U.S. patent application number 09/821950 was filed with the patent office on 2002-07-25 for fungi for improvements of wood and pulp appearance and qualities.
Invention is credited to Farrell, Roberta Lee.
Application Number | 20020096273 09/821950 |
Document ID | / |
Family ID | 27353923 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020096273 |
Kind Code |
A1 |
Farrell, Roberta Lee |
July 25, 2002 |
Fungi for improvements of wood and pulp appearance and
qualities
Abstract
The invention relates to the use of fungi stains of Ophiostoma
floccosum, Ophiostoma piceae or Ophiostoma pluruanulatum, or
mixtures on wood and pulp to improve chemical pulping processes
and/or reduce cooking tie and/or improve brightness and/or decrease
extractives.
Inventors: |
Farrell, Roberta Lee;
(Hamilton, NZ) |
Correspondence
Address: |
JACOBSON, PRICE, HOLMAN & STERN
PROFESSIONAL LIMITED LIABILITY COMPANY
400 SEVENTH STREET, N. W.
WASHINGTON
DC
20004
US
|
Family ID: |
27353923 |
Appl. No.: |
09/821950 |
Filed: |
March 30, 2001 |
Current U.S.
Class: |
162/72 ; 435/277;
435/278 |
Current CPC
Class: |
A01H 15/00 20130101;
B27K 5/00 20130101; B27K 2240/20 20130101; B27K 3/002 20130101;
C12N 1/145 20210501; C12R 2001/645 20210501 |
Class at
Publication: |
162/72 ; 435/277;
435/278 |
International
Class: |
D21C 003/20 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 7, 2000 |
NZ |
505017 |
Jun 20, 2000 |
NZ |
505316 |
Aug 30, 2000 |
NZ |
506630 |
Claims
1. An inoculum for wood comprising or including one or more forms
of a fungal culture of the species Ophiostoma floccosum and/or
Ophiostoma piceae and/or Ophiosotoma pluruanulatum effective to
reduce the amount of colour staining caused by wood staining
fungi.
2. An inoculum for wood as claimed in claim 1 wherein the fungal
culture forms are biologically pure.
3. An inoculum for wood as claimed in claim 1 wherein the fungal
culture is selected from the following strains: Ophiosotoma
floccosum F71, Ophiosotoma floccosum F80, Ophiosotoma floccosum
F93, Ophiosotoma floccosum F40, Ophiosotoma floccosum F13,
Ophiostoma piceae OPC 703, Ophiostoma piceae OPC 422, Ophiostoma
piceae OPC 580, Ophiostoma piceae OPC 194, Ophiostoma pluruanulatum
3410, Ophiostoma pluruanulatum 7073, Ophiostoma pluruanulatum 5040,
Ophiostoma pluruanulatum 4650
4. An inoculum for wood as claimed in claim 1 wherein the wood
source may be wood chips and the fungus or fungi may be applied by
spraying the wood chips.
5. An inoculum for wood as claimed in claim 1 wherein the wood
source may be logs to be cut into structural wood, and inoculation
includes inoculating at least one end of the logs, and preferably
all around the logs' surfaces.
6. An inoculum for wood as claimed in claim 1 therein the wood
source may be structural wood and inoculation includes inoculating
at least 60% of the surface area of the structural wood.
7. An inoculum for wood as claimed in claim 1 wherein the wood
source may be structural wood and inoculation includes inoculating
at least 60% of the surface area of the structural wood, which may
then be later made into chips or a subsequent fibre product.
8. An inoculum for wood as claimed in claim 1 wherein the wood to
be inoculated may be a conifer such as but not limited to Radiata
pine, Douglas fir or Loblolly pine, and it may also be a hardwood
including but not limited to eucalyptus, oak, poplar, or aspen.
9. A biologically pure culture of a strain of Ophiostoma floccosum
having all of the identifying characteristics of one of the fungi
of stains F40, F13, F71, F80 and F93.
10. A biologically pure culture of a strain of Ophiostoma piceae
having all of the identifying characteristics of one of the fungi
of strains OPC 703, OPC 422, OPC 580 and OPC 194.
11. A biologically pure culture of a strain of Ophiostoma
pluruanulatum having all of the identifying characteristics of one
of the fungi of strains 3410, 7073, 5040 and 4650.
12. A method of reducing the amount of colour staining caused by
wood staining fungi in and/or on a wood source which comprises or
includes inoculating at least a portion of the wood source with an
effective amount of at least one fungus selected from the group
consisting of the species Ophiosotoma floccosum, Ophiosotoma piceae
and Ophiostoma pluruanulatum, which is capable of reducing the
amount of colour staining caused by the wood staining fungi.
13. A method of reducing the amount of colour staining as claimed
in claim 12 wherein the fungi may be selected from the following
stains: Ophiosotoma floccosum F13, Ophiosotoma floccosum F40,
Ophiosotoma floccosum F71, Ophiosotoma floccosum F80, Ophiosotoma
floccosum F93, Ophiostoma piceae OPC 703, Ophiostoma piceae OPC
422, Ophiostoma piceae OPC 580, Ophiostoma piceae OPC 194,
Ophiostoma pluruanulatum 3410, Ophiostoma pluruanulatum 7073,
Ophiostoma pluruanulatum 5040, Ophiostoma pluruanulatum 4650
14. A method of reducing the amount of colour staining as claimed
in claim 12 wherein the wood source may be wood chips and the
fungus or fungi may be applied by spraying the wood chips.
15. A method of reducing the amount of colour staining as claimed
in claim 12 wherein the wood source may be logs to be cut into
structural wood, and inoculation includes inoculating at least one
end of the logs, and preferably all around the logs' surfaces.
16. A method of reducing the amount of colour staining as claimed
in claim 12 wherein the wood source may be structural wood and
inoculation includes inoculating at least 60% of the surface area
of the structural wood.
17. A method of reducing the amount of colour staining as claimed
in claim 12 wherein the wood source may derive from a conifer such
as but not limited to, Radiata pine, Douglas fir or Loblolly pine
and it may also be a hardwood including but not limited to
eucalyptus, oak, poplar, or aspen.
18. An inoculum for wood comprising or including one or more forms
of a fungal culture of the species Ophiostoma floccosum, and/or
Ophiostoma piceae and/or Ophiosotoma pluruanulatum effective in
reducing the pitch content of wood.
19. An inoculum for wood as claimed in claim 18 wherein the fungal
culture forms are biologically pure.
20. An inoculum for wood as claimed in claim 18 wherein the fungi
may be selected from the following strains: Ophiosotoma floccosum
F40 Ophiosotoma floccosum F13 Ophiosotoma piceae OPC 422
Ophiosotoma piceae OPC 580 Ophiosotoma piceae OPC 194
21. An inoculum for wood as claimed in claim 18 wherein the wood
source may be wood chips and the fungus or fungi may be applied by
spraying the wood chips.
22. An inoculum for wood as claimed in claim 18 wherein the wood
source may be logs to be cut into structural wood, and inoculation
includes inoculating at least one end of the logs.
23. An inoculum for wood as claimed in claim 18 wherein the wood
source may be structural wood and inoculation includes inoculating
at least 60% of the surface area of the structural wood.
24. An inoculum for wood as claimed in claim 18 wherein wood to be
inoculated may be but is not limited to a conifer such as Radiata
pine, Douglas fir or Loblolly pine and it may also be a hardwood
including but not limited to eucalyptus, oak, poplar, or aspen.
25. A biologically pure culture of a strain of Ophiostoma floccosum
having all of the identifying characteristics of one of the fungi
of stains F40 and F13.
26. A biologically pure culture of a strain of Ophiostoma piceae
having all of the identifying characteristics of one of the fungi
of strain OPC 422, OPC 580, OPC 194.
27. A method for reducing the pitch content in a wood source,
and/or improving brightness in chemical pulp resulting from the
albino treatment of wood, and/or improving pulping efficiency such
that lower kappa numbers were achieved with treatment of the wood
with the albino strains, which comprises or includes inoculating at
least a portion of the wood source with an effective amount of at
least one fungus selected from the group consisting of the species
Ophiosotoma floccosum, Ophiosotoma piceae and Ophiostoma
pluruanulatum, which is capable of reducing the pitch content.
28. A method as claimed in claim 27 wherein the fungi may be
selected from the following strains: Ophiosotoma floccosum F40
Ophiosotoma floccosum F13 Ophiostoma piceae OPC 422 Ophiostoma
piceae OPC 580 Ophiostoma piceae OPC 194
29. A milked as claimed in claim 27 wherein the method further
includes the step of maintaining the inoculated wood source under
conditions which allow fungal growth from the inoculation for a
term sufficient to effect a reduction of the pitch content of the
wood source by such inoculated fungal growth.
30. A method as claimed in claim 27 wherein the wood source may be
pulpwood, or unsterilised pulpwood, or unsterilised refined
pulpwood.
31. A method as claimed in claim 27 wherein the wood source may be
wood chips and the fungus or fungi is applied by spraying the wood
chips.
32. A method as claimed in claim 27 wherein the wood source may be
debarked or undebarked timber or logs.
33. A method as claimed in claim 27 wherein the wood source may
derive from but not limited to a conifer such as Radiata pine,
Douglas fir or Loblolly pine and it may also be a hardwood
including but not limited to eucalyptus, oak, poplar, or aspen.
34. A method as claimed in claim 27 wherein the method may include
using or applying more than one fungal strain.
35. A biologically pure culture of a strain of Ophiostoma floccosum
having all of the identifying characteristics of one of the fungi
of AGAL Accession Numbers NM00/12246, NM00/12247, NM00/12490,
NM00/12491 or NM00/12492.
36. A biologically pure culture of a stain of Ophiostoma piceae
having all of the identifying characteristics of one of the fungi
of AGAL Accession Numbers NM00/12248, NM00/12249, NM00/12250 or
NM00/12489.
37. A biologically pure culture of a strain of Ophiostoma
pluruanulatum having all of the identifying characteristics of one
of the fungi of AGAL Accession Numbers NM00/12251,
NM00/12252,NM00/12253 or NM00/12493.
