Non-enzymatic Deconstruction Systems in the Brown Rot Fungi

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Moses Strickland
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Wood Science and Technology, 2014 International

Valdeir Arantes 2, Jody Jellison 3, Steve Kelly

ICTAS II Building, Virginia Tech, Blacksburg,

USA 2 Department of Wood Science, Faculty of

1 Non-enzymatic Deconstruction Systems in the Brown Rot Fungi presented to the Society of Wood Science and Technology, 2014 International Convention Zvolen, Slovakia by Barry Goodell 1, Valdeir Arantes 2, Jody Jellison 3, Steve Kelly 4 1 Department of Sustainable Biomaterials, 216 ICTAS II Building, Virginia Tech, Blacksburg, Virginia. USA 2 Department of Wood Science, Faculty of Forestry, University of British Columbia. Vancouver. CANADA 3 VAES and the Department of Plant Pathology, Physiology and Weed Science, Blacksburg, Virginia. USA 4 Department of Forest Biomaterials, North Carolina State University, Raleigh, North Carolina. USA Courtesy G. Daniel, SLU

2 Lignocellulose biodegradation Today s topic, which will cover in-part, this mechanism.

3 Background - Basidomycota Fungal Degradation of Lignocellulose Relating fungal biodegradation of woody biomass, and climate change. A trip back into ancient history. Returning carbon to the earth, and carbon cycling CO 2 CO 2CO2 CO 2

4 400 Million Years Ago Approximately 4,000 PPM CO 2 No Trees!!

5 In Geologic Time, when did woody plants begin to form? During the late Devonian million years ago. Plants start to make lignin (lignin + cellulose = wood)

theresilientearth.com/?q=content/grand-view-4-billion-years-climate-change Floudas, D. et al. 2012. Science.

6 The production of large amounts of biomass, that could not decay pulled large amounts of carbon out of the air, driving down CO 2 levels. Essentially reducing CO 2 levels to that which we have today (slightly below 400 PPM and rising). Floudas, D. et al Science. Supported Theory: It took approximately 200 million years after plants began to produce lignin, for fungi to develop different mechanisms for degrading it.

7 What happened to all that biomass? For 200 million years it grew and collected, and grew more and collected. Ultimately getting buried under more biomass. Once buried, it slowly carbonized and formed deep seams of what we now know as COAL.

Basidiomycota Decay Fungi Background Brown Rot Only 6% of all wood-decay fungi 80% of

Lignin is also extensively attacked, modified and repolymerized.

These fungi degrade cellulose initially via a non-enzymatic oxidative process.

cellulose, hemicellulose and lignin at equal rates Selective White Rot fungi traditionally

But it is now known that most also attack at least some cellulose.

8 Basidiomycota Decay Fungi Background Brown Rot Only 6% of all wood-decay fungi 80% of these occur on conifers Degrade cellulose and hemicellulose. Lignin is also extensively attacked, modified and repolymerized. Brown rotted lignin is a major component of humic material in forest soils! These fungi degrade cellulose initially via a non-enzymatic oxidative process. White Rot Largest group of wood decay fungi Simultaneous White Rot fungi degrade cellulose, hemicellulose and lignin at equal rates Selective White Rot fungi traditionally were thought to remove lignin and hemicellulose preferentially. But it is now known that most also attack at least some cellulose. Laccase, Lignin peroxidase, and Mn peroxidase are involved in lignin degradation in different white rot fungi This is News (to many researchers), as it is a unique process in all of biological systems in nature that we have discovered.

In addition, low molecular weight metabolites (iron-reducing catechols iron reducing chelators ) contribute to important nonenzymatic processes to breakdown and modify the lignocellulose via Fenton

9 In addition, low molecular weight metabolites (iron-reducing catechols iron reducing chelators ) contribute to important nonenzymatic processes to breakdown and modify the lignocellulose via Fenton chemistry. Characteristics of brown rots Types of enzymes involved in cellulose degradation: Endoglucanases: such as endo ß-1,4- glucanase. These enzymes attack cellulose randomly along the middle of the cellulose chain. ß-glucosidases: Glucosidases in the brown rots typically act on cellobiose to convert it into glucose

10 The Fenton Reaction The basis for non-enzymatic degradation. But only one component of a complex system. Fe II + H 2 O 2 OH + OH - Hydroxyl Radical 11

