enzymatic grafting of wood extractives - American Chemical Society

Research Article pubs.acs.org/journal/ascecg

A Sustainable Treatment for Wood Preservation: Enzymatic Grafting of Wood Extractives Carmen Fernández-Costas,† Sabrina Palanti,‡ Jean-Paul Charpentier,§,∥ María Á ngeles Sanromán,† and Diego Moldes*,† †

Department of Chemical Engineering, University of Vigo, Vigo, Spain CNR IVALSA (Istituto per la Valorizzazione del Legno e delle Specie Arboree), Via Madonna del Piano 10, I-50019 Sesto F.no (Fi), Italy § AGPF, INRA, 45075 Orléans, France ∥ GénoBois, Wood Analysis Platform, INRA, 45075 Orléans, France ‡

S Supporting Information *

ABSTRACT: Natural extractives are proposed as potential sustainable bioactive compounds for wood preservative formulations, and an enzyme-mediated reaction is employed to fix them onto the wood surface. This environmentally friendly process takes place thanks to the activity of the laccase enzyme and leads to the formation of covalent bonds between these compounds and the wood itself. This then diminishes the problem of its future leaching, giving rise to long-lasting wood protection treatments. Overall, our data show suitable biocidal properties for the raw extracts of Pinus spp. and Criptomeria japonica. Phenolic compounds such as pinosylvin, pinosylvin monomethyl ether, pinocembrin, naringenin, and pinobanksin were found in the extracts of pine together with totarol and sugiol in the C. japonica extract and could explain the antifungal properties found. Fungal tests have revealed mass losses below 7% for wood treated with the toluene extracts of Pinus spp. and the ethanolic extract of C. japonica, pointing to this enzymatic green methodology as a promising alternative in the wood preservation field. Therefore, potential inclusion of these enzymatic strategies in the wood industry today is discussed and analyzed in a “green” scenario. KEYWORDS: Antifungal, Enzymatic grafting, Laccase, Leaching, Wood decay, Wood extractives

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these natural biocides.10 Arantes and Goodell11 have suggested that nonenzymatic oxidative free radical mechanisms could be involved in degradation processes, and therefore, it is reasonable to conclude that free radical scavengers could hamper fungal decay. Indeed, a study by Schultz and Nicholas12 has provided evidence of the synergistic effect of the combination of both an antioxidant and a biocide. Therefore, the antioxidant properties of heartwood extractives would also prevent free radical formation and inhibit the degradative process of microorganisms. Then, these compounds related with the natural durability in wood could be employed in the formulation of biocides in the context of the wood protection industry. However, several drawbacks are pointed out to deploy natural products for wood protection: the retention of biocides, their susceptibility for biodegradation, and the penetration efficacy.5

INTRODUCTION Wood preservation has been a concern for mankind since timber was first used for structural purposes. The increasing use of wood as a building material has implied an increasing interest in wood decay and the development of numerous strategies for the preservation of the wood against abiotic agents (e.g., chemicals, sun radiation, and fire) and biotic agents (e.g., bacteria, algae, fungi, insects, and shipworms). At present, legislation on health and environmental risks is constantly restricting substances in wood preservative formulations, which means that eco-friendly, long-lasting, and more benign treatments are desired. In fact, there is growing consideration of the use of natural products for wood protection.1−4 Particularly, natural compounds such as plant extracts, essential oils, heartwood extractives, waxes, resins, and tannins from bark seem to show good prospects for wood protection.5−8 Extractives are nonstructural wood constituents that could be obtained with organic solvents. It has been shown that wood durability has a correlation with the extractive content, although other factors could confer resistance to decay.9 Specifically, antioxidants could be responsible for enhancing the efficacy of © 2017 American Chemical Society

Received: March 7, 2017 Revised: June 27, 2017 Published: August 1, 2017 7557

DOI: 10.1021/acssuschemeng.7b00714 ACS Sustainable Chem. Eng. 2017, 5, 7557−7567

Research Article

ACS Sustainable Chemistry & Engineering

concentrated up to a final volume of 10 mL, and the recovered solvent was recycled and employed for subsequent extractions. General Procedure for Laccase-Assisted Impregnation of Wood. Wood blocks (30 mm × 10 mm × 5 mm) from sapwood of Scots pine (Pinus sylvestris) were impregnated following the enzymatic strategy presented in this study. Wood blocks were prepared after elimination of heartwood and defects and were cut in accordance with specifications of EN 113 European standard.22 Samples were weighed after oven-drying at 103 °C and reconditioned at 20 °C and 65% relative humidity until impregnation. Then, impregnation of sets of eight wood samples was performed in accordance with the EN 113. The treating solution consists of a mixture of 7 mL of the wood extract and 50 U g−1 of laccase (on dry wood basis) brought to a final volume of 35 mL with a 0.1 M phosphate buffer at pH 7 (i.e., 20% content of organic solvent). The toluene extracts were evaporated and redissolved in acetone in order to maintain a suitable enzymatic activity. After impregnation, the chamber was slowly brought back to atmospheric pressure, and samples were left for 2 h to finish the impregnation. In this last step, a temperature of 50 °C was maintained in order to boost enzymatic grafting (as the enzyme employed is a thermophilic one). Later, samples were weighed after impregnation and left to air-dry. Probes with an absorption which differed by more than 15% from the mean absorption were rejected. Then, they were weighed once more after oven-drying at 103 °C. Weight Percent Gain (WPG1) was used to assess the efficacy of the impregnation process. Samples were weighed before and after impregnation, and treating solution absorption was determined as follows:

In this work, enzymatic grafting is proposed as a strategy to overcome some of these limitations. Enzymatic grafting is based on the covalent anchoring of target compounds onto a desired substrate by an enzyme-based reaction.13 Laccases (diphenoloxidases, EC 1.10.3.2) are the preferred enzymes employed in these applications as they catalyze the oxidation of phenols and amines to their corresponding phenoxy and amino radicals.14,15 Laccases could activate both the phenolic groups from lignin and the desired target compounds and lead the target compound to be grafted onto wood. It is an environmentally friendly and simple treatment and operates under mild conditions with the advantage of allowing a covalent fixation of the substrates on the wood surface. As a consequence, leaching is avoided and long-lasting treatments are obtained. Despite its promising potential,16 there are scarce applications of enzymatic grafting for protecting wood. The work of Rättö et al.17 should be noted as it has proved the efficacy of this strategy to anchor two model extractives (vanillin and tannin) onto wood to develop sustainable preservatives. Similarly, Widsten et al.18 have used the enzymatic functionalization of lignocellulosic materials to improve their antibacterial properties. Recently, a residue of the paper industry, kraft lignin, was employed successfully to optimize the enzymatic grafting and to develop wood bioprotection strategies in combination with copper salts.19,20 Also, extractives such as condensed tannins from pine bark were also modified by a laccased-catalyzed strategy.21 In this work, the extraction of wood from several commercial species followed by the enzymatic grafting of the obtained extractives onto wood mini-blocks is proposed as a new approach for wood protection against fungi. Considering the specificity of the enzyme employed, laccase, for phenols and amines, special attention was paid to the chemical characterization of the extracts. Finally, a critical analysis of this strategy was conducted together with a consideration for feasible perspectives of the introduction of this type of methodology in the wood industry.