23TABLE 11 Extractive Analysis of Lab and Mill Research with O.
floccosum strains OF40 and OF13 % Fatty acids % Resin acids Total
on OD on % Triglicerides on Treatment % MTBE Wood OD wood OD Wood
Frozen control 1.97 0.14 0.67 0.16 F13 1/10 YM 0.71 0.08 0.13 0.03
F13 .times. YM 1.09 0.08 0.31 0.02 F13 10 .times. YM 1.32 0.09 0.42
0.01 F40 '1/10 .times. YM 1.88 0.12 0.77 0.1 F40 .times. YM 1.14
0.09 0.24 0.03 F40 10 .times. YM 1 0.02 0.41 0.02 F13 + F40 .times.
YM 1.75 0.14 0.65 0.09 F13 1/10 .times. Peptone 1.15 0.03 0.5 0.03
F13 .times. peptone 0.59 0.02 0.14 0.01 F13 10 .times. peptone 0.58
0.03 0.18 0.01 F40 1/10 .times. peptone 1.04 0.07 0.37 0.07 F40
.times. Peptone 0.9 0.03 0.28 0.01 F40 10 .times. peptone 1.28 0.04
0.5 0.02 F13 1/10 .times. powder 1.04 0.08 0.35 0.03 F13 .times.
powder 0.56 0.06 0.16 0.03 F13 10 .times. powder 1.59 0.1 0.47 0.04
F40 1/10 .times. powder 1.15 0.08 0.29 0.04 F40 .times. powder 0.91
0.08 0.20 0.03 F40 10 .times. powder 0.97 0.1 0.19 0.05 Mix F13 +
F40 1/10 0.74 0.07 0.14 0.03 powder Mix F13 + F40 .times. 1.06 0.11
0.27 0.04 powder Mix F13 + F40 10 .times. 1 0.11 0.24 0.04 powder
Mill Trial % MTBE % extractives % Fatty acids % Resin acids
Triglycerides Treatment On OD wood on OD wood on OD wood on OD wood
F40 0 Wk 1.94 0.09 0.94 0.31 F40 2 wk 2.39 0.15 1.23 0.21 F40 4 wk
1.47 0.08 0.67 0.04 F40 6 WK 1.86 0.06 0.93 0.04 F40 8 wk 1.41 0.04
0.63 0.03 F40 10 wk 1.16 0.04 0.46 0.01 F40 pulp 0.92 0.05 0.37
0.02 Chemiwasher F40 pulp Caustic 0.23 0.02 0.06 0.02 F40 pulp TWWP
0.1* 0.0 0.01 0.01 F40 pulp bleached 0.1* 0.01 0.01 0.01 F13 + F40
2 wk 1.71 0.1 0.72 0.18 F13 + F40 4 wk 1.45 0.09 0.55 0.08 F13 +
F40 6 wk 2.24 0.09 1.09 0.06 F13 + F40 8 Wk 1.15 0.05 0.38 0.03 F13
+ F40 10 wk 1.4 0.07 0.49 0.02.
Description
TECHNICAL BACKGROUND
[0001] The present invention relates to the use of certain fungi in
the enhancement of wood quality. More particularly, but not
exclusively, it relates to the use of particular fungi strains of
Ophiosotoma floccosum, Ophiostoma piceae or Ophiostoma
pluruanulatum, or mixtures of strains, to elicit a useful effect on
wood or wood products derived therefrom in or on a substrate or
locum e.g. including biocontrol; the prevention of staining of
cellulosic materials from detrimental strains or other detrimental
effects from detrimental strains, or for the reduction of pitch
components and/or their detrimental effects, the production of
positive effects achieved from the strains in regard to solid wood
with uptake of liquid materials including but not limited to glue,
preservative, varnish, paint, improvement in mechanical or chemical
pulping as achieved by increased efficiency of cooking liquor (with
concomitant lower kappa number value achieved for the equivalent
cooking liquor or less cooking liquor required for the same kappa
number achieved without use of strains); and increased steam
penetration efficiency. The kappa value is an indication of the
amount of bleaching chemical required to remove the residual
lignin.
BACKGROUND ART
[0002] Wood is a complex material composed of cellulose,
hemicellulose, lignin, and wood extractives or a resinous material
commonly called pitch. "Resin" or "pitch" (used interchangeably)
includes that complex mixture of hydrophobic substances in wood,
which are soluble in neutral organic solvents such as methylene
chloride, diethyl ether, benzyl alcohol and the like. These include
the terpenes, the diterpene ("resin") acids, fatty acids and
esters, glycerides and waxes as well as alcohols, hydrocarbons and
other compounds associated therewith.
[0003] In the production of products from wood pulps, the presence
of pitch is undesirable as it frequently forms deposits which are
difficult to remove or its presence causes detrimental properties
to the products derived from the wood.
[0004] Another problem in the timber industry derives from
staining. When trees are cut down they commonly become infected by
any one or more of a variety of fungi which can stain the wood in
any one or more of a variety of colours. A major problem in the
industry today involves loss of value in timber products, and any
products derived therefrom due to the unsightly staining caused by
so-called blue stain fungi which can colour the wood a variety of
colours including gray, dark blue and black, such staining
appearing in the wood even though the outer surfaces or regions of
the wood have been cut away in forming the lumber.
[0005] In the early 1990's workers at Sandoz Chemicals Biotech
Research Corporation and their colleagues at the University of
Minnesota developed a resin degradative and biocontrol fungus for
pretreatment of wood from an Ophiostoma species. This work resulted
in the development of the current commercial product, the fungal
inocula Catapip.RTM.97. It was made by classical mating of isolated
ascospores from various Ophiostoma piliferum isolates of the United
States. Ophiostoma piliferum is a saprophytic Ascomycete found
throughout the world, and commonly referred to as one of the
sapstain fungi.
[0006] Sapstain fungi grow mainly in wood in the ray parenchyma
cells, within resin canals, within tracheids and fibre cells and
penetrate simple and bordered pits, occasionally forming bore holes
through wood cell walls. Sapstain fungi are not capable of
degrading cellulose or lignin, but metabolise resin extractives,
starch and simple sugars. Sapstain fungi cause a characteristic
stain of sapwood resulting in a blue, black, grey or brown
discolouration of the wood, and is responsible for major economic
losses in the timber and some pulping industries. Problems with
sapstain are most prevalent in warm, humid climates and when wood
with high sapwood content is used (Kay et al, 1997).
[0007] Ascospore mating and selection procedures on loblolly pine
produced the isolate Cartapip.RTM.97 which grew rapidly on wood
chips, degraded substantial quantities of pitch and did not stain
wood (Farrell et al, 1992; Wendler et al, 1992; Hoffmann et al,
1992; Farrell et al, 1993). This colourless (albino) isolate was
shown to be blocked in the 1,8-dihydroxynaphthalene (DHN) melanin
synthetic pathway by the inability to produce the intermediate
scytalone (Zimmerman et al 1993). Later work showed that O. piceae
albino strains could also be generated by the same methodology
(White-McDougall 1998). Treatments of wood chips with the albino
fungi resulted in a biocontrol effect, such that the albino fungus
quick grew throughout the chips and suppressed the growth of
staining fungi (Farrell et al, 1993). Biocontrol of sapstain in
logs using the Cartapip 97 inoculum was demonstrated on red pine in
the lab and in th field (Benhrendt et al 1995; Behrendt et al,
1995).
DISCLOSURE OF THE INVENTION
[0008] It is an object of the present invention to provide an
inoculum and a process which enhances wood quality, having
advantages over those previously known, to provide a process which
enhances wood quality of wood (including but not limited to New
Zealand woods or woods of the Souther Hemisphere), or to at least
provide the public with a useful alternative.
[0009] Our recent studies dealing with New Zealand sapstaining
organisms have yielded surprising results. Studies particularly
with radiata pine have shown certain New Zealand Ophiostoma albino
species especially albino or very slightly pigmented in their
hyphae; even if slightly pigmented in other fungal bodies provide
improved results in biocontrol of sapstain and reduction of pitch.
These species have shown to provide an improved result over that of
Cartapip.RTM.97 on radiata pine. Cartapip.RTM.97 has proved to be
somewhat but not fully effective to reduce stain to the desired
industry target of <10% stain in 3 months. Furthermore, our
studies have shown mixtures of more than one albino fungus have
also been effective in increasing brightness of wood, and/or
brightness increase in chemical pulp resulting from albino
treatment of wood, and/or improved pulping efficiency such that
lower kappa numbers were achieved with treatment of the wood with
he albino strains.
[0010] As used hereinafter reference to specific stains means the
following:
[0011] Ophiosotoma floccosum strain F13--that strain deposited as
AGAL accession number NM00/12246 on 16/08/00
[0012] Ophiosotoma floccosum strain F40--that strain deposited as
AGAL accession number NM00/12247 on 16/08/00
[0013] Ophiosotoma floccosum strain F71--that strain deposited as
AGAL accession number NM00/12490 on 16/08/00
[0014] Ophiosotoma floccosum strain F80--that strain deposited as
AGAL accession number NM00/12491 on 16/08/00
[0015] Ophiasotoma floccosum strain F93--that strain deposited as
AGAL accession number NM00/12492 on 16/08/00
[0016] Ophiosotoma floccosum strain F3410--that strain deposited as
AGAL accession number NM00/12252 on 16/08/00
[0017] Ophiosotoma floccosum strain F5040--that strain deposited as
AGAL accession number NM00/12251 on 16/08/00
[0018] Ophiosotoma floccosum strain F4650--that strain deposited as
AGAL accession number NM00/12253 on 16/08/00
[0019] Ophiosotoma floccosum strain F7073--that strain deposited as
AGAL accession number NM00/12493 on 16/08/00
[0020] Ophiostoma piceae strain OPC 422--that strain deposited as
AGAL accession number NM00/12248 on 16/08/00
[0021] Ophiostoma piceae strain OPC 580--that strain deposited as
AGAL accession number NM00/12249 on 16/08/00
[0022] Ophiostoma piceae strain OPC 194--that strain deposited as
AGAL accession number NM00/12250 on 16/08/00
[0023] Ophiostoma piceae strain OPC 703--that strain deposited as
AGAL accession number NM00/12489 on 16/08/00
[0024] In a first aspect of the present invention there is provided
an inoculum for wood comprising or including one or more
(preferably biologically pure) forms of a fungal culture of the
species Ophiostoma floccosum and/or Ophiostoma piceae and/or
Ophiosotoma pluruanulatum effective to reduce the amount of colour
staining caused by wood staining fungi.
