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Impact of a low pulsed electric field on the fungal degradation of wood in laboratory trials
International Biodeterioration & Biodegradation 114 (2016) 244e251
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Impact of a low pulsed electric field on the fungal degradation of wood in laboratory trials Andreas Treu*, Erik Larnøy Norwegian Institute of Bioeconomy Research, Department Wood Technology, Pb. 115, 1431, Ås, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2015 Received in revised form 28 June 2016 Accepted 7 July 2016
Wood protection against fungal decay is mainly based on chemical protection. Nontoxic protection methods have become more important in Europe due to environmental concerns. A method using electric fields to inhibit wood decay by fungi has been investigated in laboratory trials and wood mass loss and moisture content after exposure to fungal attack were determined. The results show significantly reduced mass loss for wood samples exposed to a low pulsed electric field (LPEF), while wood samples connected to alternating and direct current displayed higher mass loss compared to LPEF. Changing the electrode material reduced the mass increase due to metal ion transfer into the wood samples for LPEF-exposed samples. The use of conductive polymer instead of metal electrodes and carbon fibers was preferable as no ions were transferred and the integrity of the material persisted. Decay of pre-exposed wood samples to white rot could be stopped or slowed down by means of LPEF. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Low pulsed electric field Wood decay Wood protection Fungal attack Conductive polymer
1. Introduction Extending service life of wooden products used in adverse environments is still a partially unsolved and highly topical issue for a sustainable future of wood protection. There are limitations to using untreated wood when high durability and long service life are needed. Many European wood species have low durability and need to be protected when used in outdoor applications (Van Acker et al., 2003; Plaschkies et al., 2014). Protection of wood by means of liquid chemical impregnation is dependent on time- and energyconsuming processes, and the use of non-refractory wood species (Tanikawa et al., 1994). Wood protection measures are linked to several restrictions and directives concerning biocides (Aston, 2001). Alternatives to conventional preservatives have become more important in European and North American markets, leading to the development of organic and inorganic chromium-free wood preservatives (Murphy, 1998; Hingston et al., 2001; Read, 2003). Alternative technologies that change the chemical structure of wood, such as wood modification with furfuryl alcohol (Lande, 2008), acetylation with acetic anhydride (Hill et al., 1998),
* Corresponding author. E-mail addresses: [email protected] (E. Larnøy).
(A.
http://dx.doi.org/10.1016/j.ibiod.2016.07.007 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
Treu),
[email protected]
modification with 1,3-dimethylol-4,5-dihydroxy-ethyleneurea (Xie et al., 2007) and heat treatment (Kamdem et al., 2002), are commercially available in Europe and are characterized by different non-toxic modes of action compared to traditional biocide-based wood preservatives. However, these wood modification systems are costly and the majority of today’s commercial wood protection treatments still rely on conventional wood preservatives with toxicity as the action principle (Cheremisinoff et al., 2008). The increased durability of protected wooden products is a major contributor to increasing the use of wood and wooden products, which again results in a positive contribution to the climate (Richter and Gugerli, 1996; Pajchrowski et al., 2014). The search for alternatives to existing wood protection treatments is motivated by the reduction of process costs, less interaction with dangerous substances, reduction of hazardous waste production, reduction of wood preservative leaching, and minimizing the negative influence of the treatment process on the wood material, such as reduction of strength properties. Furthermore, the possibility of using wood protection treatments that do not depend on wood species is desirable, since product performance is dependent on uptake and distribution of wood protection agents (Yildiz, 2007). A variety of electric and electromagnetic treatments have been evaluated with respect to their interaction with microorganisms in the field of food research (Aronsson et al., 2001; Ho and Mittal,
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2001; Hermawan et al., 2004; Huang et al., 2012). The efficacy of these treatments is based on thermal or non-thermal effects (Palaniappan et al., 1990). A pulsed electric field (PEF) is an effective method for killing bacteria in liquid while avoiding thermal damage. The efficacy of these treatments depends on an electricallyconductive medium. Wood, however, has a low specific conductivity and is considered a dielectric material (Torgovnikov, 1993). Water, therefore, plays a major role in the dielectric properties of wood. Dry wood has a specific resistance ranging from 1013 to 1015 Ohm-m. Electrical conduction in wood is described by the percolation theory and the existence of a continuous path of loosely bound or capillary water in wood (Zelinka et al., 2008). Applying an electric field through a natural material, such as wood, can lead to electrical transport of water and ions. The ionic transference and the electro-osmotic water flow were shown to be two independent processes (Simons et al., 1998). However, it is unclear whether the movement of ions and water can be accounted for the mode of action in preventing fungal decay. Alternative wood protection technologies, such as the use of electric fields against wood decay, were evaluated in an early study and showed that direct current had a greater effect against wooddestroying fungi compared to alternating current and highfrequency current (Hattori and Tamura, 1939). The growth of blue stain fungi in different electric fields showed that a direct current inhibits the growth and germination of the fungi. Polarity switching, however, decreased the inhibiting effect, whereas short pulses of high voltage could inhibit the growth of surface fungi (Bjurman, 1996). While high-voltage electric fields are not feasible for the area of wood protection close to human accessibility, a low-current electric system is needed in order to influence hyphal growth or wood degradation by fungi. The application of a specific low-pulsing pattern of a low voltage (LPEF) has been shown to protect Scots pine (Pinus sylvestris) samples from Coniophora puteana fungi and molds (Treu and Larnøy, 2010; Treu et al., 2011, 2014). LPEF used in these studies has a low frequency and has been proven not to be harmful to humans (Berget, 2012). Little is known in terms of the mechanism of protection, the effect on material moisture content, or the degree of protection provided when applied to larger samples. The aim of the study was to analyze the influence of a slow, low pulsed electric field (LPEF) on wood samples exposed to basidiomycetes in fungal trials in dependence on different test setups. The specific objectives were. 1) To evaluate the influence of different electric fields on the mass loss of Scots pine sapwood when exposed to the brown rot fungus Coniophora puteana. 2) To evaluate the influence of electrode material, wood species, fungal species and exposure time on the efficacy of LPEF.