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WPG1 (%) = (m1 − m0)/(m0)× 100

(1)

where m1 and m0 are the oven-dried masses (103 °C, 24 h) of the treated and untreated wood samples, respectively. Accelerated Aging of Treated Wood. A set of wood samples treated with the proposed enzymatic strategy was subjected to a leaching procedure following the EN 84 standard.23 Wood probes were submerged in distilled water. and the latter was changed at intervals of at least 1 day and no more than 3 days. Finally, samples were conditioned, oven-dried at 103 °C, and finally weighed to calculate weight percent gain after leaching (WPG2) as in eq 1 but with the oven-dried weights of the treated, leached wood specimens. Antifungal Activity of the Extracts. Antifungal activity of the concentrated extracts was evaluated on growth of the brown-rot fungus Coniophora puteana (Schumacher ex Fries) Karsten (BAM Ebw 15) and the white-rot fungus Coriolus versicolor (Linnaeus) Quélet (CTB 863A). The strains were supplied by the Spanish Type Collection (CECT) and maintained under the recommended conditions of the standard EN 113 (reisolated every 6 months and their virulence was checked in every assay). A round, paper disk filter of 1.5 cm in diameter, previously sterilized by autoclave, was impregnated with 50 μL of the corresponding concentrated extract and then placed in the center of an agar plate with agar:malt (20:40) medium. Finally, two plugs of pregrown fungi were inoculated on the plate on opposite sides of the impregnated paper disk. Later, cultures were incubated at 22 °C and 70% relative humidity in triplicate. The growth response of the fungi was observed and compared with the control plate. Accelerated Efficacy Tests against Wood-Decay Fungi. An accelerated test based on the standard EN 113 test24 and following the methodology proposed by Bravery25 was carried out to check wood durability after treatment. Treated samples were sterilized by a tyndallization process in order to protect the wood structure from a more aggressive treatment. Samples were submitted to a boiling temperature (99−100 °C) for 20 min in an autoclave with the vapor faucet open (i.e., sterilization on fluent vapor). Then, they were left overnight in the closed autoclave, and finally, a cycle of 10 min at 99− 100 °C was applied. C. puteana was employed to evaluate the wood treatment effectiveness on softwood (samples of pine). Two pieces of the fungi were previously grown on Petri dishes with an agar:malt medium (25:40). Then, when the mycelium had reached the edge of the Petri dish, sterilized wood samples were placed in the dishes on a

EXPERIMENTAL SECTION

Materials. Heartwood from Eucalyptus globulus, Pinus pinaster, and Pinus radiata, species of great commercial importance in the Northwest of Spain, was used to obtain extracts. E. globulus was collected from plantations in Vigo (Pontevedra, Spain). P. pinaster and P. radiata were felled from 28-year-old plantations in Pontecaldelas (Pontevedra, Spain). Samples were left to air-dry for five months, and then, blocks of heartwood were obtained. Pith and knotty wood were avoided. Furthermore, prunings of Cryptomeria japonica were supplied by the Fundación Sales (Vigo, Spain). Finally, all samples were milled in a Brabender laboratory mill (Duisburg, Germany), and a fraction of 40−120 mesh was selected and stored in a cool dry place. Laccase NS51003 from Myceliophthora thermophila was provided by Novozymes (Bagsvaerd, Denmark). Laccase activity was determined spectrophotometrically by monitoring the oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate at pH 7 (phosphate buffer, 0.1 M) and room temperature. One activity unit (U) was defined as the amount of enzyme that oxidized 1 μmol of ABTS per min (ε436 = 29300 M−1 cm−1). Organic solvents (toluene, acetone, and ethanol) of reagent grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). Wood Extraction. An amount of 15 g of milled wood was subjected to 50 cycles of solvent extraction on a Soxhlet extractor. Two different extractions were carried out, one with pure toluene and other with pure ethanol, for comparison reasons. Extraction was accomplished with 150 mL of solvent. Finally, extracts were 7558


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Figure 1. Fungal growth of Coniophora puteana and Trametes versicolor in the presence of disks impregnated with the ethanolic and toluene extracts of Pinus pinaster, Pinus radiata, Cryptomeria japonica, and Eucalyptus globulus. and diluted 5-fold with methanol. An aliquot of 15 μL was injected and separated using a 250 mm × 4 mm LiChrospher 100RP-18e column (5 μm) stabilized at 40 °C at a flow rate of 1 mL min−1 with the following linear elution gradient: initial conditions: 65% solvent A (acetic acid 1% in ultrapure water) in solvent B (methanol/ acetonitrile, 50:50); 0−5 min: 65% to 40%; 5−13 min: 40% to 20%; 13−28 min: 20% to 0%; 28−35 min: 0% to 65%. Compounds were characterized by their elution time and their UV absorption spectrum at 280 and 340 nm in comparison with standards (caffeic acid, cathechin, chlorogenic acid, coniferin, coniferyl alcohol, ellagic acid, ferulic acid, gallic acid, kaempferol, pinosylvin (PS), pinosylvin monomethyl ether (PSM), pinocembrin (PC), taxifolin, vanillic acid, vanillin, and totarol). Additionally, compounds were also identified with an HPLC−MS (Hewlett-Packard 5989B) equipped with a column Luna 5 μ C18 100A and an electrospray ionization (ESI) ion source. The samples were filtered through a 0.45 μm PTFE filter before injection. A flow rate of 0.4 mL min−1 was maintained during the separation. The elution gradient was as follows: initial conditions: 65% solvent A (aqueous solution of 1 nM sodium formate and 0.1% formic acid) and 35% solvent B (methanol:acetonitrile with 0.1% of formic acid, 50:50); 0−5 min: 65% solvent A; 5−20 min: 65% to 40% solvent A; 20−50 min: 40% to 20% solvent A; 50−95 min: 20% to 2% solvent A; 95− 110 min: 2% solvent A; 110−111 min: 2% to 65% solvent A; 111−120 min: 65% solvent A. Detection was carried out with an Agilent DAD G1315B at 280 nm, and the column temperature was maintained at 35 °C. The coupled mass spectrometer was a Hewlett-Packard 5989B with a detection range from 10 to 2000 Da. Compounds were ionized in the positive electrospray ion source (ESI+) of the mass-spectrometer at 4.5 kV. Moreover, identification of the compounds present in the extracts of P. radiata and C. japonica was carried out by GC-MS analysis using a 6850 Agilent GC equipped with a 5975C VL MSD detector and an HP-5-MS column (Agilent). Hydrogen was the carrier gas at a flow rate of 1.2 mL min−1, and the MS detector operated in EI mode (70 eV). Samples were dried over anhydrous Na2SO4 and filtered. Individual components were identified by comparison of the mass spectrum with those of the computer mass library NIST (2008). For C. japonica, a 1 μL hexane extract was injected directly in the splitless mode, and the temperature ramp for the oven was 5 °C/min from 80 °C up to 280 °C with a final hold stage of 5 min. For the P. radiata extract, a silanization reaction was performed; 500 μL of the methanol extract were evaporated and resuspended for its derivatization with 500 μL of bis(trimethylsilyl)-trifluoroacetamide (Panreac, Germany). Then, 0.2 μL were injected in the splitless mode with an initial