[0025] Preferably said fungal culture is selected from the
following strains:
[0026] Ophiosotoma floccosum F71,
[0027] Ophiosotoma floccosum F80,
[0028] Ophiosotoma floccosum F93,
[0029] Ophiosotoma floccosum F40,
[0030] Ophiosotoma floccosum F13,
[0031] Ophiostoma piceae OPC 703,
[0032] Ophiostoma piceae OPC 422,
[0033] Ophiostoma piceae OPC 580,
[0034] Ophiostoma piceae OPC 194,
[0035] Ophiostoma pluruanulatum 3410,
[0036] Ophiostoma pluruanulatum 7073,
[0037] Ophiostoma pluruanulatum 5040,
[0038] Ophiostoma pluruanulatum 4650
[0039] In one embodiment the wood source may be wood chips and the
fungus or fungi may be applied by spraying the wood chips. In an
alternative embodiment the wood source may be logs to be cut into
structural wood, and inoculation includes inoculating at least one
end of the logs, and preferably all around the logs' surfaces. In a
further alternative embodiment the wood source may be structural
wood and inoculation includes inoculating at least 60% of the
surface area of the structural wood. In another alternative
embodiment the wood source may be structural wood and inoculation
includes inoculating at least 60% of the surface area of the
Structural wood, which may then be later made into chips or a
subsequent fibre product.
[0040] Preferably the wood to be inoculated may be a conifer such
as but not limited to Radiata pine, Douglas fir or Loblolly pine,
and it may also be a hardwood including but not limited to
eucalyptus, oak, poplar, or aspen.
[0041] In a second aspect of the invention the is provided a
biologically pure culture of a strain of Ophiostoma floccosum
having all of the identifying characteristics of one of the fungi
of strains F40, F13, F71, F80 and F93.
[0042] In a third aspect of the invention there is provided a
biological pure culture of a strain of Ophiostoma piceae having all
of the identifying characteristics of one of the fungi of strains
OPC 703, OPC 422, OPC 580 and OPC 194.
[0043] In a fourth aspect of the invention there is provided a
biologically pure culture of a strain of Ophiostoma pluruanulatum
having all of the identifying characteristics of one of the fungi
of strains 3410, 7073, 5040 and 4650.
[0044] In a fifth aspect of the invention there is provided a
method of reducing the amount of colour staining caused by wood
staining fungi in and/or on a wood source which comprises or
includes inoculating at least a portion of the wood source with an
effective amount of at least one fungus selected from the group
consisting of the species Ophiosotoma floccosum, Ophiosotoma piceae
and Ophiostoma pluruanulatum, which is capable of reducing the
amount of colour staining caused by the wood staining fungi.
[0045] Preferably said fungi may be selected from the following
stains:
[0046] Ophiosotoma floccosum F13,
[0047] Ophiosotoma floccosum F40,
[0048] Ophiosotoma floccosum F71,
[0049] Ophiosotoma floccosum F80,
[0050] Ophiosotoma floccosum F93,
[0051] Ophiostoma piceae OPC 703,
[0052] Ophiostoma piceae OPC 422,
[0053] Ophiostoma piceae OPC 580,
[0054] Ophiostoma piceae OPC 194,
[0055] Ophiostoma pluruanulatum 3410,
[0056] Ophiostoma pluruanulatum 7073,
[0057] Ophiostoma pluruanulatum 5040,
[0058] Ophiostoma pluruanulatum 4650
[0059] In one embodiment the wood source may be wood chips and the
fungus or fungi may be applied by spraying the wood chips. In an
alternative embodiment the wood source may be logs to be cut into
structural wood, and inoculation includes inoculating at least one
end of the logs, and preferably all around the logs' surfaces. In a
further alternative embodiment the wood source may be structural
wood and inoculation includes inoculating at least 60% of the
surface area of the structural wood.
[0060] Preferably the wood source may derive from a conifer such as
but not limited to, Radiata pine, Douglas fir or Loblolly pine and
it may also be a hardwood including but not limited to eucalyptus,
oak, poplar, or aspen.
[0061] In a sixth aspect of the present invention there is provided
an inoculum for wood comprising or including one or more
(preferably biologically pure) forms of a fungal culture of the
species Ophiostoma floccosum, and/or Ophiostoma piceae and/or
Ophiosotoma pluruanulatum effective in reducing the pitch content
of wood.
[0062] Preferably said fling may be selected from the following
strains:
[0063] Ophiosotoma floccosum F40
[0064] Ophiosotoma floccosum F13
[0065] Ophiostoma piceae OPC 422
[0066] Ophiostoma piceae OPC 580
[0067] Ophiostoma piceae OPC 194
[0068] In one embodiment of the wood source may be wood chips and
the fungus or fungi may be applied by spraying the wood chips. In
an alternative embodiment the wood source may be logs to be cut
into structural wood, and inoculation includes inoculating at least
one end of the logs. In a further alternative embodiment the wood
source may be structural wood and inoculation includes inoculating
at least 60% of the surface area of the structural wood.
[0069] Preferably the wood to be inoculated may be but is not
limited to a conifer such as Radiata pine, Douglas fir or Loblolly
pine and it may also be a hardwood including but not limited to
eucalyptus, oak, poplar, or aspen.
[0070] In a seventh aspect of the invention there is provided a
biologically pure culture of a strain of Ophiostoma floccosum
having all of the identifying characteristics of one of the fungi
of strains F40 and F13.
[0071] In an eighth aspect of the invention there is provided a
biologically pure culture of a strain of Ophiostoma piceae having
all of the identifying characteristics of one of the fungi of
strains OPC 422, OPC 580, OPC 194.
[0072] In a ninth aspect of the invention there is provided a
method for reducing the pitch content in a wood source and/or
improving brightness in chemical pulp resulting from the albino
treatment of wood, and/or improving pulping efficiency such that
lower kappa numbers were achieved with treatment of the wood with
the albino strains, which comprises or includes inoculating at
least a portion of the wood source with an effective amount of at
least one fungus selected from the group consisting of the species
Ophiosotoma floccosum, Ophiosotoma piceae and Ophiostoma
pluruanulatum, which is capable of reducing the pitch content.
[0073] Preferably said fungi may be selected from the following
strains.
[0074] Ophiosotoma floccosum F40
[0075] Ophiosotoma floccosum F13
[0076] Ophiostoma piceae OPC 422
[0077] Ophiostoma piceae OPC 580
[0078] Ophiostoma piceae OPC 194
[0079] Preferably the method may further include the step of
maintaining the inoculated wood source under conditions which allow
fungal growth from the inoculation for a term sufficient to effect
a reduction of the pitch content of the wood source by such
inoculated fungal growth.
[0080] In one embodiment the wood source may be pulpwood, or
unsterilised pulpwood, or unsterilised refined pulpwood.
Alternatively the wood source may be wood chips and the fungus or
fungi is applied by spraying the wood chips. Alternatively the wood
source may be debarked or undebarked timber or logs.
[0081] Preferably the wood source may derive from but not limited
to a conifer such as Radiata pine, Douglas fir or Loblolly pine and
it may also be a hardwood including but not limited to eucalyptus,
oak, poplar, or aspen.
[0082] Preferably the method may include using or applying more
than one fungal strain.
[0083] In a tenth aspect of the invention there is provided a
biological pure culture of a strain of Ophiostoma floccosum having
all of the identifying characteristics of one of the fungi of AGAL
Accession Numbers NM00/12246, NM00/12247, NM00/12490, NM00/112491
or NM00/12492.
[0084] In an eleventh aspect of the invention there is provided a
biologically pure culture of a strain of Ophiostoma piceae having
all of the identifying characteristics of one of the fungi of AGAL
Accession Numbers NM00/12248, NM00/12249, NM00/12250 or
NM00/12489.
[0085] In a twelfth aspect of the invention there is provided a
biologically pure culture of a strain of Ophiostoma pluruanulatum
having all of the identifying characteristics of one of the fungi
of AGAL Accession Numbers NM00/1225, NM00/12252, NM00/12253 or
NM00/12493.
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLE
[0086] From the 21 sapstain fungal species isolated and identified
in New Zealand (Farrell et al, 1998), four species were chosen for
a breeding programme in order to make albinos/white fungi. These
New Zealand albinos were made as had been done with Cartapip 97,
classically mating a variety of isolates of the same species to
produce albino, non-melanising strains. Neither genetic engineering
nor mutagens were used to make the isolates. The albino isolates
were studied for their ability to biocontrol and reduce staining in
mature radiata pine logs (Farrell et al 1997a; Farrell et at
1997b), and for their ability to reduce resin components in radiata
pine wood chips.
[0087] Surprisingly and a significant advancement over what is
presently commercially available, were the effect of some of the
Ophiostoma floccosum, O. pluruanulatum, and O. Piceae albinos. In
this example these stains were isolated from New Zealand but it is
expected that these species and their subsequent albinos could
prove beneficial when isolated from any country and origin and give
beneficial effects superior to Cartapip.RTM.97, Some of the New
Zealand albinos were significantly better at maintaining biocontrol
effect and exclusion of detrimental organisms, including stain
fungi, and degrading detrimental wood components than others and
better than the commercial product. Also surprising and unobvious
according to presently granted patents was the fact that different
albinos of various species had differing degrees of effect, with
some of the albinos providing other than a positive effect on wood
with their application and incubation.
FIGURES
[0088] FIG. 1: Results of solid logs with albino Inoculation.