2. Materials and methods Different experimental configurations (test A-C) were used to investigate the effect of electric current on wood decay. For all trials (test A-C), unleached Scots pine (Pinus sylvestris) sapwood samples from eastern Norway were used. Additionally, beech wood (Fagus sylvatica) samples were used in tests B and C. An overview of samples and test setup is given in Table 1. All wood samples were sterilized including the already installed electric cabling by exposure to 25e50 kGy g-radiation using a 60Co source prior to fungal exposure. Other sterilization measures such as steam sterilization influenced negatively the cable- and electrode material and were therefore not used in this study. The wood samples were dried at
245
103 C after fungal exposure, and mass loss and wood moisture content were calculated. Mass loss of each sample caused by fungi is given by Eq. (1):
mass lossð%Þ ¼
ðm0 md Þ 100 m0
where m0 is dry mass of wood prior to test, and md is dry mass of wood after the test. Wood moisture content (MC) after fungal exposure was calculated by Eq. (2):
MCð%Þ ¼
ðmu md Þ 100 md
where mu is the mass of wood samples after fungal exposure and md is dry mass after test. 2.1. Fungal trials The brown rot fungus Coniophora puteana (BAM Ebw. 15) and the white rot fungus Trametes versicolor (CIB 863A) were used as test organisms, since basidiomycetes are the major hazard for above-ground applications and these fungal species are obligatory fungi when testing according to the European standard (CEN, 1996). The fungi were grown and maintained in agar plates (87 mm diameter and 14 mm high for test A, 87 mm diameter and 20 mm high for test C) or Kolle flasks (test B). Petri dishes contained 25 ml 4% (w/v) malt agar. A plastic mesh was used to avoid direct contact between the samples and the medium. Sterile cables (EKKX copper signal cable, Ø ¼ 0.5 mm) were introduced into the Petri dishes (tests A and C) through small holes in the lid, which were waxsealed afterwards (see Fig. 1). Kolle flasks in test B were sealed by cotton with cables passing through the cotton. Contamination of Petri dishes and Kolle flasks by mold and bacteria was reduced by disinfection of the working area and equipment between each set of samples, and by using sterile gloves. Samples that showed visible contamination after the test (less than 5% of the samples) were removed and not used for the evaluation. Four replicates per exposure time were used in test A, and eight replicates per exposure time were used in tests B and C. For both fungi, virulence samples were included to measure the vitality of the fungal strains. Virulence samples in this study were untreated wood samples exposed to fungi without the influence of an electric field. The incubation time of the fungi was between 4 and 16 weeks (see Table 1) at 22 C and 70% relative humidity. After the incubation period, fungal mycelium was removed from the samples which were dried at 103 C, and mass loss [%] was calculated (see Eq. (1)). 2.2. Electric fields The electric fields used in this study were connected to the wood material permanently or for a certain period of time. A lowintensity and low-frequency pulsed electrical current was used in all tests. The pulsed electric field described by Kristiansen (1998) was selected and was generated by a pulse generator provided by EPT AS. A pulsed electric field (0.806 Hz) with 13 V was used in test A while 40 V was used for tests B and C using the same pulse pattern. The pulse consisted of a longer positive pulse (tþ) followed by a short negative pulse (t) and a pause (t0). Amplitude and the different pulse lengths are described in Fig. 2. In addition to a pulsed electric field with 13 V in test A, an alternating and direct current was included. A TG2000 - 20 MHz DDS Function Generator (Aim TTi) was used in combination with an EL302RT Triple power supply in order to generate an alternating electric field (AC) with 13 V. A battery (Sønnak Nautilus, 56,000, 12 V, 60 Ah, 400 A) was
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Table 1 Overview of test setup used in test A, B and C. Wood species
Wood dimension (r t l) [mm]
Fungal species
Test Exposure time
Scots pine sapwood (Pinus sylvestris) 5 10 30
Coniophora puteana
Scots pine sapwood (Pinus sylvestris), 15 25 50 Beech (Fagus sylvatica) 5 10 30
Coniophora puteana, Trametes versicolor Coniophora puteana, Trametes versicolor
test A test B test C
Electrode material
4 weeks, 8 weeks, 12 weeks metal screws 6 weeks, 16 weeks, 6 weeks þ 10 weeks LPEF 4 weeks, 8 weeks, 4 weeks þ 4 weeks LPEF
carbon fibers
Electric field
Number of replicates
DC, AC, LPEF 13V LPEF 40V
4
conductive polymer, LPEF 40V metal screws
8 8
DC ¼ direct current, AC ¼ alternating current, LPEF ¼ low pulsed electric field.