plastic net to avoid direct contact with the agar and fungus. Triplicate samples were incubated for 8 weeks at 22 °C and 70% relative humidity in a Sanyo Versatile Environmental Test Chamber MLR350H (Sanyo, Japan). After the time of fungal exposure, wood samples were removed and cleaned by gently removing the fungal mycelium adhered to the wood surface. In addition, they were weighed to estimate the moisture content, and finally, samples were dried for 24 h at 103 °C and weighed to assess the effectiveness of the enzymatic grafting treatment. Validity criteria defined by the standard EN 113 were applied and a threshold limit value of 20% mass loss was considered to define the effectiveness of the different treatments with the C. puteana. Thus, only treatments with a mass loss inferior to a 20% were considered effective. Moisture content (MC) percentage was calculated as

MC (%) = (m2 − m3)/(m3)× 100

(2)

where m2 and m3 are the wet and the oven-dried mass (103 °C, 24 h), respectively, of the wood samples after fungal exposure. Effectiveness of the treatment was correlated with the mass loss (ML) of the wood samples and expressed as

ML (%) = (m0 − m3)/(m0) × 100

(3)

Statistical analyses of wood durability assays were conducted in R. Quantification of Extractives. Total extractives content was determined by measuring the dry residue of 10 mL of extract (prior to concentration) after 24 h at 103 °C. Total phenolic content was determined by means of a modified version of the Folin−Ciocalteu method.26 Absorbance was measured at 735 nm on a plate reader spectrophotometer (Multiskan Spectrum, Thermo Scientific, Waltham, MA, USA). A calibration curve was accomplished with gallic acid solutions (0−20 μg mL−1), and results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry wood. Total flavonoid content was measured spectrophotometrically with their specific reaction with 4-dimethylaminocinnamaldehyde (DMACA, Sigma) according to the protocol of Pizzo et al.27 A calibration curve was accomplished with catechin solutions (0−15 μg mL−1), and results were expressed as micrograms of catechin equivalents (CE) per gram of dry wood. Chromatographic Conditions. Wood components from the extracts of Pinus spp. were chromatographically studied by an HPLCdiode array detector system controlled by OpenLab EZChrom Elite workstation software (Chromaster, VWR International SAS, Fontenaysous-Bois, France). Samples were centrifuged (6000 rpm for 5 min) 7559


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Table 1. Weight Percent Gain (WPG) Values and Efficacy Data Against C. puteana Exhibited by Treatments at 20% Organic Solvent Content in Unleached and Leached Samples Unleached

Leached

Reference

WPG1 (%)

WPG2 (%)

N

MC (%)

ML (%)

N

MC (%)

ML (%)

Virulence control Control 1 (Buffer) Toluene extracts: Control 2 (Buffer+Acetone) Control 3 (Buffer+Acetone+Laccase) Treatment 1 (P. pinaster) Treatment 2 (P. radiata) Treatment 3 (C. japonica) Treatment 4 (E. globulus) Ethanolic extracts: Control 4 (Buffer+Ethanol) Control 5 (Buffer+Ethanol+Laccase) Treatment 5 (P. pinaster) Treatment 6 (P. radiata) Treatment 7 (C. japonica) Treatment 8 (E. globulus)

− 0.16

− −2.7

12 4

112 ± 38 174 ± 6

28 ± 26 5±1

3 3

147 ± 47 85 ± 7

43 ± 30 42 ± 6

1.85 2.92 3.51 3.77 3.31 2.83

−1.13 −1.56 −1.03 −0.89 −1.44 −1.34

4 4 4 4 4 4

207 164 175 170 180 185

± ± ± ± ± ±

41 8 21 27 16 43

5 4 3 5 7 5

± ± ± ± ± ±

2 1 1 2 2 2

2 2 3 2 2 3

89 121 56 85 116 82

± ± ± ± ± ±

2 14 16 18 6 9

33 57 4 8 55 44

± ± ± ± ± ±

21 1 1 1 10 8

1.40 3.05 5.20 4.44 4.97 3.18

−2.25 −1.03 0.15 −0.43 −0.62 −0.84

4 4 4 4 4 4

177 193 174 170 170 159

± ± ± ± ± ±

33 26 15 13 18 15

7 5 4 9 5 5

± ± ± ± ± ±

2 2 1 2 1 2

3 2 3 3 3 3

86 85 141 103 66 138

± ± ± ± ± ±

7 1 40 34 50 42

50 52 25 20 4 26

± ± ± ± ± ±

5 1 26 14 1 32

temperature of 100 °C for 2 min and the following oven temperature ramp: 5 °C min−1 up to 200 °C, 2 °C min−1 up to 280 °C, 5 °C min−1 up to 310 °C, where there was a final hold stage of 10 min. FTIR Spectroscopic analysis. Solvent was removed from the wood extracts with the aid of an RVC 2-25 CHRIST rotational vacuum concentrator. The residue obtained was analyzed by the CACTI laboratory (University of Vigo) with a Nicolet 6700 (Thermo Scientific) equipped with a DTGS KBr detector and via attenuated total reflection technique (ATR). A resolution of 4 cm−1 was employed from 500 to 4000 cm−1.