[0089] FIG. 2: 80,000 ton trial--Extractives of chips as a function
of incubation time in the chip piles.
[0090] FIG. 3: Chips inoculated with both OF40 and OF13.
Example 1
[0091] Competition Assays
[0092] Albinos were constructed by single ascospore isolation and
mating, according to Zimmerman, W. C., Blanchette, R. A., Burnes,
T. A., Farrell, R. L. (1993). Melanin and perithecial development
in Ophiostoma piliferum. Mycologia 87: 857-863.
[0093] Albinos were tested in the laboratory for presence of
pigment under a variety of incubating conditions and in competition
assays. The competition assays were conducted by inoculating either
sterilised, gamma irradiated or some other method, or
non-sterilised radiata pine cubes or chips with each or more than
one of sapstaining species, including but not limited to
Sphaeroides sapinea, Ophiostoma piliferum or Leptographium species,
prior to, simultaneously or after inoculation with the albino or
more than one albino fungus. In most assays the staining
fungus/fungi was introduced to the chips as actively growing
vegetative cells at one-tenth the cell density as the albino(s), an
extreme situation with respect to challenge.
[0094] An example below shows typical conducted lab competition
assays, which consisted of albinos made from New Zealand
ascomycetes, and the commercial product Cartapip.RTM.897.
1 TABLE 1 Intensity Comments F71 0 F71 + Stainer same day 0.8
successful F71 + Stainer next day 0.5 successful F73 0 F73 +
Stainer same day 1 successful F73 + Stainer next day 1.3 Not
successful F74 0.5 Not successful F74 + Stainer same day 1.5 Not
successful F74 + Stainer next day 1.5 Not successful F77 0 F77 +
Stainer same day 2 Not successful F77 + Stainer next day 0.5
successful F80 0.5 Successful F80 + Stainer same day 1 Successful
F80 + Stainer next day 1 Successful F81 0 Successful F81 + Stainer
same day 2 Not Successful F81 + Stainer next day 2 Not Successful
F87 0 Successful F87 + Stainer same day 1.2 Not successful F87 +
Stainer next day 1.5 Not successful F93 0 Successful F93 + Stainer
same day 0.3 successful F93 + Stainer next day 0.5 successful
OPC703 0.2 Not successful OPC703 + Stainer same day 0.3 successful
OPC703 + Stainer next day 0.7 successful OPC422 0.5 Not successful
OPC422 + Stainer same day 0.8 successful OPC422 + Stainer next day
1 successful Intensity % coverage OPC194 0 successful OPC194 +
Stainer same day 3 Not successful OPC194 + Stainer next day 3 Not
successful OPC580 0 successful OPC580 + Stainer same day 2 Not
successful OPC580 + Stainer next day 1.8 Not successful 3410 0
Successful 3410 + Stainer same day 0.2 Successful Intensity
Comments 3410 + Stainer next day 0.5 Successful 7073 0 Successful
7073 + Stainer same day 0.5 Successful 7073 + Stainer next day 0.7
Successful Cartapip 97 0.2 Not successful Cartapip + Stainer same
day 0.7 successful Cartapip + Stainer next day 1.7 Not successful
Stainer same day 3.0 Stainer next day 3.3 Uninoculated sterile
control 0
[0095] Albino concentration was 10.sup.6 blastospore/ml
[0096] Stainers (fungi which cause stain including but not limited
to Leptographium, or Diplodia (Sphaeropsis sapinea) or any other
organism that leaves a sin on wood) concentration was 10.sup.6
blastospore/ml
Example 1
[0097] Results
[0098] Surprisingly not all of the albinos had the same effect for
prevention of stain. This was an unexpected and unobvious result as
previously it was considered that any albino or white fungus, or
almost albino would constitute a biocontrol effect on wood, and
prevent staining fungi from staining the wood, Successful
competitive albino fungi were judged as those with less than 0.5
stain score on their own, and that maintained a stain score when
challenged with one of the staining fungi, in this example
Leptographium or Diplodia (Sphaeropsis sapinea) of 1 or less than
1. As shown in the results, the preferred organisms were F71, F80,
F93 (all albinos, with regard to hyphae but not with regard to
synnemata, of Ophiosotoma floccosum), OPC 703, 422 (albinos, with
regard to hyphae but not with regard to synnemata, of the species
Ophiostoma piceae) and 3410 and 7073 (albinos, with regard to
hyphae but not with regard to synnemata, of the species Ophiostoma
pluruanulatum). The commercial Product Cartapip.RTM.97. had too
much stain and was not successful.
Example 2
[0099] In order to teach the mechanism of positive effect upon
wood, the albino fungi have been tested in the laboratory for
growth characteristics and enzyme production, By standard
biochemistry assays the albino fungi have been tested for their
ability to deplete starch in growth media, and units of activity in
media of amylase and lipase enzymes.
2TABLE 2 Albino Characteristic Chart Key Characteristics of New
Zealand Ophiostoma albinos: Nutrient depletion Cube Competition
Albino % Starch Amylase Liplo O.pilaf Growth in Species ID #
Depletion/hr Lipase Lepto(L) (D) (Op) Blocks pluruanulanim 3410
92/24 700 342 3440 98/24 364 5040 2226 486 4630 1620 4650 540 4680
1119 673 4880 738 4890 88/24 413 5980 97/24 576 7052 100/24 840 643
1 1 1 7058 92/24 917 701 1 1 1 7060 85/100 843 566 1 1 1 7073 721 1
1 1 7076 98/48 1527 384 1 1 1 7078 1041 2 1 floccosum F3 74 1 1 P
F13 293 1 1 P F29 270 496 2 1 P F36 44/24 226 653 1 1 P F61 92/24
230 526 P F63 21/24 235 365 P F30 45/24 388 482 1 1 P F20 16/24 502
562 1 1 1 P F35 100/48 959 604 1 1 P F40 17/24 462 728 1 1 1 P
piccae 1 156 piccae 194 14/24 180 442 P 422 25/24 177 421 1 P 542
13/24 1091 782 1 1 P 400 95/24 1055 702 P 12 553 1 P 580 11/24 960
614 2 P
[0100] Stain during competition is marked on a scale of 1-5 with 0
being white wood and 5 black stain. Each competitive experiment is
marked individually.
Example 2
[0101] Results
[0102] There is a variety of responses of the albino fungi to these
diagnostic tests. Not all the albinos have the same
characteristics.
Example 3
[0103] Field Results on Solid Logs with Albino Inoculation
[0104] Albino fungi were grown in liquid cultures of growth media
consisting of sufficient carbon, nitrogen and trace elements and
vitamins to promote cell biomass accumulation (standard and
non-standard mycological media are acceptable), either used in the
spent growth media, or harvested by centrifugation and resuspended
in water or buffer, with or without washing, were sprayed onto
radiata pine logs, Either a pure single strain or more than one
strain can be used simultaneously or consecutively. The typical
cell concentration of the fungus in spent media, water or buffer
was from 10e2 to 10e6 colony forming units per millilitre. The
fungal cell suspensions were applied onto the logs by back-pack
spray systems, or commercial sprayers, either variety such as used
by commercial agricultural spray systems or commercial log spray
systems. Fungal cell suspensions could also be applied to logs by
dipping the logs into a bath suspension containing the cells in
water, buffer or spent growth media. Fungal application can be done
only one time or more than one time. Mouldicides may be used after
a few days of the fungal application to improve surface appearance.
After application of the fungal cell suspension to the logs, the
logs were let in the field, or in an enclosed container such as
warehouse, boat etc, and after 1, 2, 3 or longer months the amount
of in was assessed on the logs visually. Results of one such
application of multiple fungi are given in FIG. 1.
Example 3
[0105] Results
[0106] Some albinos can have a positive effect on wood quality,
biocontrol and keep wood from staining, better than the commercial
fungus and better than commercial anti-sapstain chemicals. Some
albinos have detrimental effects and increase the amount of stain,
therefore not all white fungi behave in the same manner with regard
to their effect on wood.
EXAMPLE 4
[0107] Resin Component Degradation
[0108] Solid wood or wood chips, either previously sterilised by
gamma irradiation or some other method, or non-sterilised, was
treated with more than two dozen albino fungi, a bacteria (for
example Pseudomonas resinovorans) or the commercial product
Cartapip 97. The inoculated wood was either left to stand at
ambient temperature or incubated from 0 degrees Centigrade to plus
65 degrees Centigrade. The wood was analysed anytime from 2 days to
16 weeks, most typically between 1-8 weeks) for full extractive
analysis.
[0109] Extractives Analysis
[0110] The wood extractives content of treated and untreated chips
was determined on freeze dried chip samples which had been ground
to <0.5 mm. The wood meal was extracted by solvent extraction
with acetone using a Soxtec extractor. Gas chromatography of the
methylated extract was performed using a DB-1 5 m.times.0.32 mm
capillary column fitted with a 0.5 m.times.0.5 mm deactivated
silica retention gap and using an on column injection technique.
Detection was by flame ionisation detector and quantitation was
performed using the internal standard technique on the
chromatography data system.
Example 4
[0111] Results
[0112] Control, non-fugal inoculated radiata pine wood chips were
analysed as a reference for resin acids, fatty acids and
glycerides, and the results are given in Table 3,
3TABLE 3 Analysis of Wood Extractives of Control Chips from Lab
Experiment Sample % Acetone % Free % Resin % Control 1.26 0.227
0.411 0.130 Control 1.27 0.156 0.282 0.081 Average: 1.26 0.19 0.35
0.11
[0113] Results for the fungal treated samples can be most easily
compared as to the effect of one fungal isolate as compared to
another fungal, rather than compared to the reference control,
non-fungal treated samples.
[0114] Dozens of albino isolates were treated for their ability in
the laboratory to degrade wood extractives. Results given in Table
2 show in duplicate some of the better and the lesser fungal
overall effects for wood extractives decrease.