Fig. 1. Examples of electrodes attached in different ways to test blocks in decay test A, B and C.
50 40 30
pulse amplitude (V)
20 10 0 LPEF -10 -20 -30 -40 -50 0
500
1000 1500 pulse length (ms)
2000
2500
Fig. 2. Pulsing pattern of LPEF used in this study; tþ ¼ 856 ms, t ¼ 128 ms, t0 ¼ 242 ms.
used as a source for direct current (DC). Wood samples in all tests were connected in parallel to the electric source. 2.3. Element analysis by IPC-AES The determination of metal components in wood samples from test A was performed by a simultaneous ICP-AES technique with axial or radial viewing of plasma (Skoog et al., 1998) on a Thermo Jarell Ash ICP-IRIS HR Duo. For sample preservation 0.05 ml of HCL were added to 7 ml sample. The following elements were determined: Chromium (Cr), Copper (Cu), Iron (Fe), Manganese (Mn), Molybdenum (Mo), Nickel (Ni) and Lead (Pb). Wood samples were divided into three equally sized parts in axial direction prior to analysis.
2.4. Test A Unleached Scots pine sapwood samples with dimensions 5 10 30 mm were connected to different electric fields by drilling holes that were 5 mm deep and 2.1 mm in diameter in the center of each cross section of the samples. Acid-resistant and rustfree nickel-chromium plated screws, 15 mm in length, were inserted on both sides of the samples. Isolated copper cables (0.5 mm) were connected to the screws to transfer the different electric fields to the wood samples. The samples were exposed to C. puteana and harvested after 4, 8 and 12 weeks, in accordance with Bravery (Bravery, 1978). Each plate in test A contained one test sample connected to an electric field and one untreated control sample (see Fig. 1).
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Additionally, virulence samples were used, which were exposed to fungal attack without LPEF or a neighboring LPEF- connected wood sample. Additionally, wood samples connected to screws and cables, but without LPEF, were used as reference. Petri dishes in test A consisted of compartments that divided the dishes into two separate sections, in order to avoid neighbor effects. The higher mass loss of AC- and DC-connected samples after fungal exposure compared to LPEF samples resulted in the exclusion of these fields and the use of LPEF for further trials (tests B and C). 2.5. Test B Scots pine sapwood (P. sylvestris) and beech wood (F. sylvatica) samples with dimensions 15 25 50 mm exposed to a low pulsed electric field were examined according to the European standard CEN/TS 15083-1 (CEN, 2005) for 6 or 16 weeks using the brown rot fungus C. puteana and the white rot fungus T. versicolor as test fungi. Additionally, samples were exposed to fungal attack for 6 weeks without LPEF and connected to LPEF for the remaining 10 weeks. Kolle flasks were used for test B, with two LPEF-connected samples per Kolle flask. Two untreated wood samples per Kolle flask were used as virulence. Additionally, Kolle flasks with one LPEF-connected and one untreated control sample were used per Kolle flask and exposed to decay for 16 weeks according to EN 113 (CEN, 1996) (see Table 4, marked with *) in order to analyze any neighbor effects. Carbon fibers provided by Miljøteknologi AS were used as electrodes and introduced into the samples by gluing them in a 10 mm deep and 2.1 mm wide hole in both cross sections of the sample, using a two-component resin (S&P resin 220 epoksilim, S&P Clever Reinforcement Company AG). The 50 mm long carbon fibers were connected to splices and further connected to insulated copper cables. 2.6. Test C Scots pine sapwood (P. sylvestris) and beech wood (F. sylvatica) samples with dimensions 5 10 30 mm were exposed to C. puteana and T. versicolor for 4 or 8 weeks. Additionally, wood samples were exposed to fungal attack for 4 weeks without LPEF and connected to LPEF for the remaining 4 weeks. Petri dishes in test C contained two LPEF-connected samples or two untreated virulence samples. Additionally, for wood samples exposed for 8 weeks, one LPEF-connected and one untreated control sample per Petri dish were used, (see Table 5, marked with *). Petri dishes without compartments were used in test C. Metal-free conductive solid polymer (Elektroplast AS), 45 mm in length and 2 2 mm in lateral dimensions, was used in order to connect LPEF to the wood samples. The conductive polymer was analyzed for its antifungal behavior prior to test C and did not inhibit fungal growth. Additionally, acid-resistant and rust-free screws plated with nickel chromium, 15 mm in length, were used for the exposure of P. sylvestris to C. puteana in order to study the influence of metal ion transfer. Electrodes were inserted into wood samples by drilling holes that were 5 mm deep and 2.1 mm diameter in the center of each cross section of the wood samples. Conductive polymer was pressed into the each hole in the cross sections and connected to splices and insulated copper cables, which led to the LPEF generator. 2.7. Statistical analysis Analysis of variance and Tukey-Kramer HSD tests were performed by JMP Pro software version 10.0 in order to analyze the differences in mass loss and wood moisture content after fungal
