Ascomycetes, Basidiomycetes and Zygomycetes: Fungal Treatment for Commercial Applications in the Pul

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• Ascomycetes, Basidiomycetes and Zygomycetes
  Fungal Treatment for Commercial Applications in
  the Pulp and Paper and Solid Wood Industries

Roberta L. Farrell
Department of Biological Sciences, University of
Waikato, Private Bag 3105, Hamilton, New Zealand.
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Outline of IAWS presentation

• Why fungi, versus enzymes etc?
• Basidiomycetes - Fungal pre-treatment of wood
  chips for biopulping
• Basidiomycetes Zygomycetes - Bioremediation
  Current Research on Eucalyptus with Fungi
• Ascomycetes - Fungal pre-treatment of wood chips
  and solid wood for pitch removal, biocontrol,
  biopulping
• Challenges, critical variables, conclusions

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Why Fungi versus Enzymes?

• Fungi alter wood constituents cell architecture
• Different fungi have different resultant effects
  key point is the application and colonisation of
  the fungi and selectivity of effect i.e.
  delignification, extractives removal
• Fungi can produce unique results for processed
  fibres with reduced environmental impact

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Enzymes

• Enzymes protein catalysts, do not penetrate wood
  cells. More interesting to pulp or fibres acting
  on surfaces.
• Application Concerns
• Extent of Activity should be greater than the
  mill fluctuations i.e. gt20 effect.
• Range of Activity pH, temperature, salt ranges
  of activity important i.e. an enzyme that has
  activity between pH 6-8 is not effective at pH9.5
• Commercial
• Xylanase prebleaching
• Lipases triglycerides hydrolysis
• Amylases drainage
• Not Commercial
• Laccases, ligninases, Mn dependent peroxidases
• Cellulases fibre modification

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Select fungus with right activities to achieve
required aims

• Ascomycetes - Primary Coloniser little or no
  preparation of wood
• Pitch removal with biopulping effects
• Ophiostoma sp. (Cartapip)
• Rapid colonisers, penetrate rays resin canals,
• degrade extractives
• Fibre bonding modifications
• Basidiomycetes - Secondary Coloniser wood
  sterilisation required
• Classic biopulping and bioremediation
  applications
• Phanerochaete chyrosporium Ceraporiopsis
  subvermispora
• lignin removal and/or modification
• Pitch removal
• Fibre and cellular modifications
• Zygomycetes - Secondary Coloniser wood
  sterilisation required
• Bioremediation applications
• Soil inhabiting

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Basidiomycetes - Classic biopulping and
bioremediation applications

• In 1950s, Westvaco began biopulping studies, to
  find fungi that could be applied to logs and in
  transit would initiate pulping by
  delignification.
• In 1970s, Eriksson and colleagues at STFI
  continued work but had 2 problems
• Having the fungus effectively grow on wood chips
  without complicated pre-treatment
• Cellulase activity and loss of yield and strength
  of fibre

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Basidiomycetes - Classic biopulping and
bioremediation applications

• In 1987, Kent Kirk and colleagues in
  collaboration with Bob Blanchette, with the
  Biopulping Consortium achieved most significant
  breakthroughs with Basidiomycetes, particularly
  Ceraporiopsis subvermispora , and continuing with
  Masood Akhtar but had 1 problem
• Having the fungus effectively grow on wood chips
  without complicated pre-treatment.
• In 2002, in New Zealand Biomechanical Pulping
  Consortium with Papro/ensis, University of
  Waikato, Norske Skog and PanPac

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Basidiomycetes - Classic biopulping and
bioremediation applications Lignin degrading
fungi can degrade pesticides
PCP
PCP
Dioxin
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Basidiomycetes - Classic biopulping and
bioremediation applications

• Rich Lamar and Rich White of EarthFax Development
  Corp has shown effective degradation of PCP and
  dioxins with Basidiomycetes Phanerochaete sordida
  and Pleurotus sp.

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Bioremediation fungus in New Zealand Zygomycetes

• Gongronella (Absidia) butleri
• Isolated from PCP contaminated soil in Whakatane,
  New Zealand
• Inoculum grown in hyphal mats in liquid medium
• Prior to field use inoculated onto intermediate
  substrate mushroom spawn
• Grows well on sterilised Pinus radiata chips

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Bench scale bioremediation

• Soils had high levels of boron
• Dioxin/furan congeners also decreased to
  lt 10 ppb

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Biopiles applying lab knowledge to industrial
application

• First, obtain good growth B101 fungus from
  mushroom spawn onto wood chips
• Second, mix fungus on wood chips with
  contaminated soil
• Third, ensure biopile provides favourable
  environment for fungus and enzymes to work
• Fourth, fate of bioremediated soil

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Biopile results 50 and 800 tonne biopiles
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Ascomycetes - Primary Coloniser little or no
preparation of wood. Pitch removal with
biopulping effects Ophiostoma sp. (Cartapip)

• Investigated brightness loss and resin (pitch)
  decrease in loblolly pine at request of TMP mill
  in USA.
• Identified fungus, O. piliferum, responsible
  for resin decrease but in native form also
  caused stain.
• Fungus had good biotech product characteristics
  if we could eliminate stain
• - Rapid growth when applied to chips at mill
• - Significant reduction of extractives in 2
  weeks
• - Ferment and process to stable inoculum
  product
• Started classical mating to select for albino

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Culture O. piliferum
Sapstained pine
Isolate ascospores in perithecial stalk
Albino O. piliferum
Classical breeding
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Past Practices - Seasoning of wood chips

• Seasoning practiced for decades, holding logs or
  wood chips for weeks for natural organisms to
  reduce pitch/resin components.
• Disadvantages
• non-uniformity,
• brightness loss,
• yield loss.