solvent content. Parameters of the impregnation were followed and are summarized in Table 1. Dramatic low values were obtained for WPG1 and WPG2; the aqueous nature of the treatment and the fact that only covalently fixed compounds are retained are the feasible explanations for these low values. Nonetheless, interesting conclusions could be extracted from these values. WPG1 in the treatment with buffer solution (control 1) is nearly negligible; however, there are constant increases in buffer solution with 20% of acetone (control 2) and buffer solution with 20% of acetone and laccase (control 3). This was tentatively attributed to a better fixation of the salts of the buffer in the organic solvent, in the case of control 2, and also to the attachment of the laccase to the wood surface, for control 3. With regard to the treatments, in general, a greater WPG1 was found with respect to controls, a fact which reveals the attachment of substances from the extracts onto the wood surface. The smaller WPG1 corresponds to the enzymatic treatment with E. globulus. Values of WPG2, almost all of negative value, have proved that most of these compounds are removed after leaching. In control 3 (samples submerged in buffer solution), the more negative value for WPG2 was found; this fact is due to the leaching of the compounds naturally present in the wood during the 14 days of the leaching procedure. This leaching of the soluble compounds in wood will affect all the samples equally. Notwithstanding, as mentioned in the objectives of this work, only phenolic extractives are supposed to remain covalently bonded to the wood surface. Then, the treated wood would suffer from the same loss of naturally present substances, but on the other hand, it is expected to have an increase in weight in case of the formation of covalent bonds and anchoring of the phenolic substrates onto the wood surface. In fact, it is possible to observe that the values of WPG2 of the toluene extracts treatment are lower (absolute value) than those obtained for control 3, and the same pattern is found in the ethanolic extract treatments with respect to control 5. This fact suggests that laccase was able to bind phenolic compounds present in the extracts onto the wood surface, and this linkage should be stable as it has survived a harsh leaching procedure for 14 days. Formation of these stable bonds could be confirmed by

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RESULTS AND DISCUSSION Biocidal Activity of the Extracts. Preliminarily, antifungal activity of the extracts was evaluated in Petri dishes inoculated with C. puteana and T. versicolor. In addition to heartwood from species of economic interest, prunings of C. japonica were used for comparison because of the well-known antifungal28,29 and mosquito larvicidal30 properties of some of its compounds. Fungal inhibition results are shown in Figure 1. A similar behavior was found for both fungi, although C. puteana has appeared to be more sensitive to the wood extracts than T. versicolor. Extracts from Pinus spp. have provoked a significant growth inhibition in the tests, while the effect of C. japonica extracts was slightly lower and almost negligible in the case of the E. globulus extracts. With regard to the C. puteana fungus, a similar behavior was found for the ethanolic and the toluene extracts. However, for T. versicolor, the effect of the toluene extracts was significantly inferior to that of the ethanolic extracts. These qualitative results have enabled assessment of the degree of antifungal activity of the wood extractives content in the specimens. For the enzymatic grafting method described in this work, these results are only illustrative as, after the enzymatic polymerization, compounds could undergo structural changes and lose their properties or acquire new ones. Also, enzymatic grafting will only take place for phenolic or aromatic amine substrates, in which case they will be the main compounds that will remain strongly attached onto wood and will resist leaching. This means that the biocidal activity in the raw extracts could differ from the biocidal activity found after the enzymatic grafting. Impregnation and Enzymatic Grafting. Several impregnation treatments of the wood were performed at 20% organic 7560


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Figure 2. Quantity of the various types of extractives (based on wood dry weight) obtained with ethanol (gray stripped bar) and toluene (red bar): (a) total extractives content (%), (b) total phenolic content (expressed as gallic acid equivalents, GAE), and (c) total flavonoid content (expressed as catechin equivalents, CE).

so deleterious for fungi growth, and only extract of C. japonica wood has a noticeable antifungal effect. Significant differences among the above-mentioned effective treatments and control experiments were obtained by means of Tukey’s test (statistical analysis available in Supporting Information S6). Relationship between Extractive Content and Durability. In this work, ethanol and toluene were used as extraction solvents for comparison. Ethanol was selected due to its low toxicity and also for its expected potential to remove compounds such as condensed tannins, flavonoids, and phenolics from within the wall structure.32 Furthermore, toluene was employed due to its low polarity and aromatic nature. Different families of compounds were then expected to be removed with these two solvents. It is important to bear in mind that there is no single organic solvent which allows the total removal of extractives from wood. Generally, dichloromethane leads to lower amounts of extractives with respect to the mixture ethanol/benzene. The latter mixture appears to obtain the most complete removal of solvent-extractable substances due to the solution of low molecular weight carbohydrates and polyphenols.33 Notwithstanding, because of health, safety, and regulatory concerns associated with the use of these solvents, acetone has been chosen as an alternative for the determination of extractives in some test methods.34 Correlation of the best antifungal activity of the extracts (toluene extracts of Pinus spp. and ethanolic extract of C. japonica) with their composition was studied. Figure 2(a) shows that total extractives content follows the same trend for both ethanolic and toluene extracts. P. pinaster wood extract has the highest amount of total extractives, followed by the extracts of C. japonica, P. radiata, and E. globulus, respectively. Also, in all cases, ethanol leads to a greater yield of recovery of extractives thanks to its polarity. This higher extractive removal of the ethanol was also obtained for the total phenolic and flavonoid content (Figure 2(b) and (c)). Nevertheless, biocidal activity is related not only with the extractive content but also with the type of extractives. Indeed, although P. pinaster has proved to have a larger extractive content for the ethanol extract than for the toluene extract, the efficacy against fungi in the accelerated tests has appeared to be successful only with toluene extract. On the other hand, the effectiveness of the ethanolic extract of C. japonica could be tentatively ascribed to the high flavonoid content found for this extract, as it represents the main difference respect to the toluene extract (Figure 2(c)). Chromatographic Separation of Wood Extract Compounds. In order to identify some of the main compounds, chromatographic separations of the constituents were carried out for those extracts which have led to suitable durability in