247
exposure. Mean mass loss values without common letters are significantly different according to Tukey Kramer HSD test on a significance level of a ¼ 0.05. 3. Results 3.1. The influence of different electric field sources on fungal attack (test A) No significant differences in mass loss were observed after the exposure to C. puteana in any of the wood samples connected to alternating or direct current in test A (see Table 2). Virulence samples experienced more than 26% mass loss after 8 weeks of fungal exposure. However, LPEF-exposed samples showed low mass loss (<1%) during the entire exposure. Moreover, the LPEF samples showed a mass increase from 8 weeks onward due to the migration of metals from the screws. The analysis of wood samples confirmed the uptake of metals during the exposure (see Table 3). Especially the high amount of copper and chromium in LPEFconnected samples could explain the anti-fungal properties of the wood samples and the mass increase after 12 weeks of exposure. Metals which were analyzed in wood were most likely transported from the screws into the wood samples. Some of the samples protected by LPEF showed some discoloration on the negative pole side. Comparable to this was the accumulation of a substantially higher amount of chromium and copper on the negative side of the sample (see Table 3). DC-connected samples experienced significantly lower mass loss compared to untreated control samples and virulence samples after 8 weeks. The analysis of DC-connected wood samples revealed the uptake of a large amount of chromium and copper aswell. AC-connected samples showed no significant mass loss differences compared to their untreated control samples and virulence samples after 8 weeks and did not show a high uptake of metals. The non-connected, untreated control neighbor samples of alternating current (AC), direct current (DC) and low pulsed electric field (LPEF) in test A showed no significant difference in mass loss compared to mass loss of virulence samples after 12 weeks of exposure. A neighbor effect can therefore be excluded. The wood moisture content (MC) of samples in test A exceeded 40% and should therefore have created a favorable environment for fungal growth. However, MC exceeded 80% after 12 weeks. ACconnected samples had significantly higher moisture content compared to virulence and LPEF-connected samples. Wood moisture content after 8 weeks in test A was above literature value (80%) of optimal fungal growth according to the standard EN 113 (CEN, 1996). AC showed significantly lower MC compared to virulence and untreated controls (LPEF-neighbor samples). Significant differences in MC could not be observed in any samples after 12 weeks of exposure to fungi. 3.2. The influence of different wood- and fungal species in LPEF experiments on fungal attack (test B) Virulence samples in test B showed a mass loss above the threshold values described in EN 113 (CEN, 1996), making the test results valid. All LPEF-connected pine and beech wood samples showed significantly lower mass loss after 6 and 16 weeks of exposure compared to untreated controls and virulence samples (see Table 4). The influence on wood mass loss was higher for the exposure of white rot fungus on hardwood and for brown rot fungus on softwood. Mass loss after six weeks of non-LPEF connected wood samples to fungi could not be significantly inhibited by subsequent 10
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Table 2 Mass loss and wood moisture content of Scots pine miniblock sapwood samples in test A, after 4, 8 and 12 weeks of exposure to C. puteana. Samples were connected to different electric fields and tested with non-connected reference and virulence samples. Metal screws were used as connection material. Values in parenthesis represent one standard deviation. Values followed by the same letter do not differ significantly at a ¼ 0.05 for a given incubation time. Exposure time (weeks)
Mass loss (%)
4
AC untreated controls (AC) DC untreated controls (DC) Cable with no LPEF Virulence LPEF untreated controls (LPEF) AC untreated controls (AC) DC untreated controls (DC) Virulence LPEF untreated controls (LPEF) AC untreated controls (AC) DC untreated controls (DC) Cable with no LPEF Virulence LPEF untreated controls (LPEF)
8
12
MC (%)
5.6 7.5 12.3 6.5 13.1 6.1 0.2 4.8 12.8 17.5 9.9 27.8 26.4 1.2 35.9 24.0 28.6 21.4 31.5 23.9 22.5 3.0 23.9
(3.9) (8.2) (9.8) (2.8) (3.2) (5.4) (0.7) (4.2) (5.4) (9.9) (6.2) (6.6) (2.8) (0.3) (4.6) (11.2) (4.3) (17.4) (8.7) (10.7) (8.1) (1.1) (5.8)
A A A A A A A A CD BC CD AB ABC D A A A A A A A B A
93.7 57.9 67.9 60.4 69.4 57.0 50.6 59.2 64.9 84.1 93.3 85.7 131.7 73.6 130.9 110.2 106.0 93.7 125.7 132.9 120.1 108.3 165.5
(33.7) (12.6) (12.9) (8.8) (3.5) (13.3) (7.5) (20.1) (19.54) (11.10) (28.71) (20.51) (11.96) (23.42) (22.08) (50.35) (7.24) (40.65) (7.79) (17.69) (15.94) (49.25) (69.5)
A AB AB AB AB B B AB C ABC ABC ABC AB BC A A A A A A A A A