11 Fiber Cell Wall OH Fe III H 2 O 2 + Fe II High ph (5.5) + Chemical, biological, catechol Fungal and spatial Chelator/ Catechol relationships between Oxidized Fungal fungal hyphae and the Chelator fiber cell wall in the brown rot degradation process. Fungal Chelator / FeIII complex Fungal Chelator: dimethoxy When released within the fiber cell wall, Fe(III) binds to cellulose where fungal chelators can reduce it again to promote cellulose degradation. Fiber Cell Lumen Low ph (2.0) Fe III oxalate Fe oxy(hydr)oxides Fungal Hyphae x-section Oxalate secretion (crystals) + Extracellular matrix Iron (III) oxy(hydr)oxides in the environment are solubilized and sequestered by oxalate. When oxalate-bound iron moves to higher ph locations outside the cell lumen, iron can then be pulled from oxalate by fungal iron-reducing chelators.

Fungal extracellular matrix partially peeled away from the wood cell

12 Oxalate Crystal embedded in fungal extracellular matrix Atomic Force Microscopy of Oxalate Crystal Embedded in Fungal Extracellular Slime. Fungal extracellular matrix partially peeled away from the wood cell wall when the sample was split open. Bordered pit in cell wall of spruce wood

We wanted to explore the relationship between enzymatic and non-enzymatic attack on cellulose We set up an experiment where we treated ground wood with: 1) Cellulolytic degrading enzymes 2) Mediated

13 We wanted to explore the relationship between enzymatic and non-enzymatic attack on cellulose We set up an experiment where we treated ground wood with: 1) Cellulolytic degrading enzymes 2) Mediated Fenton 3) Fenton alone 4) Fenton and Cellulolytic enzymes Then, we analyzed using: A) Pyrolysis Mass-Beam, Mass Spectrometry to look for specific lignin and cellulose fragmentation patterns that would occur if both of these components were being deconstructed. B) And 13 C- tetramethylammonium hydroxide (TMAH) thermochemolysis to look specifically for lignin fragments generated during the brown rot decay process.

14 The Chelator mediated Fenton system (CMF) changes the carbohydrate and lignin chemistry of the wood, mimicking natural degradative processes. PCA score plot of the py-mbms spectra for biomimetic samples: 2 = Enzyme only treatment 8 = Mediated Fenton treatment 11 = Enzyme plus Fenton 14 = Fenton plus enzyme The enzyme treatment (#2s) shows a reduction in carbohydrate signal, suggesting that the Fenton systems are more in the liberation of low MW cellulosics as opposed to enzyme treatments.

15 13 C-TMAH assessment of lignin oxidation Spruce wood residue before (control), and after Fenton, and Chelator-mediated Fenton (CMF) treatment. [Changes in G4 (3,4-dimethoxybenzoic acid) indicate alteration of ß-O-4 linkages in lignin.] RESULTS: Nothing happens!! at least to the Wood residue

16 13 C-TMAH assessment of lignin oxidation: Supernatant before (control), and after treatment, with Fenton, or CMFS Note the change in G4 (3,4-dimethoxybenzoic acid) which is associated with alteration of ß-O-4 linkages in lignin. This decrease was not seen in the Fenton or CMFS-treated wood residues, only in the supernatants from Fenton and CMFS treatments.

SUMMARY Proposed mechanism employed by brown rots Data for deconstruction of both holocellulose and lignin continue to support the important role of a non-enzymatic, chelator-mediated

Additional research is still needed to assess: 1) oxalate concentrations at the nanoscale, and also 2) ph at the nanoscale in the vicinity of the fungal hyphae, the extracellular

17 SUMMARY Proposed mechanism employed by brown rots Data for deconstruction of both holocellulose and lignin continue to support the important role of a non-enzymatic, chelator-mediated Fenton (CFM) mechanism in 3 Orders of brown rot fungi. Additional research is still needed to assess: 1) oxalate concentrations at the nanoscale, and also 2) ph at the nanoscale in the vicinity of the fungal hyphae, the extracellular glycoprotein matrix, and within the wood cell wall Research on these aspects will allow further refinement of the brown rot model, and particularly in enhancing our understanding of the mechanism which permits the generation of oxygen radicals deep within the wood cell wall, and away from the fungal hyphae, to avoid damage to the fungus.

18 -- Thank You -- QUESTIONS??

Oxidative Degradation of Wood by Brown-Rot Fungi

Active Oxygen/Oxidative Stress and Plant Metabolism, E Pell, K Steffen, eds, 1991, American Society of Plant Physiologists Oxidative Degradation of Wood by Brown-Rot Fungi Barbara L. Illman Forest Products