Extractives as a source of potential wood-protectors for a grafting application Carmen Fernández-Costas 1, Sabrina Palanti 2, Mª Ángeles Sanromán 1, Diego Moldes 1 1 Department of Chemical Engineering. University of Vigo, Vigo, 36310, Spain mcarmenfc@uvigo.es, sanroman@uvigo.es, diego@uvigo.es 2 Preservation and Biodegradation Laboratory, CNR IVALSA, Via Madonna del Piano 10, Sesto Fiorentino, 50019, Florence, Italy palanti@ivalsa.cnr.it
Extractives as a source of potential wood-protectors for a grafting application VIGO FIRENZE
INTRODUCTION GRAFTING: Promising methodology for modification of polymers Polymer Monomer Grafted Polymer Advantage: Covalent bonding of the monomers onto the polymer chain
INTRODUCTION TECHNIQUES OF GRAFTING: Grafting initiated by chemical means Grafting initiated by radiation technique Photochemical grafting Plasma radiation induced grafting Enzymatic grafting
INTRODUCTION ENZYMATIC GRAFTING: An enzyme initiates the chemical/electro chemical grafting reaction Substrates: Diphenols Methoxy-substitued monophenols Aromatic and aliphatic amines LACCASES: Mechanism of action: OH OH O Laccase/O 2 R R R
INTRODUCTION Wood + Functional compounds enzyme Modified wood WOOD MODIFICATION: Functionalization of wood New properties
INTRODUCTION SUBSTRATES: Phenolics compounds from the extractives of wood Extractives: Related with the natural durability of wood High concentration in heartwood Triglycerides, steryl esters, fatty acids, sterols, terpenoids or phenolic compounds Laccase PHENOXY RADICAL OH
INTRODUCTION PHENOXI RADICAL Laccase
INTRODUCTION ENZYMATIC GRAFTING DESIRED: Selective anchoring of the phenolic extractives onto the wood Wood lignin Phenolic Extractives Laccase Wood lignin + + Phenoxy radical Phenoxy radical O Self coupling reactions Further oxidations Wood lignin? Wood with fungal resistance
AIM OF THE WORK To modify wood surface through an enzymatic grafting and introduce new functional groups from natural sources in order to confer it biocidal activity against fungi
MATERIALS & METHODS OBTENTION OF THE WOOD EXTRACTS: Eucalyptus globulus Milling/Meshing of the wood Pinus pinaster Pinus radiata 40-120 mesh Cryptomeria japonica Soxhlet extraction/concentration
MATERIALS & METHODS ASSESSMENT OF INHIBITION OF THE FUNGAL GROWTH : Impregnation of the extracts in paper disks FUNGI INOCULATION: Coniophora puteana Trametes versicolor T. versicolor C.puteana
MATERIALS & METHODS ENZYMATIC GRAFTING OF EXTRACTIVES: Impregnation of mini-blocks of Pinussylvestris (EN113) Aqueous solutions with extracts at 20% (V/V) Enzyme dosage: 50 U/g (odw) Assessment of the grafting (EN84) Aqueous leaching procedure 14 days, 9 water changes
MATERIALS & METHODS ACCELERATED TEST AGAINST FUNGAL DECAY: C. puteana 8 weeks 22 ºC, 70% RH Effectiveness of the grafting treatment against wood destroying basidiomycetes was evaluated in term of mass loss (%)
RESULTS & DISCUSSION ASSESSMENT OF INHIBITION OF THE FUNGAL GROWTH : TOLUENE EXTRACT ETHANOL EXTRACT Control P. pinaster P. radiata C. japonica E. globulus Both Pinus spp. have shown good biocidal activity in in vitro assays against C. puteana
RESULTS & DISCUSSION ASSESSMENT OF INHIBITION OF THE FUNGAL GROWTH : TOLUENE EXTRACT ETHANOL EXTRACT Control P. pinaster P. radiata C. japonica E. globulus Both Pinus spp. have shown certain degree of biocidal activity in in vitro assays against T. versicolor
RESULTS & DISCUSSION ACCELERATED TEST AGAINST FUNGAL DECAY: Probes NOT submitted to washing procedure Buffer has shown to blocked the fungus activity CONTROL 1 (Buffer) CONTROL 2 (Acetone+ Buffer) CONTROL 3 (Acetone+ Buffer+ Enzyme) Treatment 1 (P. pinaster) Treatment 2 (P.radiata) Treatment 3 (C. japonica) Treatment 4 (E. globulus) Final moisture (%) Mass loss (%) Standard deviation (Mass loss) N (replicates) 174 5.0 0.6 4 207 5.2 2.4 4 164 3.7 0.8 4 175 3.3 0.9 4 170 4.8 1.8 4 181 7.1 2.4 4 185 5.1 1.9 4
RESULTS & DISCUSSION ACCELERATED TEST AGAINST FUNGAL DECAY: Probes treated with toluene extracts and submitted to the washing procedure Both treatments with Pinus spp. have shown to increase the wood durability against C. puteana CONTROL 1 (Buffer) CONTROL 2 (Acetone+ Buffer) CONTROL 3 (Acetone+ Buffer+ Enzyme) Treatment 1 (P. pinaster) Treatment 2 (P.radiata) Treatment 3 (C. japonica) Treatment 4 (E. globulus) Final moisture (%) Mass loss (%) Standard deviation (Mass loss) N (replicates) 85 41.8 5.6 3 89 33.1 20.5 2 121 56.5 0.8 2 56 3.8 0.1 3 85 8.4 0.2 2 116 54.6 10.2 2 82 44.3 7.6 3
RESULTS & DISCUSSION ACCELERATED TEST AGAINST FUNGAL DECAY: Probes treated with ethanolic extracts and submitted to the washing procedure For ethanolic extracts, only the treatment with C. japonica has demonstrated to be effective against fungal decay CONTROL 4 (Ethanol+ Buffer) CONTROL 5 (Ethanol+ Buffer+ Enzyme) Treatment 1 (P. pinaster) Treatment 2 (P.radiata) Treatment 3 (C. japonica) Treatmnet 4 (E. globulus) Final moisture (%) Mass loss (%) Standard deviation (Mass loss) N (replicates) 86 50.1 4.6 3 85 52.4 1.0 2 95 54.6 0.0 1 95 26.8 5.0 2 37 3.9 0.3 2 94 62.1 0.0 1
CONCLUSIONS P. pinaster and P. radiata have resulted to contain extractives with biocidal activity against fungal decay Enzymatic grafting of extractives was successfully achieved onto miniblocks of P. sylvestris Enzymatic treatments with the toluene extracts from P. pinaster and P. radiata and the ethanolic extract from C. japonica seem to be effective wood treatments against C.puteana
FUTURE PERSPECTIVES Identification of the phenolic extractives responsible of the biocidal activity would be crucial to explain the new properties conferred to wood Optimization of the enzymatic grafting conditions will be the next step in order to achieve optimal results Treatments should must be checked with the standard EN 113
Acknowledgements: This research has been financially supported by the Xunta de Galicia and by ERDF Funds (Projects EM2014/041 and GRC2013/003). C. Fernández-Costas is grateful to Universidade de Vigo by the financial support of the stay in the CNR-IVALSA.