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Wood Treatment with Albino Ophiostoma improves
seasoning

• Albino Ophiostoma sp. used for acceleration of
  seasoning (resin decrease).
• Resulting in maintenance of brightness levels in
  transportation and storage of wood chips prior to
  pulping.

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Ophiostoma piliferum growing
on loblolly pine wood chip
enzymes (lipases to catalyse hydrolysis
triglycerides oxidases to catalyse oxidation
of sterols, resin fatty acids etc.)
SEM
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Requires 7- 14 days chip storage or Treat logs
prior to chipping.
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Anti-sapstain Project Goalsin New Zealand

• Project started 1996 in NZ with
• Bob Blanchette, Univ of Minnesota,
• Tom Harrington, Iowa State Univ,
• Yitzhak Hadar, Hebrew Univ of Jerusalem
• Survey and identify sapstain organisms
• Identify major causes sapstain radiata pine
• Develop albino fungus anti-sapstain
• product and diagnostics

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More than One Fungus

• Important - there are several fungal isolates
  developed with varied ability to decrease pitch
  and improve brightness maybe unfortunately only
  one was marketed 1990-2004.
• In NZ, Australia, Asia we have different
  Ophiostoma species, degrade pitch and biocontrol.
• Ophiostoma piliferum, O. floccosum, O. piceae,
  O. pluruanulatum, O.querci

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Biocontrol with Albino Fungi
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Albino strains of O. pluriannulatum, O sp E,O.
piceae and O. floccosum growing on radiata pine
Held, B.W., Thwaites, J.M., Farrell, R.L.,
Blanchette, R.A. (2003). Albino Strains of
Ophiostoma Species for Biological Control of
Sapstaining Fungi. Holzforschung, 57, 237-242.
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NZ Albinos on Radiata pineField Study after 6
months
Target 0 stain, 2000
2005 - Target 5? in 4 months
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Albino fungi strains
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Exportation of Ophiostomatoid Fungi Japan
Peeler Trials followed colonisation of blue
stain fungi in radiata pine from harvest, to NZ
port, to ship (dataloggers), to port. Dominating
NZ fungi on logs exported to Japan O. piceae,
O. quercus, O. floccosum, O. setosum 6 more
species
Thwaites, J.M., Farrell, R.L., Hata, K., Carter,
P., Lausberg, M. (2004). Sapstain fungi on Pinus
radiata logs from New Zealand Forest to Export
Destination in Japan. Journal of Wood Science
50 459-465.
Eucalyptus globulus Brightness Improvement
Project 2005 Aim Produce unbleached kraft pulp
with improved brightness and reduced pitch.
Decrease in brightness caused by sapstaining
fungi growing on wood chips prior and during
shipping from Australia to Asia.
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• Export Eucalyptus wood chips E1 (higher pulp
  yield grade) and E2 grade

E2 Grade
E1 Grade
Staining Ophiostoma species (O. quercus, O.
pluriannulatum, lots others) colonising
Eucalyptus E1 E2 grades
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After 2 weeks inoculation
Presently testing Eucalyptus globulus and mixed
species from Western Australia.
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Fungal Inoculation Methodology

• Wood
• Eucalyptus camaldulensis harvested from Kukeng
  plantation of Taiwan Forestry Research Institute.
  
• Fungal treatment of wood chips
• Cultures of individual fungal strains added to
  wood chips and incubated at 25? for 2 weeks.
• Solvent extraction of wood chips
• Wood chips extracted with acetone for 48 hr,
  three times. After evaporating solvent to
  dryness, amounts of extractives determined.
• Lipophilic extractives fractionated from
  chloroform soluble fractions of acetone
  extractives.
• Extractives and lipophilic fractions obtained
  were examined GC and GC-MS.

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Albino Fungi Path Forward

• Research
• Enhancement of effect classical genetics or GE
• Understand full effects in Pulping
• Define Impact
• Wood Cellular architecture
• Wood chemistry
• Nutrients, toxins
• Diffusible metabolites
• Fungi Bacteria Synergism/inhibitory
• Commercial success
• Efficacy
• Cost
• Shelf life
• Registration

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Challenges and Critical Variables

• Inoculation spray applications
• Maintenance of fungus on wood chips and/or soil
• Length of time to incubate chips
• Our Current Research
• Genetic transformation of Ophiostoma sp to
  achieve selective lignin and extractives
  degradation when fungus applied to wood.
• Use of Basidiomycetes, Zygomycetes, Ascomycetes
  for treatment of wood, hemp, NZ flax for
  biocomposites.
• Use of fungi and/or enzymes for treating NZ flax
  for haute couture high fashion!

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In the year 2100

• Harvest trees in the forest while harvesting
  spray with GE fungus that modifies lignin and
  removes extractives in 4 days such that when logs
  arrive at mill they are pre-pulped.
• Fungi and/or enzymes used
• to selectively remove hemicelluloses and/or
  lignin to obtain cellulose fibres to strengthen
  plastics or contains, improve adhesion or the
  long term bondability of high density hardwoods

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Acknowledgements

• NZ Foundation for Research Science Technology

K. Hata, S. Yamanami
R. Blanchette, B. Held T. Harrington, D.
McNew, R. Lamar, R. White
Y. Su, E. Wang, C. Ho
Y. Hadar, E. Hadar
S. Duncan, A. Ram, S.Reay L. Robson, S. Kay,
A.Schirp P. Cooper, CHH, FCF
K021
J. Navarette
M. Wingfield