Table 3 Amount of metal analyzed on three different positions (negative and positive electrode side and center of the sample), in wood samples after 12 weeks of exposure to brown rot decay and different electric fields; AC ¼ alternating current; cable ¼ wood samples connected to screws and cables, but without electric field; DC ¼ direct current; LPEF ¼ low pulsed electric field. Electric field
Position
N
Mean Cr (mg/kg)
Mean Cu (mg/kg)
Mean Fe (mg/kg)
Mean Mn (mg/kg)
Mean Mo (mg/kg)
Mean Ni (mg/kg)
Mean Pb (mg/kg)
AC
center negative positive center negative positive center negative positive center negative positive negative positive
4 4 4 1 1 1 4 4 4 4 4 4 4 4
10.56 (8.63) 22.48 (29.33) 59.31 (105.45) 2.72 (0) 3.59 (0) 2.69 (0) 3054.4 (3030.9) 5163.6 (6795.5) 4855.9 (6188.3) 540.1 (178.3) 2852.2 (2793.1) 809.8 (1242.6) 2.29 (0.51) 1.95 (0.36)
11.48 (13.90) 30.78 (41.15) 25.32 (15.66) 7.47 (0) 17.45 (0) 10.40 (0) 178.6 (177.4) 996.6 (913.1) 2774.9 (1844.7) 1852.4 (1730.9) 5169.9 (8285.9) 533.7 (367.4) 8.53 (5.55) 7.63 (7.19)
0.58 (0.29) 2.73 (3.48) 5.84 (10.60) 0.19 (0) 0.28 (0) 0.19 (0) 292.9 (279.4) 439.4 (297.8) 457.5 (440.5) 60.03 (94.57) 271.0 (191.4) 82.43 (150.2) 0.163 (0.04) 0.14 (0.02)
1.19 1.36 1.25 0.52 0.53 0.60 0.85 3.67 9.16 5.45 2.32 5.68 0.66 0.65
16.89 (6.33) 43.85 (60.26) 87.06 (146.29) 11.33 (0) 14.99 (0) 11.21 (0) 3459.6 (3825.6) 9804.8 (13409.1) 5558.8 (11086.9) 56.15 (70.0) 3952.1 (3515.0) 948.7 (1819.8) 9.47 (2.26) 8.06 (1.41)
0.17 (0.191) 0.31 (0.50) 0.66 (1.22) 0.046 (0) 0.061 (0) 0.05 (0) 14.84 (18.22) 43.76 (67.23) 105.5 (96.6) 52.99 (32.62) 15.43 (2.68) 23.28 (5.39) 0.04 (0.01) 0.03 (0.01)
5.68 (1.45) 698.32 (1384.0) 784.12 (1556.57) 1411.16 (0) 6.94 (0) 5.19 (0) 709.6 (1370.9) 39.56 (26.56) 783.5 (1395.5) 22.47 (19.59) 44.37 (46.79) 11.91 (11.89) 4.38 (1.05) 3.73 (0.65)
cable
DC
LPEF
untreated
weeks of LPEF connection in test B compared to virulence samples. Some of the carbon fibers in test B were destroyed during the exposure period, especially after 16 weeks of exposure. It cannot be determined at which point in time the failures occurred. High standard deviation of average mass loss values was observed for 6 and 16 weeks as well as 6 þ 10 weeks LPEF connected samples. All wood samples displayed wood moisture content between 40 and 120%.
(0.35) (0.33) (0.17) (0) (0) (0) (0.85) (5.28) (8.33) (5.11) (1.63) (2.7) (0.21) (0.21)
subsequent connection to LPEF after 4 weeks for the white rot fungus T. versicolor compared to mass loss of virulence samples after 8 weeks (see Table 5). However, this inhibition effect cannot be seen when wood samples are exposed to the brown rot C. puteana. Conductive polymer electrodes performed without any visible failure. Electrode material from metal screws in test C led to a mass increase as observed in test A. All wood samples displayed a wood moisture content between 40 and 120%.
3.3. The influence of electrode material in LPEF on fungal attack (test C)
4. Discussion
All virulence samples showed sufficient mass loss after 8 weeks to validate the test, except beech samples exposed to C. puteana, which showed very low mass loss. All LPEF samples showed significantly lower mass losses after 4 and 8 weeks of exposure compared to untreated controls and virulence samples. In contrast to the results of test B, mass loss of non-LPEF connected wood samples in test C was significantly reduced for pine wood samples and slightly reduced for beech wood samples after 8 weeks by
The effect of LPEF on wood decay was more pronounced for samples used in test C (see Table 5), which were smaller in dimension compared to samples used in test B (see Table 4). However, the simplified and more rapid test method in test A and C was deployed because it gave similar results compared with standardized tests, when toxic values were determined (Bravery, 1978). Nevertheless, the influence of sample dimension on wood mass loss cannot be compared directly since different electrode material was
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Table 4 Mass loss of Scots pine sapwood and beech wood samples after 6 and 16 weeks of exposure to C. puteana and T. versicolor connected to a pulsed electric field using carbon fibers as electrodes (test B).* Untreated control sample and LPEF-connected sample (control 16 w* þ LPEF 16 w*) were exposed together in the same Kolle flask, in accordance with EN 113 (1996). Values in parenthesis represent one standard deviation. Values followed by the same letter do not differ significantly at a ¼ 0.05 for a given wood and fungal species combination. Wood species
Fungi species
Fagus sylvatica
Coniophora puteana
Trametes versicolor
Pinus sylvestris
Coniophora puteana
Trametes versicolor
Mass loss (%) 6 weeks þ 10 weeks control 16 weeks* LPEF 16 weeks LPEF 16 weeks* LPEF 6 weeks virulence 16 weeks virulence 6 weeks 6 weeks þ 10 weeks control 16 weeks * LPEF 16 weeks LPEF 16 weeks * LPEF 6 weeks virulence 16 weeks virulence 6 weeks 6 weeks þ 10 weeks control 16 weeks* LPEF 16 weeks LPEF 16 weeks* LPEF 6 weeks virulence 16 weeks virulence 6 weeks 6 weeks þ 10 weeks control 16 weeks* LPEF 16 weeks LPEF 16 weeks* LPEF 6 weeks virulence 16w virulence 6w
