Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W ~ /13960 2 ~ O ~ ~ ~ 7 PCT/US92/00871
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~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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WO92/13960 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
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~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
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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
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~ nutrient solutions:supportive of ~ungal growth and :
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wos~13960 PCT/US92/0087~
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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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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
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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 ~
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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
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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
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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
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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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w092/~39~0 PCT/U~92/00871
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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
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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
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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. ~
~amPl.Q4 - . ' ~ ::
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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:
. .:
,
. . .
: .
.
: .
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
18 0
05 17 25 . 17 44
24 86 37 107
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.
~ .
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 -
,
: .
WO92/13960PCT/US92/00871
~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. . . .
.
~m~:z "
' .' :''
Production of Mn peroxidase .
usin~ 5;31L~QIY~ :
. .
.
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:
.
,: :
.
. :~
:,..
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
-22-
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.
.. - ....
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
.~ - .
. ..
... . . .
:
:
,
WO 9~t13960 2 1 0 1 0 ~ 7 PCT/US92/00~71
23-
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. ~ ~
~':
:
WO92/13960 ~ 1 0 1 ~ ~ 7 P~T/~S92/0087l
-24-
:~: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
'....
: . ,: . - .
':
::
WO92/13~60 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 :
--26--
CONCENTRATION IN PPM
Plot ID Contaminant InitialFinal
Conc. Conc.
.. ..
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.
: .
','''~
:':
WO92/13960 PCT/US~2/00871
27-
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: . :
' ~
'
: : ~ .
:,:~ : : ~: , . .
W~92/13960 ~ 1 0 ~ PCT/~S92/00871
-28-
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 `.
:; ~ ~ ~ . '. .,
. ,.
, ::
. .
:
:
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:
.
.,
' .'
.
~ "..
:
~ ~ .
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. .
: ' '.
.
~ . .
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.
,.
.. ..
:..
, ~,
~.
WO92tl396~ ~ 1 0 1 0 ~ ~ PCT/US~2/00871
-32-
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
-33
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
-36-
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
. . .
. ~
.
:
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
7 236
l00 1~ 122 ;- :
140 l9 66 .::~
275 26 35 ~ :
315 35 20 ~:
12
335 55 less than l0
-- - .
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W092/139fiO 2 1 0 1 0 ~ 7 PCT/US92/00~7~ .
-38-
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:
';~,..
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
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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
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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
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Plot 2 130 110 100 95 87 12
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wog2/l3s60 i i~ 10 J ~ PCT/US92/0087
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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
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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
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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
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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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W092/13960 PCT/US92/00871
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~
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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
-47
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
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150% 13 :
200% . 8 : :
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WO92/13~0 2 1 0 1 0 $ 7 PCT/lJS92/00871
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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
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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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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
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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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