techniques such as X-ray photoelectron spectroscopy, as has already been demonstrated by Fernández-Fernández et al.13 Accelerated Efficacy Tests against Wood-Decay Fungi. This work’s accelerated methodology to assess the efficacy of the treated wood against fungi was first proposed by Bravery25 and has shown toxic values after 12 weeks of incubation in accordance with the results derived from the standard EN 113 test.24 Palanti et al.31 have alerted of the risk of the overestimation of effects with these tests due to the deeper impregnation of these mini-blocks and the higher dispersion found in the results. However, these accelerated tests allow fast screening and were selected to enable a quick evaluation of the proposed enzymatic treatments. Table 1 displays the results obtained for the efficacy tests of the enzymatic treatment at 20% of organic solvent content against C. puteana. Although a dramatic standard deviation between the different replicates has been found for some treatments, the tests have been considered valid as virulence controls have exhibited mass losses superior to 20%. On the other hand, the accelerated aging of the treated samples was done to evaluate the loss of effectiveness against fungi of these samples in comparison with samples that have not suffered any leaching procedure. Unfortunately, nonleached samples presented a high and nonexpected durability, probably due to the buffer solution used in the treatments. In fact, in Table 1, it is possible to appreciate that for unleached samples, even controls, really low mass losses were reported. This was considered a false positive, as when examining the moisture content, it was found to be higher than 150% for all cases. In addition, abnormally high moisture content (waterlogging) was visually recorded in the test specimens. Figure S1 shows an exudate or a liquid droplet coming from the mycelium of C. puteana at the end of the efficacy test in the presence of the control with the acetone and buffer solution. This high humidity is detrimental for the fungi and could be the reason why fungi barely attack wood samples. As all controls followed the same behavior, the buffer salts were identified as possibly being responsible for this effect. Fungi require an acidic pH to develop their wood degrading mechanism, and these salts could be hindering this action. Particularly, optimal pH for fungal enzymes has been reported between pH 3.6 and 5.2.14 Indeed, once leached, all controls have led to mass losses over 20%, and the moisture content has been considerably decreased. Therefore, enzymatic treatments which show no mass loss after the leaching procedure could be considered suitable for wood preservation. In Table 1, it can be noticed that once leached both toluene treatment with P. pinaster and P. radiata wood extracts reduce the mass losses in the wood specimens provoked by the fungal attack. However, in the case of the ethanolic extracts, wood pine extracts are not 7561


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ACS Sustainable Chemistry & Engineering Table 2. Chemical Structure of Phenolic Compounds Identified in This Worka

a

Analytical techniques employed have been included. *Components which had been verified with pure standards. 7562


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ACS Sustainable Chemistry & Engineering Table 3. Composition of Hexane Extract of C. japonicaa Retention time (min)

Compound

Probability (%)

9.8 10.4 10.7 11.4 13.1 13.2 13.3 13.7 13.9 14.0 14.4 16.2 16.3 16.5 16.7 16.8 18.3 20.1 22.7 23.0 24.0 24.8 27.0 27.4 29.5 29.6 34.2

α-cubebene copaene β-cubebene caryophyllene epi-bicyclosesquiphellandrene isoledene α-muurolene β-cadinene δ-cadinene 1,2,3,4,6,8a-hexadydro-1-isopropyl-4,7-dimethyl-naphtalene elemol β-guaiene (±) cadinene cubenol β-eudesmol γ-eudesmol 3,7,11-trimethyl-dodeca-2,4,6,10-tetraenol α-eudesmol juniper camphor kaur-15-ene (5α, 9α, 10β) kaur-16-ene (8β,13β) 7-isoproppyl-1,1,4a trimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene pimaral podocarpa-6,13-diene, 13-isopropyl podocarpa-6,8,11,13-tetraen-12-ol, 13-isopropyl-acetate totarol* sugiol

36 19 16 12 16 6 35 11 60 45 17 7 12 9 24 16 33 17 16 72 42 91 67 30 41 39 90

Identification was accomplished by comparison of the mass spectrum with the NIST mass library of the GC-MS. *Components which have been verified with pure standards. a

Scots pine heartwood, although these stilbenes alone are not the only causes.37 Other flavonoids such as taxifolin and kaempferol were not detected. The presence of these compounds was additionally confirmed with LC-MS in a sample of the toluene extract of P. radiata (Figure S3). Also, other compounds were tentatively identified comparing the mass fragmentation spectra with the positive-ion ESI mass spectra from database MS. Profiling of peak 2* has produced an ion at m/z 213.09 [M + H]+, revealing the presence of pinosylvin. Peak 3* and 4* were confirmed to be pinocembrin and pinosylvin methyl ether due to the ion at m/z 257.08 [M + H]+ and m/z 227.11 [M + H]+, respectively. All of them were also identified by coelution with authentic standards. Peak 1 has presented an ion at m/z 295.05 [M + Na]+, which could be assigned as pinobanksin. This is an antioxidant bioflavonoid which is biosynthesized from pinocembrin and, therefore, is expected to be in the extract. These flavonoids could play an important role in durability as they have proved to have antioxidant and biological properties.38,39 Tentatively, peaks 5, 6, 7, 8, and 9 have an ion at m/z 301.21 [M + H]+, which could be attributed to dehydroabietic acids (resin acids). Also, abietic-type acids have appeared to be present due to the ion at m/z 303.23 [M + H]+, corresponding to peak 10. The toluene extract of P. radiata was also silanized and analyzed in the GC-MS (Figure S4). The presence of resin acids (such as isopimaric, pimaric, abietic, and dehydroabietic acid) and β-sitosterol has been reported together with other phenolic compounds: 3-hydroxy-4-methoxybenzyl alcohol, vanillic acid, and the flavonoid naringenin (Table 2). Flavonoid

the wood blocks. Extractable phenolic compounds (phenolic stilbenoids, flavonoids, and lignans) are the bioactive components of interest for this work (as they could be substrates for the laccase). Furthermore, some other nonphenolic compounds were also studied. Table 2 summarizes the phenolic compounds found in the target extracts, their structure, and also the analytical technique which has allowed their identification. HPLC-DAD was applied to identify the extractives present in the extracts of Pinus spp. A long gradient was necessary because of the difference in polarity of the extractive compounds present in the extract, although most of them were of low polarity. Despite the different behavior found for the enzymatic treatments with the fungal resistance tests, HPLC chromatograms of P. pinaster and P. radiata extracts were found to be quite comparable (Figure S2(a)). Additionally, both ethanolic and toluene extracts have turned out to have numerous similarities (Figure S2(b)). So, concentration of the compounds in the extracts or some characteristic compounds could be playing an important role in their biocidal activity. These chromatograms were compared with the corresponding standards and have allowed the identification of some major components. The flavonoid pinocembrin (PC) and the phenolic stilbenoid pinosylvin monomethyl ether (PSM) were identified in the toluene extract of P. pinaster together with a weak signal of pinosylvin (PS). These compounds where also found in the toluene extract of P. radiata but with a higher signal for PS. The latter is interesting because of its potential biocidal properties,35,36 and some studies support the idea that PS and PSM contribute to the differences in the decay rate of 7563


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Figure 3. FTIR spectra of (a) toluene and (b) ethanolic extracts of the different wood specimens.