4TABLE 4 Analysis of Wood Extractives after Fungal Treatment % %
Free Acetone Fatty % Resin Sample Extractives Acids Acids %
Glycerides OPC 542 1.46 0.092 0.730 0.025 " 1.70 0.096 0.797 0.018
F63 1.19 0.108 0.481 0.020 " 1.02 0.080 0.375 0.021 7060 0.73 0.039
0.226 0.012 " 0.74 0.040 0.222 0.015 7073 0.72 0.073 0.184 0.039 "
0.68 0.069 0.168 0.036 7076 1.19 0.120 0.268 0.093 " 1.15 0.120
0.265 0.089 OPC 422 0.57 0.044 0.133 0.033 " 0.61 0.048 0.150 0.038
F13 0.62 0.036 0.100 0.016 " 0.64 0.036 0.095 0.017 OPC 580 0.65
0.038 0.165 0.010 " 0.70 0.050 0.217 0.015 F40 0.37 0.028 0.129
0.017 " 0.58 0.043 0.156 0.023
[0115] As shown in Table 4, the various isolates tested on wood
chips varied considerably in their ability to degrade wood
extractives. The range of extractives values for differing fungal
treatments was from 0.37% to 1.70% by weight of wood; obviously
some much more efficient at degrading the wood extractives than
others. Fungi such as the isolate O. floccosum F40 on the average
decreased total acetone extractives to 0.47% versus OPC 542, with
the same length of time incubating on the chips, had an average
acetone extractives value of 1.58% by weight of wood. Analogously,
some of the fungi had significant decreases in free fatty acids,
for example O. floccosum F40 with free fatty acids of 0.035% and
free resin acids of 0.143% whereas O. floccosum isolate F63 did not
decrease the free acids as much with average % free fatty acids of
0.094% and % free resin acids of 0.428% by weight of wood.
[0116] The free fatty acids and free resin acids were further
analysed by gas chromatography. Examples of the data for isolate
F13 are given in Table 5.
[0117] All the data obtained to date on wood extractive decrease
was sorted for the possible contenders for further evaluation on
the basis that their resin acid contents were below 0.2% by weight
of wood; These isolates were O. floccosum F40 and F13, and O.
piceae C OPC 422, OPC 580, and OPC 194.
5TABLE 5 Analysis of Free acids and extractive components after F13
Lab Fungal Treatment-duplicate analysis % Extractives 0.62 0.64 mg
Analysed 0.546 0.574 C16:0 found (ug) 4.76 4.96 C18:2 found (ug)
5.94 5.51 C18:1 found (ug) 19.88 20.53 C18:0 found (ug) 4.01 4.09
Free Fatty Acids Sum (ug) 34.59 35.09 % Free Fatty Acids in Extract
6.34 6.11 Corrected % FFA in Extract 5.82 5.60 % Free Fatty Acids
in Sample 0.039 0.039 Corrected % FFA in Sample 0.036 0.036 Pimaric
found (ug) 15.31 14.73 Sandara. found (ug) 3.19 3.13 Isopimaric
found (ug) 8.34 7.59 Levo. + Pal. found (ug) 16.53 16.02 Dehydro.
found (ug) 44.26 42.96 Abietic found (ug) 2.94 3.04 Neoabietic
found (ug) 5.81 5.59 Resin Acids Total (ug) 96.38 93.06 % Resin
Acids in Extract 17.65 16.21 Corrected % RA in Extract 16.20 14.85
% Resin Acids in Sample 0.109 0.104 Corrected % RA in Sample 0.100
0.095 C16:0 found (ug) 3 Unidentified (ug) 14.62 16.47 C18:1 found
(ug) C18:2 found (ug) C18:0 found (ug) Glycerides Sum (ug) 14.62
16.47 % Glycerides in Extract 2.68 2.87 Corrected % Glyc. in 2.52
2.68 % Glycerides in Sample 0.017 0.018 Corrected % Glyc. in 0.016
0.017 % Extract Identified 24.5 23.1
Example 5
[0118] Effect of Albinos on Processed Pulp
[0119] Fungal cell cultures as described in Example 1 were applied
either to logs or to chips at a ratio of 1000 gallons cell
suspension in water to 200 tons wet weight chips--this cell
suspension to wood weight ratio could be varied by several orders
of magnitude.
[0120] In this Example are given the results from the use of two O.
floccosum albinos mixed together and applied onto chips, or three
O. piceae albino fungi mixed together and sprayed onto wood chips.
Control pile of chips sprayed just with water were also
established.
[0121] OF Pile: in this example consisted of chips sprayed with
water plus a mixture of two Ophiostoma albinos, O. floccosum 40,
and O. floccosum 13, but other combination of albino fungi could
also be used. OPC Pile in this example consisted of chips sprayed
with water plus mixture of three O. piceae C albino isolates, OPC
580, OPC 422 and OPC 194 cell concentrate, but other combination of
albino fungi could also be used.
[0122] Sprayed in 4500 litre water solution onto 200 tons wet
weight chips. Temperature or rain/moisture does not affect the
spray trials.
[0123] Materials and Methods
[0124] Survey, Culture Isolations and Identification, and
Competition Experiments.
[0125] Sampling, isolations, identifications, mating, culture
growth and competition experiments on wood cubes and chips were
done as described by Farrell, et al, 1998
[0126] Analysis of the Mill Trial
[0127] Spray Inocula for the Mill trial
[0128] The O. piceae 540, O. piceae 422, O. piceae 194 culture
suspensions were mixed together and applied to one pile, and O.
floccosum 40 and O. floccosum 13 were mixed together and applied to
another pile, as described in the Materials and Methods and details
in Appendix 2. From the spray tank, a sample of the inocula was
taken at start middle and end of the spraying of the 200-ton wet
weight of chips, to verify the mixing and dosage of the
blastospores per volume spray was consistent throughout the
trial.
[0129] Fungal Growth during Eight Week Trial
[0130] Growth of the fungi at the mill piles was monitored by
standard mycological culturing of chips taken from the four piles
every two weeks during the trial and with many isolates checking
with molecular markers for DNA analysis. Consistently, the albino
O. floccosum grew from the OF pile. There was little if any growth
from the OPC pile of O. piceae and there was O. floccosum, growing
in this pile, the native O. floccosum of the site The Wet Pile
showed positive Ophiostoma identification of native O. quercus, O.
floccosum Rhizopus; Trichoderma, Fusarium and yeasts. Therefore the
Tantanoola Mill site consistently has the presence of moulds,
yeasts and three sapstain organisms O. quercus, O. floccosum and
Graphium species C on the radiata pine wood chips.
[0131] Analysis of the Pulps of the Mill Trial
[0132] DNA: The wood chips of the piles were made into pulp at the
mill by normal procedures. Samples of pulp were taken for further
analysis from the Chemiwasher, Twin wire wash press (TWWP), and
bleached pulp (M57). DNA extractions were performed on the pulps to
show whether there was any trace of the New Zealand-origin fungi;
these analyses showed that there was no residual DNA left in the
pulps. Polymerase chain reaction (PCR) of specific DNA probes for
O. piceae and O. foccosum on the extractions confirmed that no DNA
could be isolated from the pulps, and hence no PCR reaction was
observed; therefore there was no residual DNA in the pulps from the
applied albino fungi.
[0133] EXTRACTIONS: Soxtec dichloromethane extractions of the pulps
from the chips of the 8 week mill trial (Dry pile not processed)
were compared to the mill data from May 1999 to February 2000, with
seasoning of chips routinely during this period being 12-16 weeks
and results are given in Table 6.
6TABLE 6 Wood Extractives of Pulps from Mill (average values based
on wood weight) SampleChemiwasherTWWPM57Mill data May '99-Feb 2000
0.90% 0.10% 0.07% OF Pile pulp 0.92 0.12% 0.05% OPC Pile pulp 0.90%
0.12% 0.06% Wet Pile pulp 0.88% 0.10% not done
[0134] TWWP=Twin wire wash press
[0135] Interestingly, as evident in the data of Table 6, it can be
observed for commercial utility that the amounts of wood
extractives after fungal treatment (OF Pile Pulp or OPC Pile Pulp)
did not decrease relative to the Wet Pile pulp, nor from the normal
mill wood extractives of pulp data as collected from May
'99-February 2000. Therefore, contrary to the laboratory results of
the fungi (Example 4 showing resin component degradation by the
organisms of choice), in the true setting of the utility of the
fungi the resin component numbers are not decreased from the
controls or normal mill running without fungal use.
[0136] BRIGHTNESS: Brightness's of the pulps from the fungal
treated pulps showed that the OPC pile pulp from the TWWP had 54.0%
ISO brightness, and after bleaching 78.3% ISO brightness. The OF
pile, which during the growth assays showed significant growth of
the albino O. floccosum, and less growth of staining species showed
improved brightness with the OF pip from the TWWP having a
brightness of 66.8% ISO brightness and after bleaching a 85.5% ISO
brightness, a significant improvement in brightness.
7TABLE 7 BLEACHING RESULTS: O. floccosum O. piceae Mill seasoning
Caustic 66.8% 54.0% 59% extracted Bleached 85.5% 78.3% 80-82%
[0137] Finally a further experiment comprising inoculating 6000
tons of fungi with O. floccosum 13 and 40 was performed. In
processing the pulp through chemical pulping after the fungal
incubation, the mill again experienced an upward brightness shift
of approximately 5 points. The pulp was processed through the mill
without any problems.
[0138] The other unexpected benefit which became clear during the
extended trial was a Kappa shift downwards of 5 points. This result
may result in a huge cost saving as it means the mill can do more
for less with regard to the cooking liquor charge on wood. Cooking
liquor is one of the major manufacturing expenses. This benefit of
the 5 point downwards shift of kappa (the kappa test is made from
the reaction of pulp with acidic permanganate solution and the
calculated number resulting is indicative of the amounts of
non-cellulosic components (especially lignin) residing in the pulp,
used in mill control work to indicate the degree of delignification
occurring during cooking and the chemical requirement for bleaching
(G. A. Smook, Handbook for Pulp & Paper Technologists)) to pulp
mills is a significant achievement of increased chemical pulping
efficiency. The increased pulping efficiency has been suggested in
a research publication (M B Wall, G Stafford, Y Noel, A Fritz, S
Iverson, R Farrell, Treatment with O. piliferum Improves Chemical
Pulping Efficiency, presented at the 6th international Conference
an Biotechnology in the Pulp and Paper Industry, Vienna, Jun.