LPEF
LPEF
LPEF
LPEF
10.3 14.3 3.1 3.5 1.3 22.8 9.4 31.0 35.6 18.4 10.9 3.8 30.0 13.2 22.8 32.4 9.9 3.8 5.4 32.0 20.0 18.4 26.2 5.4 5.7 0.8 23.3 5.8
MC (%) (5.9) (10.6) (4.4) (4.9) (2.2) (5.1) (2.8) (5.3) (3.6) (8.9) (6.8) (2.4) (1.0) (0.5) (12.7) (3.2) (3.8) (0.4) (3.7) (2.8) (2.5) (3.3) (2.2) (1.8) (2.5) (0.3) (2.2) (0.9)
AB B BC BC C A B A A B BC C A B A AB C C C A B A B C C D A C
49.7 79.3 64.9 59.3 51.6 71.0 46.5 96.9 50.6 68.4 62.8 44.7 48.3 37.8 111.6 87.9 83.5 70.6 58.9 86.6 55.5 92.3 58.6 69.4 91.7 63.1 58.2 43.7
(7.3) (2.1) (9.8) (3.3) (7.0) (7.7) (4.6) (31.7) (6.4) (41.4) (13.2) (4.7) (6.6) (1.5) (53.2) (4.4) (16.9) (6.5) (19.1) (6.6) (4.9) (35.9) (7.1) (7.4) (15.8) (29.9) (12.0) (3.6)
C A AB BC C AB C A B AB AB B B B A AB AB AB B AB B A ABC ABC AB ABC BC C
Table 5 Mass loss of Scots pine sapwood and beech miniblock samples after 4 and 8 weeks of exposure to C. puteana and T. versicolor connected to a pulsed electric field using conductive polymer electrodes and metal screws (only for P. sylvestris exposed to C. puteana) with non-connected control and virulence samples (test C). * Untreated control samples and LPEF-connected samples (control 8 w* þ LPEF 8 w*) were exposed together in the same Petri dish. Values in parenthesis represent one standard deviation. Values followed by the same letter do not differ significantly at a ¼ 0.05 for a given wood and fungal species combination. Wood species
Fungi species
Fagus sylvatica
Coniophora puteana
Trametes versicolor
Pinus sylvestris
Coniophora puteana
Trametes versicolor
Mass loss (%) 4 weeks þ 4 weeks LPEF control 8 weeks* LPEF 4 weeks LPEF 8 weeks LPEF 8 weeks* virulence 4 weeks virulence 8 weeks 4 weeks þ 4 weeks LPEF control 8 weeks* LPEF 4 weeks LPEF 8 weeks LPEF 8 weeks* virulence 4 weeks virulence 8 weeks 4 weeks þ 4 weeks LPEF control 8 weeks* LPEF 4 weeks LPEF 4 weeks metal LPEF 8 weeks LPEF 8 weeks metal LPEF 8 weeks * virulence 4 weeks virulence 8 weeks 4 weeks þ 4 weeks LPEF control 8 weeks* LPEF 4 weeks LPEF 8 weeks LPEF 8 weeks* virulence 4 weeks virulence 8 weeks
4.6 0.4 0.0 0.4 0.0 1.0 2.0 25.8 38.3 0.2 0.5 0.8 17.2 36.8 25.1 19.1 0.4 0.9 0.2 2.6 0.1 21.1 27.1 6.8 21.1 0.3 0.1 0.6 4.8 19.5
MC (%) (3.4) (0.7) (0.3) (0.5) (0.5) (1.4) (1.3) (4.8) (8.1) (0.4) (1.3) (0.7) (2.6) (3.0) (5.5) (2.9) (0.4) (1.4) (0.2) (3.8) (0.6) (3.2) (4.4) (0.8) (3.9) (0.6) (0.5) (0.3) (1.5) (3.4)
A
44.9 79.3 33.2 53.8 47.6 75.4 96.1 86.7 48.5 46.6 63.7 38.7 70.9 67.9 53.1 115.5 42.5 50.5 50.4 63.0 51.1 110.1 113.1 58.4 103.0 54.7 44.2 54.3 97.0 130.0
(5.8) (3.4) (1.5) (24.1) (19.6) (8.5) (10.7) (39.2) (10.6) (17.2) (31.6) (4.5) (18.1) (8.9) (13.0) (28.3) (19.1) (32.9) (39.5) (33.2) (42.2) (11.2) (19.1) (55.0) (19.1) (32.8) (21.3) (39.9) (22.2) (32.8)
A
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used in the tests and some of the carbon fibers in test B failed. Furthermore, exposure time was different between the tests. Test methods deployed in the field of wood protection are often focused on obtaining toxic values of protective treatments. The protection of wood by means of an electric field, however, is an installation rather than a treatment. Further modification of the test setup could be utilized in the future. As C. puteana mainly attacks softwood, an overall low mass loss was achieved when beech samples were exposed in test C. However, this effect could not be observed in test B (see Table 4). Different pre-exposure times and qualitative analysis of fungal DNA would further explain the colonization pattern of fungi and the mode of action of LPEF. 4.1. The role of wood moisture content Although the mass loss of LPEF-connected samples was significantly lower compared to those samples which had AC or DC applied fields (see Table 2), wood moisture content after 12 weeks of exposure was around 100% for all wood samples exposed to electric fields. Unfavorably low wood moisture content for fungal decay was not reached for any wood sample in test A-C. Elevated wood moisture content in fungal tests is normally due to fungal activity or growth of hyphae (Schmidt, 2006). Optimum wood moisture content for C. puteana is 30e70% (Schmidt, 2006; Stienen et al., 2014). Higher wood moisture content could lead to reduced availability of oxygen and therefore inhibit fungal activity (Boddy, 1983). However, the high wood moisture content in after 12 weeks exposure in test A (see Table 2) cannot explain the efficacy of LPEF on fungal decay, since untreated control and virulence samples showed in general over 20% mass loss and displayed even higher wood moisture content. Due to high variation, wood moisture content of untreated control and virulence samples was not significantly different from other wood moisture contents. Wood held under 20% moisture content can be considered to be protected from basidiomycete decay (Dix and Webster, 1995). However, none of the tested samples from test A-C displayed such low wood moisture content. Wood moisture content differences are therefore not a plausible explanation for the mode of action of LPEF. 4.2. The influence of metal ion transfer and the