrading at least one aromatic compound in soil or water, the method comprising the step of mixing an essentially cell-free enzyme preparation derived from a fungal culture including a sugar beet pulp substrate with the soil or water containing at least one aromatic compound, at a concentration and temperature sufficient to degrade enzymatically at least a portion of the aromatic compound in the soil or water.

25. The method of claim 24, wherein the white-rot fungus is selected from the group of genera consisting of Phanerochaete, Phlebia, Trametes, Pleurotus and Bierkandera.

26. The method of claim 24, wherein at least one of the aromatic compounds is a chlorinated aromatic compound.

27. A bioremediation method for degrading at least one aromatic compound in soil or water, the method comprising the step of mixing with the soil or water containing said at least one aromatic compound a fungal culture, said fungal culture comprising a ligninase-producing white-rot fungus in admixture with a solid substrate comprising sugar beet pulp at a concentration and temperature sufficient to degrade enzymatically at least a portion of the aromatic compound in the soil or water.

28. The method of claim 27, wherein the white-rot fungus is selected from the group of genera consisting of Phanerochaete, Phlebia, Trametes, Pleurotus and Bjerkandera.

29. The method of claim 28, wherein the white-rot fungus is selected from the group consisting of P. chrysosporium and B. adusta.

30. The method of claim 27, wherein the at least one aromatic compound is a chlorinated aromatic compound.

31. A bioremediation method for degrading at least one chlorinated aromatic compound or polynuclear aromatic hydrocarbon in soil or water, the method comprising the step of mixing with the soil or water containing the at least one aromatic compound a fungal culture comprising ligninase-producing P.
chrysosporium in admixture with a solid substrate comprising sugar beet pulp at a concentration and temperature sufficient to degrade enzymatically at least a portion of the chlorinated aromatic compound or the polynuclear aromatic hydrocarbon in the soil or water.

32. A bioremediation method for degrading at least one chlorinated aromatic compound or a polynuclear aromatic hydrocarbon in soil or water, the method comprising the step of `
mixing with the soil or water containing the at least one aromatic compound a fungal culture comprising ligninase-producing B.
adusta in admixture with a solid substrate comprising sugar beet pulp at a concentration and temperature sufficient to degrade enzymatically at least a portion of the chlorinated aromatic compound or the polynuclear aromatic hydrocarbon in the soil or water.

33. A method of degrading a polyhalogenated biphenyl compound in a material, comprising mixing the material with a culture of white-rot fungus under conditions sufficient to degrade the polyhalogenated biphenyl compound in the material, said culture comprising a white-rot fungus producing an aromatic compound-degrading enzyme and a substrate of sugar beet pulp.

34. A method of claim 33, wherein the polyhalogenated biphenyl compound is a polychlorinated biphenyl.

35. A method of claim 33, wherein the material is soil or water contaminated with a polyhalogenated biphenyls.

36. A method of claim 33, wherein the white-rot fungus is selected from the genus Phanerochaete or Bierkandera.

37. A method of claim 36, wherein the white-rot fungus is Phanerochaete chrysosporium

38. A method of claim 36, wherein the white-rot fungus is Bjerkandera adusta.

39. A method of degrading a polychlorinated biphenyl compound in contaminated soil, comprising mixing the soil with a solid state fungal culture comprising Bierkandera adusta in admixture with a substrate of sugar beet pulp, under conditions sufficient to degrade the polychlorinated biphenyl in the soil or water.

40. A method of degrading a polychlorinated biphenyl compound in contaminated soil, comprising mixing the soil with a solid state fungal culture comprising Phanerochaete chrysosporium in admixture with a substrate of sugar beet pulp, under conditions sufficient to degrade the polychlorinated biphenyl in the soil or water.

41. A method of culturing white-rot fungus, comprising:
combining a white-rot fungus and a substrate forming a solid state fungal culture;
and subjecting the solid state fungal culture to growth-supportive conditions such that at least one aromatic degrading enzyme is produced by the white-rot Fungus.

42. A method of claim 19, wherein the white-rot fungus is grown under growth-supportive conditions such that the lignin-degrading enzyme is produced at least during a portion of the primary metabolic phase.

43. A fungal culture comprising an aromatic compound degrading enzyme-producing white-rot fungus in admixture with and grown on a solid substrate comprising sugar beet pulp.

44. The fungal culture of claim 43 wherein the white-rot fungus is producing a lignin-degrading enzyme.

45. The culture of claim 42 or 43 wherein the white-rot fungus is selected from the group of genera consisting of Phanerochaete, Phlebia, Trametes, Pleurotis and Bjerkandera.

46. A method of claim 9, wherein the enzyme is manganese peroxidase.

47. The method of claim 3, wherein at least one aromatic compound is a polynuclear aromatic hydrocarbon.

48. The method of claim 12, wherein at least one aromatic compound is a mixture of polynuclear aromatic hydrocarbons.

49. The method of claim 9, wherein at least one aromatic compound is a polynuclear aromatic hydrocarbon.

50. The method of claim 14, wherein at least one aromatic compound is a mixture of polynuclear aromatic hydrocarbons.

51. A method of claim 1 wherein the material is soil.

52. A bioremediation method for degrading a polyhalogenated biphenyl compound in a material, comprising mixing the material with an essentially cell-free, aromatic compound-degrading enzyme preparation derived from a fungal culture under conditions sufficient to degrade the polyhalogenated biphenyl compound, said fungal culture comprising a white-rot fungus producing an aromatic compound-degrading enzyme in admixture with a substrate comprising sugar beet pulp.

53. A method of claim 10 wherein the polyhalogenated biphenyl compound is a polychlorinated biphenyl.

54. A method of claim 10 wherein the material is soil or water.

55. A method of claim 1 wherein the white-rot fungus is a ligninase producing white-rot fungus.