11-15, 1995), but this publication showed in the laboratory only a
downward shift of 1.25 kappa points and this work has never been
demonstrated in a mill, and in a true commercial setting. The
increased chemical pulping efficiency described here has practical
significance to mills in several ways, including but not limited
to, decreased amount of cooking liquor to be added to fungal
treated wood chips to reach equivalent kappa (lignin) content at
the same amount of cellulosic content, and as measured at the same
viscosity, greater yield of pulp from equivalent cooking liquor as
the cooking liquor works with greater efficiency to remove lignin
and not cellulosic contents, improved pulp and fibre properties as
the reduced cooking liquor does not act as much non-specifically on
cellulosic components, improved benefit to the environment because
less cooking liquor, and less concomitant effluent, to be used, and
reduced cooking time as the cooking liquor is working with greater
efficiency on fungal treated wood chips.
Example 5
[0139] Results
[0140] The albino O. floccosum sprayed wood chips produced a
brighter pulp than had been observed in the mill, and which
required less bleach chemical to reach a higher brightness than the
standard mill pulps.
[0141] The extractives analysis indicated that the O. floccosum
pulps, after 8 weeks incubation, had less extractives than normal
seasoning without sprayed fungus requiring 12-16 weeks.
Example 6
[0142] 80,000 ton wood chip experiment Our fungal of New Zealand
Ophiostoma floccosum OF40 and OF13 were researched in this 80,000
ton wood chip trial. The fungal inocula were provided as freeze
dried powder in vacuum-sealed bags, packaged as approximately 1000
tonnes wood chips inocula per bag of each fungus strain.
[0143] A day of inoculation was typically considered about 000
tonnes wood chips. [tonne refers to 1,000 kg or 2200 lb. The wood
chips used for trial purposes were all green wood with no
additional water added prior to inoculation.]
[0144] The trial was established as follows:
[0145] Stage 1: OF 40 sprayed 10,000 tons (10 days)
[0146] Stage 2: OF 40 & OF 13 for 60 days, about 60,000 tons,
and
[0147] Stage 3: OF 13 at the end sprayed onto 7000 tonnes,
[0148] Specifics of the chips and the spraying inocula were as
follows:
[0149] Average chip size=25 mm.times.25 mm.times.2 mm
[0150] Density of wood=350-550 (average 400 kg/cub meter) (400
micro gram/cub.millimeter)
[0151] In order to estimate in the future for inocula-loading
efficiency, one chip had a surface area of 1450 square. mm, volume
of 1250 cub. mm and a mass of 5 gram.
[0152] 1 tonne of wood occupies 2.5 cub.meters, & chipped has
an average surface area of 290 squ.meter. Fifty tonne of wood was
sprayed with 2 cub. meters Water/Fungi
[0153] i.e. 2 cubic meters of water is sprayed over an average
surface area of 14500 squ.meters.
[0154] i.e. 1 cubic meters of water is sprayed over an average
surface area of 7250 squ.meters.
[0155] i.e. 1 cubic centimetre is sprayed over an average surface
area of 7250 squ.milimeters.
[0156] Since there were about 10,000 (10E+4) to (10E+5) colony
forming units of fungi per millilitre, each square millimetre of
chip surface was sprayed with 0.725 to 7.25 fungi spores.
[0157] Results of the Trial
[0158] Mill Specifics
[0159] During the trial the mill was constantly making observations
regarding performance of the fungal inoculated chips. A successful
outcome of the trial was decided at the beginning to have no pitch
shutdowns when fungal inoculated chips were run through the mill.
This was the case. There were no shutdowns at the mill because of
pitch during the three months of fungal use.
[0160] Importantly, the pulp was clean leaving the mill.
[0161] Analysis Method for Fungal Trial Extractives
[0162] The samples were freeze dried and then ground on a Wiley
mill to pass a 40 mesh sieve and then extracted using the Soxtec
apparatus with t-buthymethyl ether as solvent. The total
extractives are expressed on the basis of oven dried starting
wood.
[0163] Gas Chromatography
[0164] (According to Procedure of Orsa and Holmbom, J. Pulp Pap.
Sci., 20(12) J361, 1994)
[0165] The internal standard and synthetic mixture solutions were
prepared. The internal standard (50 .mu.L) was added to the extract
vial containing approximately 0.6 mg of material, and then treated
with an excess of ethereal diazomethane. The solvent and excess
diazomethane were evaporated under a stream of nitrogen
dichloromethane (400 .mu.L) added and the vial was sealed.
[0166] A Hewlett Packard 5890 series II GC with on-column injection
and FID detector was used, with 1 .mu.L injected onto the column,
which was a BP1, 10 m.times.0.54 mm.times.2 mm.
[0167] GC Conditions
[0168] Injector temperature=80.degree. C. initially, held for 2
minutes and ramped to 340.degree. C. at 20.degree. C./minute and
then held at 340.degree. C. Detector temperature 340.degree. C.
[0169] Oven temperature=40.degree. C. initially, held for 2
minutes. Then it was increased to 100.degree. C. at 60.degree.
C./minute, held for 2 minutes, ramped to 200.degree. C. at
4.degree. C./minute and then ramped to 340.degree. C. at 5.degree.
C./min and held for 10 minutes.
[0170] The helium carrier gas flow rate was constant at 12 kPa.
[0171] All retention times were determined by comparison to
authentic standards.
[0172] Soxtec Method
[0173] The finely ground wood (.about.2 g) was accurately weighed
in duplicate into small cellulose thimbles (26 mm.times.60 mn). At
the same time sample (.about.1 g) was accurately weighed in
duplicate for moisture content. The thimble was put into the Soxtec
apparatus and 60 mL t-butylmethyl ether added to a pre-weighed
aluminium cup containing anti bumping granules. The thimble was
placed in the boiling position for 30 minutes and then raised to
the rinsing position for 60 minutes. After this time, the solvent
was removed by evaporation, and the last trace of solvent removed
under vacuum. The dry cup was re-weighed and the % extract
calculated.
[0174] GC Chromatography
[0175] (a) Extract Preparation
[0176] The extract was washed into a volumetric flask with small
volumes of acetone, and made up to the mark. An aliquot was removed
to yield approximately 0.6 mg of material and transferred to a
vial. The acetone was removed by a gentle stream of nitrogen, and
the vial flushed with nitrogen prior to sealing. The vials were
stored sealed under nitrogen in the freezer before analysis.
[0177] (b) Solutions
[0178] The internal standard solution contained pentadecanoic acid
(0.8472 mg/mL), betulin (0.7782 mg/mL), cholesteryl heptadecanoate
(0.94 mg/mL) and dipalmitoyloleoyl glycerol (0.4154 mg/mL) in
dichloromethane. The pentadecanoic acid is a check for complete
derivitisation, and the dipalmitoyloleoyl glycerol is a check for
triglyceride recovery. The betulin is used as internal standard for
resin and fatty acids and the cholesteryl heptadecanoate is used as
internal standard for triglycerides. The ratio of the 2 internal
standards is used to monitor column performance.
[0179] A synthetic resin mixture, containing 3 fatty acids, 2 resin
acids and a triglyceride was also prepared in dichloromethane to
check response factors.
[0180] Percentage component is determined by adding together the
areas of peaks of the gas chromatagram that fall within a retention
time range. This range is determined by analysis of authentic
standards. However, there is no guarantee that peaks in the range
are the same as the standards. Some verification work has been done
on GCMS (gas chromatography/mass spectroscopy) to check peak
assignments, however this was not done for triglycerides, where the
samples can not be put on the GCMS. The pitch and pulp samples
especially may not actually contain triglycerides at all.
[0181] The Mill sent samples of chips every two weeks of the trial
to The University of Waikato for analysis. These chips had done the
following:
[0182] Chips were assayed for the type of fungi growing on them by
the Waikato lab's standard mycological procedures and assessed for
stains an indication of the albino fungal dominance over other
microorganisms on the chips and biocontrol effect.
[0183] Chips were analysed for extractives.
[0184] Fungal Isolation from Chips
[0185] The fungal isolates from the chips used were isolated and
purified on selected media, and identified by morphology and in
some cases by DNA probe. The purpose of these isolations is first,
to detect whether the inoculated fungi sprayed onto the chips were
growing, and second, to see what else might be growing on the
chips. The two albino O. floccosum strains, called OF 40 and OF13,
had a reversion of melanin formation such that when growing on
chips, these fungi produced light brown synnema hairs that stick up
from the chip. The hyphae of the fungi are non-melanised i.e.
non-staining. The brown synnema proved useful for the mill and lab
to be able to see when the albino was well growing on the chip. The
data given in Table 9 is from all the research mill trials.
8TABLE 9 Identification of Fungi from Chips Isolates from 800
tonnes wood chips Mill Expt. (Nov '1999) Piles: Wet Pile (water),
Dry Pile (not sprayed), O. piceae pile and O.floccosum pile No Site
From ID 1 Wet pile O. querci 2 Wet pile O. querci 3 Dry pile Mucor
4 Wet pile Graphium sp. C 5 Wet pile Graphium sp. C 6 Wet pile O.
querci 7 O. piceae O. floccosum/Graphium sp. C pile 8-31 O.
floccosum O. floccosum albino O. floccosum/Graphium sp. C, pile O.
querci, Pesotum sp. C and Y
[0186] Isolates from 6000 tonnes wood chips Mill Expt. (March
2000). Pile sprayed with 50:50 mixture of O. floccosum 13 and O.
floccosum 40
9 No ID 1A Mucor 2B O. floccosum albino 3C Mucor 4D O. floccosum
albino 5E O. floccosum 6F O. floccosum 7G O. floccosum 2a O. querci
2c O. querci 2d Mucor 2e O. querci 7f O. floccosum Pesotum sp Y
Isolates from 80,000 tonnes wood chips Mill Expt. (August,
2000-February 2001). Pile sprayed with 50:50 mixture of O.
floccosum 13 and O. floccosum 40 or each alone
[0187] F40 Treatment
[0188] 0 week: Chips were clean, no stained chips observed.