electrode material The mass increase of LPEF-connected samples during test A is assumed to be a result of ion uptake from the metal electrodes which exceeds a possible mass loss of wood substance (see Tables 2 and 3). It was found that electric fields can be used for in-situ impregnation of wood (Ottosen et al., 2011). A similar effect could explain the weight gain of LPEF-connected samples after exposure in this test. Some of the samples protected by LPEF showed discoloration on the negative pole side, which could be confirmed by the analysis of large amounts of metals on the negative sample side (see Table 3). Even pulse length in AC fields did not lead to substantial accumulation of metals in the wood samples. At this point, the experimental focus was drawn to the possibility that the transfer of ions into the wooden matrix was the sole source of the mode of action. Copper from the wires, as well as nickel and chromium from the screws, were the main concerns (see Table 3). Metal-free electrodes were therefore used in tests B and C. Some of the carbon fibers in test B were destroyed during the exposure period, especially after 16 weeks of exposure. It cannot be determined at which point in time the failures occurred. Despite the failure of some carbon electrode material in test B, the mass loss of all LPEF-connected wood samples is significantly lower than control and virulence samples. However, the mass loss of LPEFconnected samples in test B exceeds the threshold value of 3%
mass loss according to standard EN 113 (CEN, 1996). The carbon fibers as electrode material need to be optimized if used for the installation of LPEF. Conductive polymer electrodes performed without any visible failure during lab trials. Other possible materials could be titanium and platinum; however, a more costefficient material is desired. Earlier trials by the authors on the same wood material showed that already previously LPEF- and fungi exposed samples, which had been shown mass increase after 8 weeks due to the ion transfer, lost 20% of the mass after disconnecting from LPEF and exposure to C. puteana for an additional 8 weeks (Treu and Larnøy, 2010). Movement of metal ions into the wood samples during 8 weeks of exposure in earlier trials was therefore not causing a potentially toxic protective effect in later unprotected (not connected to LPEF) exposure. However, the analysis of the distribution and the amount of introduced metal ions in this study within different exposure time periods has not been part of the investigation, but is assumed to have reached a possible protective level after 12 weeks. Levels of introduced metals need to be compared in relation to toxicity threshold levels for different metals (Wazny and Kundzewicz, 2008). The highest value for copper content in wood samples from test A showed DC and LPEF connected samples with 4e5 g/kg, while literature values describe similar amounts for copper from copper azole treatment (Green and Clausen, 2005). However, the distribution of copper in three different positions in the wood samples was uneven with much lower values on the positive side of the wood samples (Table 3). Ion transport into the wood samples is therefore not the main reason why a protective effect of LPEF was observed, which could be proven by using non-metal electrodes. A long-term effect of LPEF from disconnected samples is not likely and could not be seen in earlier trials. However, it has not been investigated how interrupted pulse dosage would influence the degradation of wood by fungi and if the exposure to LPEF has a short-term effect. The protective effect of pulsed electric fields (PEF) in food protection is mainly based on effects on the intracellular organization of microorganisms or membrane damage. High temperature as the mode of action is unwanted, and non-thermal preservation of food is desired. High voltage is therefore used in small dosage and short exposure times (Aronsson et al., 2001; Van Loey et al., 2001). The LPEF used in this study consists of long repeating pulses and low voltage. Wood samples are exposed to the electric field during the whole test. An influence of LPEF on the wood temperature could not been measured. Differences between the virulence samples (a pair of untreated samples exposed to fungi) and the untreated control samples for both mass loss and MC in test A were not significant, proving that there were no neighbor effects due to electric fields. However, the Petri dishes used in test A consisted of two compartments (see Fig. 1 A) that divided the Petri dish into a compartment with an untreated control and an electric field-exposed wood sample. Tests B and C were therefore designed using non-divided flasks or dishes and proved no influence of an electric field on the neighboring untreated control wood sample (see Fig. 1 B and C). 5. Conclusion Our study analyzed a large influence of a low pulsed electric field (LPEF) on wood samples when exposed to basidiomycetes in laboratory fungal trials. LPEF was able to protect beech and Scots pine sapwood against brown and white rot. Other electric fields showed a significantly lower or no effect against fungal degradation of wood. The test setup, in particular the electrode material, influenced the outcome of the fungal trials. Metal screws and carbon fibers