4) in the framework of a circular economy. This potential closed cycle is intended to be a process to treat wood at all

naringenin has recently been pointed out as a possible indicator of resistance in the Aleppo pine.40 The composition of a hexane extract of C. japonica wood was studied with GC-MS, and the possible compounds found are listed in Table 3. They included δ-cadinene, isoledene, and γmuurolene, which were previously correlated with the antifungal activitity of the C. japonica heartwood essential oil by Cheng et al.28 As none of them are phenolic compounds, and this work found antifungal activity after leaching of the treated samples, it is expected that the antifungal activity in our treatments should be due to other grafted compounds. In fact, phenolic compounds such as totarol and sugiol seem to be present in the extract (Table 2) and could be responsible for the effectiveness against fungi. In the literature, both of them have been reported to have biological activity. Totarol has proved to have antifungal activity against fungi from cereal crops,41 and sugiol has also exhibited inhibitory effects on different Candida species. 42 Therefore, they could be responsible for the effectiveness of the enzymatic treatment. However, these extracts may contain other compounds that could be involved. Thus, further investigation is needed to elucidate the mechanism of these compounds against fungal growth. FTIR Spectra of the Extracts. Figure 3 displays the FTIR spectra obtained for both toluene and ethanolic extracts. Although it is not possible to obtain precise structural information from these spectra, they allow fast identification of the main differences between the samples and with regard to the presence or absence of certain functional groups. Band assignment was accomplished according to FTIR data of Salem et al.43 In the toluene extracts (Figure 3(a)), it is possible to appreciate the main difference as the presence in the C. japonica extract of a more intense and broad band at 3360 cm−1 together with a stretch one at 1026 cm−1, which can correspond to alcohols/phenols groups. Meanwhile, in the Pinus spp. extracts a little peak at 2650 cm−1 was found, corresponding to carboxylic acids and also a more intense signal at 1690 cm−1 than for the other extracts, corresponding to the carbonyl group and confirmed by the signal at 1280 cm−1 due to the C−O vibration. With regard to the ethanolic extracts (Figure 3(b)), signals at 3360 and 1026 cm−1 are greater for the E. globulus extract, a fact that could be revealing a higher content of aliphatic alcohols in this extract with respect to the aromatic alcohol content. Furthermore, a carbonyl signal found in these extracts is not as intense as for the toluene extracts. Development of a Sustainable Closed Cycle for Wood Treatment. On the basis of the above studies, an environmentally friendly cycle for wood treatment is proposed (Figure

Figure 4. Schematic presentation of the proposed closed cycle for wood treatment and manufacturing.

stages. First, it would affect the raw material, i.e., wood without any processing. From this wood, it would be possible to remove the naturally present extractives. After this step, the extracts could be used as described in this study for a wood protection treatment by means of a “green” wood preservative strategy. Also, other value-added products could be obtained from these extracts (and used for developing wood composites), and solvents would be recycled and reinserted in the extraction stage. With regard to the extracted wood, this material would be more suitable for the paper industry. Resinous materials (“pitch”) in wood are undesirable for paper pulp processing because they affect papermaking machines and cause the appearance of paper spotting. Traditionally, the pitch problem is controlled by seasoning wood, although new approaches are constantly appearing.44,45 The extraction of the wood would probably reduce and/or avoid the “pitch problem”. However, other uses such as wood composites would also be suitable. The viability of this proposal was assessed in terms of a SWOT analysis (Figure 5). Strengths, weakness, opportunities, and threats were identified in order to evaluate the industrial perspectives of this enzymatic treatment and enable an 7564


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Figure 5. SWOT matrix for the closed cycle proposed for wood treatment and manufacturing.

objective critique of the sustainable cycle proposed for the wood industry. Regarding the internal factors, one of the main strengths of this strategy is that it represents an eco-friendly alternative to current wood preservatives. The enzymatic strategy proposed is based on a green chemistry process as the enzymatic reaction is conducted in an aqueous medium and under mild conditions of temperature and pH. However, the inclusion of these laccase-based strategies into the industry requires a cost-effective production of them in conjunction with high catalytic activity and stability. Fortunately, many efforts are being conducted to improve the production levels at bioreactor scale through genetic tools such as directed evolution.46 Furthermore, laccase inmobilization47 or the use of a membrane reactor48 are proposed in order to reuse the enzyme. In addition, natural compounds, extractives, are being put forward as a source of bioactive compounds with antifungal properties. Their peculiar features make them simple to handle and a low risk to workers. Finally, the enzyme provokes a covalent linkage of the bioactive compounds onto the wood surface and reduces their leaching. As a consequence, the service life of the wood is increased and, in the case of wood for outdoor use, migration of the compounds to the surroundings is reduced. The weakness of this method is the limited knowledge of the enzymatic grafting for wood protection. As mentioned before, although enzymatic grafting is increasingly being applied in lignocellulosic materials, few attempts have been made in the wood protection field. In fact, there is still much research to be done in order to acquire more in-depth knowledge of the reactions taking place and to gain the knowhow needed to apply this strategy in an optimal way. Laccases oxidize phenols, and then, together with the anchoring of the extractives to the lignin of the wood surface, other reactions such as self-coupling and further oxidations occur. Further research into enzymatic grafting is required to boost the target reaction and reduce undesired reactions. Also, the protein nature of laccases means they are affected by pH, temperature, and organic solvents. Consequently, these strategies operate in mild conditions and with low solvent content, which is good for the environment but goes against some traditional, widespread formulations.