[0189] The microorganisms that grew on media 4 and 6 after 3-6 days
incubation are: A,niger, Trichoderma, Penicillium, White fungi,
Bacteria, Yeast. The albino O40 with brown synnema (O. floccosum)
dominated on plates media 6.
[0190] 2 week: Chips were clean, no stain, Albino O40 with Brown
synnema can be seen under microscope/naked eyes.
[0191] After 3-6 day incubation on media 4/6: Mucor, Bacteria, and
Albino O.floccosum (Brown synnema) dominated on media 6.
[0192] 4 week and 6 week; chips were clean no stain at 4 weeks, but
at 6 weeks some stained chips observed; After 3-6 day incubation of
4/6 week samples : Mucor (fungus with an obvious Black dot),
Bacteria, Trichoderma, Albino O. floccosum (brown synnema) and
darker O. floccosum dominated on media 6.
[0193] 8 and 10 week samples: Chips little bit darker, Albino O.
floccosum brown synnema fungi growing well and black dot fungi can
be seen under microscope After 3-6 day incubation on media 4/6:
Mucor, Rhizopus, bacteria yeast and albino O. floccosum were
dominated on media 6.
[0194] F13+F40 Treatment
[0195] 2 week: Chips were clean no stain. Brown synnema on chips
can be seen under microscope. After 3-6 days on media 4/6, Mucor,
Rhizopus, Bacteria and Albino brown synnema (O. floccosum) on media
6.
[0196] 4 and 6 Week Sample:
[0197] 4 week sample still clean, no stained chips, but on 6 week
sample chips become darker. Brown synnema and black hairy fungi cm
be seen under microscope.
[0198] On media 4/6: Mucor, Trichoderma bacteria yellowish hypae
fungi, albino O. floccosum and darker O. floccosum.
[0199] 8 and 10 week. Some chips were darkened. Black dot fungi on
chips and albino O. floccosum growing well.
[0200] After 3-6 days incubation on media 4/6. Mucor (black dot),
Bacteria, Rhizophus. Albino O. floccosum dominated.
[0201] F13 Treatment
[0202] 2 week: Clean, but stain on some chips. Under microscope:
black hairy fungi, Trichoderma, and albino brown synnema (O.
floccosum).
[0203] Under microscope observation there were no difference
between sample from surface, and bottom of pile.
[0204] After 3-6 days: Mucor, Aspergillus, brown synnema
(floccosum).
[0205] 4 week: Chips look stained. Brown synnema on all chips,
Black synnema, Black hairy fungi, Trichoderma, Penicillium.
[0206] After 3-6 days: Mucor, bacteria, Trichoderma. Floccosum
(dominated).
[0207] 6 and 8 week sample: Chips were darkened and stained, under
scope,: Brown synnema (floccosum), Trichoderma, black hairy
fungi.
[0208] After 3-6 days: Mucor, bacteria, Trichoderma, Penicillium.
Brown synnema/floccosum dominated.
[0209] 10 week sample: Less stain than samples 6 and 8 weeks. Under
microscope observe the brown synnema on chips, after 6-8 days on
media 4 and 6: Penicillium, Mucor, yeast, bacteria, Albino brown
synnema (O. floccosum), black synnema (O. querci?)
[0210] Untreated Chips Sample
[0211] 30 week Stock pile.
[0212] Mostly clean no stain, but some chips were colonised with
black hairy fungi.
[0213] 30 wk stock pile: Trichoderma, penicillium, Bacteria, Black
synnema (Querci).
[0214] Chips: Black synnema (querci?), blach hairy fungi,
Penicillium, Mucor, bacteria
[0215] Truck: Penicillium, Mucor, Yeast.
[0216] Analysis
[0217] Statistics Data from Lab and Trial Extractives Data
[0218] Tables A to L present statistical data.
[0219] Statistic extractives for Lab Trial with the parameters
Strains: 1=F13 2=F40
[0220] Doses 1=0(control), 2={fraction (1/10)}.times., 3=.times.,
4=10.times.
[0221] Media 1=YM 2=Peptone
10TABLE A Analysis of Variance for % MTBE (Two-way analysis of
Variance between Strain vs doses) Source DF SS MS F P Strain 1
0.3180 0.3180 4.26 0.050 Doses 3 2.1335 0.7112 9.52 0.000**
Interaction 3 0.1972 0.0657 0.88 0.465 Error 24 1.7922 0.0747 Total
31 4.4409 **Significantly different (P<0.05) doses level
significantly affected to % MTBE
[0222]
11TABLE A Analysis of Variance for % MTBE (Two-way analysis of
Variance between Strain Vs Media) Source DF SS MS F P Strain 1
0.318 0.318 2.30 0.140 Media 1 0.250 0.250 1.81 0.189 Interaction 1
0.003 0.003 0.02 0.884 Error 28 3.870 0.138 Total 31 4.441 Strain,
media and the interaction between them did not significantly
affected to % MTBE
[0223]
12TABLE C Analysis of Variance for % MTBE (Two-way analysis of
Variance between Doses vs Media) Source DF SS MS F P Strain 3
2.1335 0.7112 9.96 0.000** Doses 1 0.2503 0.2503 3.50 0.073
Interaction 3 0.3426 0.1142 1.60 0.216 Error 24 1.7144 0.0714 Total
31 4.4409 *Significantly different (P<0.05), Strain
significantly affected to % MTBE
[0224]
13TABLE D Analysis of Variance for % Fatty Acids (Two-way analysis
between Doses and Media) Source DF SS MS F P Dose 3 0.1959 0.0653
2.07 0.131 Media 1 0.0968 0.968 3.06 0.093 Interaction 3 0.1080
0.0360 1.14 0.353 Error 24 0.7587 0.0316 Total 31 1.1594 % FA did
not significantly influenced by doses, media or interaction.
[0225]
14TABLE E Analysis of Variance for % FA (Two-way analysis between
Strain Vs Doses) Source DF SS MS F P Strain 1 0.0242 0.0242 0.70
0.412 Media 3 0.1959 0.0653 1.88 0.160 Interaction 3 0.1056 0.0352
1.01 0.404 Error 24 0.8336 0.0347 Total 31 1.1594
[0226]
15TABLE F Analysis of Variance for % Fatty acids (Two-way analysis
between Strain vs doses) Source DF SS MS F P Strain 1 0.242 0.0242
0.70 0.412 Doses 3 0.1959 0.653 1.88 0.160 Interaction 3 0.1056
0.352 1.01 0.404 Error 24 0.8336 0.347 Total 31 1.1594
[0227]
16TABLE G Analysis of variance for % Resin Acids (Two-way analysis
between Strain and Doses) Source DF SS MS F P Strain 1 0.0957
0.0957 3.60 0.70* Doses 3 0.5150 0.1717 6.45 0.02** Interaction 3
0.0792 0.0264 0.99 0.413 Error 24 0.6386 0.0266 Total 31 1.3286
**Significantly diff (P<0.05), % RA significantly affected by
doses
[0228]
17TABLE H Analysis of Variance for % Resin Acids (Two-way analysis
between Strain Vs Doses) Source DF SS MS F P Strain 1 0.0957 0.0957
2.23 0.147 Doses 1 0.0205 0.0205 0.48 0.495 Interaction 1 0.0088
0.0088 0.20 0.655 Error 28 1.2036 0.0430 Total 31 1.3286
[0229]
18TABLE I Analysis of variance for % Resin Acids (Two-way analysis
between Doses Vs Media) Source DF SS MS F P Doses 3 0.5150 0.1717
7.37 0.001 Media 1 0.0205 0.0205 0.88 0.357** Interaction 3 0.2343
0.0781 3.35 0.036* Error 24 0.5587 0.0233 Total 31 1.3286
**Significantly diff(P<0.5), Media affected to % RA
[0230]
19TABLE J Analysis of variance of % Triglycerides (Two-way analysis
between Doses Vs Media) Source DF SS MS F P Doses 3 0.048838
0.016279 52.09 0.000** Media 1 0.005512 0.005512 17.64 0.000**
Interaction 3 0.007937 0.002646 8.47 0.001** Error 24 0.007500
0.000313 Total 31 0.069788 **Significantly different (P < 0.05),
Doses and media affected to % TG.
[0231]
20TABLE K Analysis of variance of % Triglycerides (Two-way analysis
between Strain and Media) Source DF SS MS F P Strain 1 0.00245
0.00245 1.11 0.300 Media 1 0.00551 0.00551 2.50 0.125 Interaction 1
0.00020 0.00020 0.09 0.765 Error 28 0.06163 0.00220 Total 31
0.06979
[0232]
21TABLE L Analysis of variance of % Triglycerides (Two-way analysis
between Strain vs Doses) Source DF SS MS F P Strain 1 0.002450
0.002450 4.01 0.057* Doses 3 0.048838 0.016279 26.67 0.000**
Interaction 3 0.003850 0.001283 2.10 0.126 Error 24 0.014650
0.000610 Total 31 0.069788 **Significantly diff (P < 0.05),
Doses and strain affected to % TG.
[0233] Definitions and Explanations
[0234] When varying the dose of the fungi, the mill trial fungal
dose was called 1.times. (or one times) and in the lab this was
varied to one-tenth the mill dose ({fraction (1/10)}.times.) or 10
times the mill dose (10.times.).
[0235] % MTBE means the total extractives as removed by methyl
tert-butyl ether (MTBE) from the wood chips. The components of
fatty acids, resin acids and triglycerides were identified on the
gas chromatogram. In Table 1, for the Lab data, the terms "Peptone"
and "YM" are the growth medium that the lab uses to prepare the
fungi. The term "powder" means the freeze-dried powder inocula of
the fungi that was provided to the mill for the 80,000 tonnes wood
chip trial.
[0236] The components of radiata pine extractives that cause pitch
accumulations are not known. Some suspect it might be the fatty
acids and/or resin acids that cause pitch to start to accumulate.
It does not appear to be the overall total extractives or the
triglyceride content, but this has not been proven.
[0237] From Table 11 there are the following conclusions (following
from the statistical analysis of data provided above):
[0238] Lab Results: Total Extractives (All Lab Results are 4 Week,
Fungal Incubation on Chips)
[0239] 1. In the lab, the uninoculated chips of the mill (called
frozen control as these chips were maintained frozen until the
extractives were run on them) had sing total extractives of between
1.97% of oven dry weight of wood. Extractives of 1.97% may not seem
much, but consider that of 1000 tonnes wood chips processed per
day, it could represent 19.7 tonnes of extractive `pitch` (if the
sulfite cook process extracted like the MTBE solvent.)
[0240] 2. In the lab, the best fungal reduction of overall
extractives was to about 0.56% using OF 13 either grown freshly in
peptone or as produced in the powder form for the mill trial. That
represents more than two-thirds reduction of overall extractives by
the fungus OF13. The best reduction for OF40 of overall extractives
in the lab was reduction by little more than a half of overall
extractives by the fungus.
[0241] Lab Results Fatty Acids and Resin Acids
[0242] 3. The best reduction of % fatty acids by fungi was OF40 at
10 times inocula in YM, or OF13 at 1 times inocula in peptone;
0.14% starting control fatty acids were reduced in each case to
0.02% (reduction of 86%).
[0243] 4. The best reduction of % resin acids by fungi was OF13
(with several different doses and medium) and the OF40 and OF13
powder inocula; 0.67% starting control resin acids were reduced to
0.14% (reduction of 80%).
[0244] 5. The best reduction of % triglycerides by fungi was
achieved by both OF13 and OF40 and, 0.15% starting control
triglycerides were reduced to 0.01% (reduction of 94%).
[0245] With both OF40 and OF13 powder, the 1.times. dosage used in
the lab, same dosage as used in the mill, was better in total
extractives, fatty acid, resin acid and triglycerides decrease than
the 10 times, or one-tenth dosage.
[0246] Mill Results; Extractives OF 40 (Mill Results are Generally
10 Weeks Fungal Incubation on Chips)
[0247] For OF40, the 0 week chips had total extractives of 1.94%
and after 2 weeks the total extractives had risen to 2.39%. Since
this type of shift in total extractives makes it difficult to try
to analyse what was happening, it is best to look at FIG. 2 as the
overall trend in the extractives of the chips as a function of
incubation time in the chip piles. In FIG. 2 it is clear that
first, there was reduction of the four extractive parameters, and
second the greatest reduction was after 10 weeks than at any other
time point, though % MBTE Total extractives and % triglycerides
have decreased to their lowest levels by 8 weeks.
[0248] For the chips inoculated with both OF40 & OF13, see FIG.
3, it appears at 6 weeks that there was a peak of "resin"
extractives. Overall again the numbers decrease by the time 10
weeks incubation is achieved, but not as much reduced as with OF13
alone, though very close.
[0249] As the data of the OF13 will not be completed until end of
March we have to wait until then to understand by these parameters
what is the most effective fungal inoculation. Again the caution is
that no one knows the extractive parameters that cause pitch
shutdowns and they may not be one of the four criteria tested in
this analysis.
[0250] The pulps resulting from the OF40 and OF40& OF13
combined inoculated and incubated chips were analysed also for
extractives, given in Table 11 and graphed in FIG. 4 and 5.
[0251] From this data, the OF40 pulp at the Chemiwasher stage still
contains a significant amount of extractives The chips of OF40 at
10 weeks had 1.16% MBTE total extractives, and after the sulfite
cook, the pulp at the Chemiwasher has 0.92% MBTE--in other words
the cook only removed 20% of the total extractives. This pulp
mainly contained resin acids, (20% less resin acids than were in
the chips) and the fatty acids and triglycerides in the pulps were
about the same amounts as present in the chips, but one-seventh and
one-twentieth the amounts respectively as the amount of resin acids
present in the pulps. Resin acid is a bigger component of
extractives in chemiwasher pulp than anything else we tested. Note
that the components of resin acids, fatty acids and triglycerides
are only about half of the total extractives--the other half is not
identified.
[0252] The data of the pulps resulting from the OF40& OF13
combined inoculated chips follows the same trends as the OF 40
pulps, though there is 7% more total extractives in the OF40&
OF13 chemiwasher pulp and 11% less resin acid in the OF40 & OF
13 chemiwasher pulp than the OF40 chemiwasher pulp. Again, it looks
like resin acids are more present in the chemiwasher pulp than the
other components identified, and there is little extractive in the
other pulps.
[0253] The analysis of the OF13 & Of40 pitch deposit showed
that 89% of the pitch deposit was MBTE solvent exactable. Of the
extractables we have identified only about one-third of the
components. The deposit contains three times the amount of fatty
acid as resin acids, and not much triglycerides. That deposited
pitch has 22% fatty acids versus 7% resin acids.
[0254] Lipase Assay on Trial Organisms
[0255] Fungi, in order to colonise wood effectively, have to be
able to secrete specific enzymes to readily metabolise the
essential compounds. One such extracellular enzyme, lipase is
particularly important in this study to understand better the
mechanism of resin degradation. Lipase catalyses the hydrolysis of
triglycerides to glycerol, fatty acids and resin acids. Below is a
brief outline of a lipase assay which uses p-nitrophenyl palmitate
(PNP) as a substrate. An enzyme unit is that amount of enzyme that
will cause release of 1 .mu.mole of PNP per minute under assay
conditions.
[0256] The fungi used in this assay were isolated off the 8 weeks
incubated wood chips, inoculated with either OF40, OF40&13 or
OF13. Overnight cultures of the fungi were standardised to 10.sup.7
cells/ml and inoculated into Peptone and YM media
[0257] Assay
[0258] Substrate p-nitrophenyl palmitate; Assayed at 40.degree. C.
for 20 minutes.
[0259] Cooled on ice and reaction stopped with 0.1 M
Na.sub.2CO.sub.3+10% Triton.
[0260] Absorbance read at 400 nm. Activity:
.DELTA.A.sup.400/0.011.times.1- 0/Time=mU/ml
22 Lipase Activity (mU/ml/10.sup.7 cells) Organism Peptone Media YM
Media 8 weeks samples 467 131 (Mixture of O. floc 40 & O. floc.
13) O. floccosum 40 475 107 O. floccosum 13 365 62
[0261] It can be concluded that these strains of Ophiostoma
floccosum, as isolated of the wood chips, produced a very
significant amount of lipase enzyme, This explains its ability to
invade sapwood so effectively and to be able to use resin as a
nutrient source. The University of Waikato lab is now looking for
other resin degrading enzymes to remove more of the resin
components.
Example 6
[0262] Results
[0263] 1. In the lab, looking at the statistics of the extractives
decrease, strain, media and the interaction between them did not
significantly affected to % MTBE
[0264] 2. Use of fungi at the mill went smoothly with fungi inocula
sprayed evenly to inoculate wood chips as conveyed by the system as
evidenced by the good growth of the albino fungi on chips assayed
from 2 weeks chip pile incubation to 10 weeks incubation.
[0265] 3. The mill had no shutdowns when using the fungal
inoculated chips with less seasoning than routinely done.
[0266] References
[0267] Farrell, R. L., Blanchette, R. A., Brush, T. S., Hadar, Y.,
Iverson, S., Krisa K., Wedler, P. A., Zimmerman, W. (1993). Cartpip
TM: A biopulping product for control of pitch and resin acid
problems in pulp mills. J. Biotechnol. 30: 115-122.
[0268] Farrell R. L., Blanchette, R. A., Brush, T. S., Gysin, B.,
Hadar, Y., Perollaz, J. J., Wendler, P. A., and Zimmerman W.
"Cartapip.RTM." A Biopulping Product for Control of Pitch and Resin
Acid problems in Pulp Mills in Biotechnology in the Pulp and Paper
Industry, Kyoto, Japan Editor: M. Kuwahara and M. Sbimada, Uni
Publishers Co., Ltd., Tokyo, Japan 1992, p. 27-32.
[0269] Hoffmann, G. C., Brush, T. S., Farrell, R. L., "A Biopulping
Product for Control of Pitch and Resin Acid Problems in Pulp and
Paper Products", Naval Stores Review, 102(3) 10-12 (1992).
[0270] Kay, S. J., Farrell, R. L., Hadar, E., Hadar, Y.,
Blanchette, R. A., Harrington, T. C. (1997). Sapstain in New
Zealand--the cause and a potential anti-sapstain solution.
Proceedings on the 11th Biennial Conference of the Australasian
Plant Pathology Society, pp.21.
[0271] Wendler, P. A., Brush, T. S., Iverson, S., Krisa, K.,
Zimmerman, W., and Farrell, R. L., "Biological Control of Pitch
Problems in Paper Mills", Kemia Kemi 19, 262-264 (1992).
[0272] White-McDougall, W. J., Blanchette, R. A., Farrell, R. L.
(1998). Biological control of the stain fungi on Populus
tremuloides using selected Ophiostoma isolates. Hozforschung,
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[0273] Behrendt, C. J., R. A. Blanchette and R. L. Farrell 1995. An
integrated approach, using biological and chemical control, to
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[0274] Behrendt C J, Blanchette, R A and Farrell, R L. (1995)
Biological Control of Blue Stain Fungi in Wood: Investigation of
Fungal Interactions in the Laboratory and Field. Phytopathology 85,
92-97.
[0275] Zimmerman, W. C., Blanchette, R. A., Bumes, T. A., Farrell,
R. L. (1993). Melanin and perithecial development in Ophiostoma
piliferum. Mycologia 87. 857-863.
[0276] Wall, M. B., Stafford, G., Noel, Y., Fritz, A., Iverson, S.,
Farrell, R. (1995) Treatment with O. piliferum Improves Chemical
Pulping Efficiency; 6th International Conference on Biotechnology
in the Pulp and Paper Industry
* * * * *