With regard to the external factors, the main opportunity resides in the development of a closed cycle in the wood industry. In the northwest of Spain, where this study was made, the land is highly productive in forest biomass but fails with regard to the valorization of the raw material, and the industry is limited to little more than woodland exploitation. This strategy would allow a simple flow chart where the raw material is extracted and destined to the paper industry with the added value of being suitable by avoiding “pitch”. In this age of social concern for the environment, a “green” solution would have the acceptance of society. However, mistrust of these mild protectors could arise among wood manufacturers, who are used to a different concept of preservatives. Another major threat found for using extracts in wood preservation was the variability of the extractive content of the tree specimens and also the low concentration of the bioactive compounds. Notwithstanding, this variability could be corrected with periodic monitoring of the extracts and by selecting a forest resource with genotypes rich in extracts for use in new plantations.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00714. Photo of incubation process of wood blocks showing liquid exudate, HPLC chromatograms of several wood extracts, total ion current chromatogram from LC-MS analysis of toluene extract of P. radiata, GC-MS chromatogram of silanizated toluene wood extrat of P. radiata, total ion current chromatogram from GC-MS analysis of the toluene wood extract of C. japonica, and statistical analysis of durability assays after laccaseassisted grafting of wood extractives. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 7565


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ACS Sustainable Chemistry & Engineering ORCID

(13) Fernández-Fernández, M.; Sanromán, M. A.; Moldes, D. Potential of laccase for modification of Eucalyptus globulus wood: A XPS study. Wood Sci. Technol. 2014, 48, 151−160. (14) Madhavi, V.; Lele, S. S. Laccase: Properties and applications. BioResources 2009, 4, 1694−1717. (15) Arora, D. S.; Sharma, R. K. Ligninolytic fungal laccases and their biotechnological applications. Appl. Biochem. Biotechnol. 2010, 160, 1760−1788. (16) Kudanga, T.; Nyanhongo, G. S.; Guebitz, G. M.; Burton, S. Potential applications of laccase-mediated coupling and grafting reactions: A review. Enzyme Microb. Technol. 2011, 48, 195−208. (17) Rättö , M.; Ritschkoff, A. C.; Viikari, L. Enzymatically polymerized phenolic compounds as wood preservatives. Holzforschung 2004, 58, 440−445. (18) Widsten, P.; Heathcote, C.; Kandelbauer, A.; Guebitz, G.; Nyanhongo, G. S.; Prasetyo, E. N.; Kudanga, T. Enzymatic surface functionalization of lignocellulosic materials with tannins for enhancing antibacterial properties. Process Biochem. 2010, 45, 1072− 1081. (19) Fernández-Costas, C.; Palanti, S.; Sanromán, M. Á ; Moldes, D. Enzymatic grafting of kraft lignin as a wood bioprotection strategy. Part 1: factors affecting the process. Holzforschung 2017, DOI: 10.1515/hf-2016-0109. (20) Fernández-Costas, C.; Palanti, S.; Sanromán, M. Á ; Moldes, D. Enzymatic grafting of kraft lignin as a wood bio-protection strategy. Part 2: effectiveness against wood destroying basidiomycetes. Effect of copper entrapment. Holzforschung 2017, DOI: 10.1515/hf-2016-0110. (21) Filgueira, D.; Moldes, D.; Fuentealba, C.; García, D. E. Condensed tannins from pine bark: A novel wood surface modifier assisted by laccase. Ind. Crops Prod. 2017, 103, 185−194. (22) European Committee for Standardization; EN 113 Wood preservatives. Test method for determining the protective effectiveness against wood destroying basidiomycetes. Determination of the toxic values, 1996. (23) European Committee for Standardization; EN 84 Wood preservatives. Accelerated ageing of treated wood prior to biological testing. Leaching procedure, 1997. (24) European Committee for Standardization; EN 335-1 Durability of wood and wood-based products. Definition of use classes. Part 1: General, 2006. (25) Bravery, A. F. A Miniaturized Wood-Block Test for the Rapid Evaluation of Wood Preservative Fungicides; IRG/WP 2113; International Research Group on Wood Protection: Stockholm, Sweden, 1978. (26) Pâques, L. E.; Charpentier, J. P. Perspectives for genetic improvement in heartwood size and extractive content in relation to natural durability and aesthetics in interspecific hybrid larch (Larix eurolepis). Eur. J. For. Res. 2015, 134, 857−868. (27) Pizzo, B.; Pometti, C. L.; Charpentier, J. P.; Boizot, N.; Saidman, B. O. Relationships involving several types of extractives of five native argentine wood species of genera. Ind. Crops Prod. 2011, 34, 851−859. (28) Cheng, S. S.; Lin, H. Y.; Chang, S. T. Chemical composition and antifungal activity of essential oils from different tissues of Japanese cedar (Cryptomeria japonica). J. Agric. Food Chem. 2005, 53, 614−619. (29) Matsushita, Y. I.; Hwang, Y. H.; Sugamoto, K.; Matsui, T. Antimicrobial activity of heartwood components of sugi (Cryptomeria japonica) against several fungi and bacteria. J. Wood Sci. 2006, 52, 552−556. (30) Gu, H. J.; Cheng, S. S.; Huang, C. G.; Chen, W. J.; Chang, S. T. Mosquito larvicidal activities of extractives from black heartwood-type Cryptomeria japonica. Parasitol. Res. 2009, 105, 1455−1458. (31) Palanti, S.; Feci, E.; Predieri, G.; Vignali, F. Copper complexes grafted to amino-functionalized silica gel as wood preservatives against fungal decay: Mini-blocks and standard test. BioResources 2012, 7, 5611−5621. (32) Laks, P. E. The Chemistry of Wood Bark. In Wood and Cellulosic Chemistry; Hon, D. N. S., Shiraishi, N., Eds.; Marcel Dekker: New York, 1991.

Diego Moldes: 0000-0001-6745-4320 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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 the Universidade de Vigo and COST FP 1006 for the financial support for stays at the CNR-IVALSA. Also, financial support from the Transnational Access to Research Infrastructures activity in the seventh Framework Programme of the EC under the Trees4Future project (No. 284181) for conducting the research at the INRA GénoBois Platform (Orléans, France) is gratefully acknowledged. C. Fernández-Costas would like to express her gratitude also to Benedetto Pizzo and Luigi Fiorentino from CNR-IVALSA for their scientific advice and technical support. The authors are also grateful to Enrique Moldes, Fundación Sales, and Novozymes for providing raw materials and CACTI-Vigo for its technological support.

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REFERENCES

(1) Binbuga, N.; Ruhs, C.; Hasty, J. K.; Henry, W. P.; Schultz, T. P. Developing environmentally benign and effective organic wood preservatives by understanding the biocidal and non-biocidal properties of extractives in naturally durable heartwood. Holzforschung 2008, 62, 264−269. (2) Terzi, E.; Nami Kartal, S.; Ibáñez, C. M.; Köse, C.; Arango, R.; Clausen, C. A.; Green, F., III Biological performance of Liquidambar orientalis Mill. heartwood. Int. Biodeterior. Biodegrad. 2012, 75, 104− 108. (3) Yang, D. Q. Potential utilization of plant and fungal extracts for wood protection. For. Prod. J. 2009, 59, 97−103. (4) Schultz, T. P.; Nicholas, D. D. Development of environmentallybenign wood preservatives based on the combination of organic biocides with antioxidants and metal chelators. Phytochemistry 2002, 61, 555−560. (5) Singh, T.; Singh, A. P. A review on natural products as wood protectant. Wood Sci. Technol. 2012, 46, 851−870. (6) Laks, P. E.; McKaig, P. A.; Hemingway, R. W. Flavonoid Biocides: Wood Preservatives Based on Condensed Tannins. Holzforschung 1988, 42, 299−306. (7) Tondi, G.; Hu, J.; Thevenon, M. -F. Advanced tannin based wood preservatives. For. Prod. J. 2015, 65, S26−S32. (8) Nemli, G.; Gezer, E. D.; Yildiz, S.; Temiz, A.; Aydin, A. Evaluation of the mechanical, physical properties and decay resistance of particleboard made from particles impregnated with Pinus brutia bark extractives. Bioresour. Technol. 2006, 97, 2059−2064. (9) Kirker, G. T.; Blodgett, A. B.; Arango, R. A.; Lebow, P. K.; Clausen, C. A. The role of extractives in naturally durable wood species. Int. Biodeterior. Biodegrad. 2013, 82, 53−58. (10) Chen, P. S.; Chen, Y. H.; Yeh, T. F.; Chang, S. T. Mechanism of decay resistance of heartwood extracts from Acacia conf usa against the brown-rot fungus Laetiporus sulphureus. Wood Sci. Technol. 2014, 48, 451−465. (11) Arantes, V.; Goodell, B. Current Understanding of Brown-Rot Fungal Biodegradation Mechanisms: A Review. In Deterioration and Protection of Sustainable Biomaterials; Schultz, T. P., Goodel, B., Nicholas, D. D., Eds.; ACS Symposium Series 1158; American Chemical Society: Washington, DC, 2014; pp 3−21, DOI: 10.1021/ bk-2014-1158.ch001. (12) Schultz, T. P.; Nicholas, D. D. Naturally durable heartwood: Evidence for a proposed dual defensive function of the extractives. Phytochemistry 2000, 54, 47−52. 7566


Research Article

ACS Sustainable Chemistry & Engineering (33) TAPPI Test Method Solvent Extractives of Wood and Pulp. T 204 cm-07, 2007. (34) TAPPI Test Method Acetone Extractives of Wood and Pulp. T 280 pm-99, 1999. (35) Plumed-Ferrer, C.; Väkeväinen, K.; Komulainen, H.; Rautiainen, M.; Smeds, A.; Raitanen, J.; Eklund, P.; Willför, S.; Alakomi, H.; Saarela, M.; von Wright, A. The antimicrobial effects of woodassociated polyphenols on food pathogens and spoilage organisms. Int. J. Food Microbiol. 2013, 164, 99−107. (36) Celimene, C. C.; Micales, J. A.; Ferge, L.; Young, R. A. Efficacy of pinosylvins against white-rot and brown-rot fungi. Holzforschung 1999, 53, 491−497. (37) Venäläinen, M.; Harju, A. M.; Saranpäa,̈ P.; Kainulainen, P.; Tiitta, M.; Velling, P. The concentration of phenolics in brown-rot decay resistant and susceptible Scots pine heartwood. Wood Sci. Technol. 2004, 38, 109−118. (38) Neacsu, M.; Eklund, P. C.; Sjöholm, R. E.; Pietarinen, S. P.; Ahotupa, M. O.; Holmbom, B. R.; Willför, S. M. Antioxidant flavonoids from knotwood of Jack pine and European aspen. Holz Roh Werkst 2007, 65, 1−6. (39) Soromou, L. W.; Chu, X.; Jiang, L.; Wei, M.; Huo, M.; Chen, N.; Guan, S.; Yang, X.; Chen, C.; Feng, H.; Deng, X. In vitro and in vivo protection provided by pinocembrin against lipopolysaccharideinduced inflammatory responses. Int. Immunopharmacol. 2012, 14, 66−74. (40) Romeralo, C.; Witzell, J.; Diez, J. J. Aleppo pine provenances vary in susceptibility and secondary chemical response to Gremmeniella abietina infection. Plant Pathol. 2016, 65, 664−672. (41) Wang, H.; Wang, J.; Peng, X.; Zhou, P.; Bai, N.; Meng, J.; Deng, X. Control efficacy against rice sheath blight of Platycladus orientalis extract and its antifungal active compounds. Eur. J. Plant Pathol. 2014, 140, 515−525. (42) Bajpai, V. K.; Baek, K. H.; Kang, S. C. Inhibitory effects of sugiol, a biologically active abietane type diterpenoid from Metasequoia glyptostroboides. Rom. Biotechnol. Lett. 2012, 17, 7287−7294. (43) Salem, M. Z. M.; Zidan, Y. E.; El Hadidi, N. M. N.; Mansour, M. M. A.; Abo Elgat, W. A. A. Evaluation of usage three natural extracts applied to three commercial wood species against five common molds. Int. Biodeterior. Biodegrad. 2016, 110, 206−226. (44) Xiao, H.; Zhao, G. I.; Li, J. R.; He, B. H. Hydrophobically associating polyethylenimine for controlling dissolved and colloidal substances of alkaline peroxide mechanical pulp. BioResources 2013, 9, 1121−1131. (45) Zasadowski, D.; Strand, A.; Sundberg, A.; Edlund, H.; Norgren, M. Selective purification of bleached spruce TMP process water by induced air flotation (IAF). Holzforschung 2014, 68, 157−165. (46) Mate, D. M.; Alcalde, M. Laccase engineering: From rational design to directed evolution. Biotechnol. Adv. 2015, 33, 25−40. (47) Fernández-Fernández, M.; Sanromán, M. T.; Moldes, D. Recent developments and applications of immobilized laccase. Biotechnol. Adv. 2013, 31, 1808−1825. (48) Pilz, R.; Hammer, E.; Schauer, F.; Kragl, U. Laccase-catalysed synthesis of coupling products of phenolic substrates in different reactors. Appl. Microbiol. Biotechnol. 2003, 60, 708−712.

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enzymatic grafting of wood extractives - American Chemical Society

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