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influenced the test results negatively by either transporting metal ions into the wood or by defibration of the carbon fibers. Metal-free conductive polymer electrodes were more suitable for the installation in laboratory trials. Pre-exposure of wood samples to white rot decay could be somewhat stopped by subsequently connecting them to LPEF. Fungal species are assumed to have a larger impact on the efficacy of LPEF compared to the influence of wood species. Wood moisture content and accumulation of metals in wood samples was excluded as a possible mode of action of LPEF. Acknowledgments The authors would like to thank the financial support of Innovation Norway and The Norwegian Research Council (199325/O10). References € nner, U., 2001. Inactivation of miAronsson, K., Lindgren, M., Johansson, B.R., Ro croorganisms using pulsed electric fields: the influence of process parameters on Escherichia coli, Listeria innocua, Leuconostoc mesenteroides and Saccharomyces cerevisiae. Innov. Food Sci. Emerg. Technol. 2 (1), 41e54. Aston, D., 2001. The Biocidal Products Directive (98/8/EC) - its consequences for the wood preservation industry. In: The International Research Group on Wood Protection. IRG/WP 01-50166-04, Nara, Japan. Berget, R., 2012. Målerapport - Eksponering for Elektromagnetiske Felt, 196248 TRF Survey. Nemko AS. Bjurman, J., 1996. Growth inhibitory effects on blue-stain fungi of applied electricity fields. In: The International Research Group on Wood Preservation. IRG/WP96e10167, Guadeloupe, France. Boddy, L., 1983. Effect of temperature and water potential on growth rate of woodrotting basidiomycetes. Trans. Br. Mycol. Soc. 80 (1), 141e149. Bravery, A.F., 1978. A miniaturised wood-block test for the rapid evaluation of wood preservative fungicides. In: The International Research Group on Wood Preservation. IRG/WP 78e2113, Peebles, Scotland. CEN, 1996. Wood preservatives - Test Method for Determining the Protective Effectiveness against Wood Destroying Basidiomycetes - Determination of the Toxic Values, CEN 113: 1996. CEN 113: 1996. CEN, 2005. Durability of Wood and Wood-Based Products - Determination of the Natural Durability of Solid Wood against Wood-destroying Fungi, Test Methods - Part 1: Basidiomycetes, CEN/TS 15083-1: 2005. CEN/TS 15083-1: 2005. CEN European Committee for Standardization, Brussels. Cheremisinoff, N., Rosenfield, P., Davletshin, A., 2008. Responsible Care: a New Strategy for Pollution Prevention and Waste Reduction through Environment Management. Elsevier Science. Dix, N.J., Webster, J., 1995. Fungal Ecology. Chapman & Hall, London. Green, F., Clausen, C.A., 2005. Copper tolerance of brown-rot fungi: oxalic acid production in southern pine treated with arsenic-free preservatives. Int. Biodeterior. Biodegrad. 56 (2), 75e79. Hattori, T., Tamura, T., 1939. On the effect of electricity upon the growth of wooddestroying fungi. Jpn. J. Phytopathol. 9 (4), 211e222. Hermawan, N., Evrendilek, G.A., Dantzer, W.R., Zhang, Q.H., Richter, E.R., 2004. Pulsed electric field treatment of liquid whole egg inoculated with Salmonella enteritidis. J. Food Saf. 24 (1), 71e85. Hill, C.A.S., Jones, D., Strickland, G., Cetin, N.S., 1998. Kinetic and mechanistic aspects of the acetylation of wood with acetic anhydride. Holzforschung 52 (6), 623e629. Hingston, J.A., Collins, C.D., Murphy, R.J., Lester, J.N., 2001. Leaching of chromated copper arsenate wood preservatives: a review. Environ. Pollut. 111 (1), 53e66. Ho, S.Y., Mittal, G.S., 2001. Non-thermal microbial inactivation in waste brine using high-voltage low-energy electrical pulses. Innov. Food Sci. Emerg. Technol. 2 (4), 251e259.
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Huang, K., Tian, H., Gai, L., Wang, J., 2012. A review of kinetic models for inactivating microorganisms and enzymes by pulsed electric field processing. J. Food Eng. 111 (2), 191e207. Kamdem, D.P., Pizzi, A., Jermannaud, A., 2002. Durability of heat-treated wood. Holz als Roh- Werkst. 60 (1), 1e6. Kristiansen, H., 1998. A Method for Dehydrating Capillary Materials. Electro Pulse Technologies of America, Inc., Greenwich, CT. Lande, S., 2008. Furfurylation of Wood - Wood Modification by the Use of Furfuryl Alcohol. Doctoral Thesis Thesis. Norwegian University of Life Sciences, Ås, Norway. Murphy, R., 1998. In: Chromium in Timber Preservation, vol. 5. International Chromium Development Association, p. 9. Ottosen, L.M., Block, T., Nymark, M., Christensen, I.V., 2011. Electrochemical in situ impregnation of wood using a copper nail as source for copper. Wood Sci. Technol. 45 (2), 289e302. Pajchrowski, G., Noskowiak, A., Lewandowska, A., Strykowski, W., 2014. Wood as a building material in the light of environmental assessment of full life cycle of four buildings. Constr. Build. Mater. 52 (0), 428e436. Palaniappan, S., Sastry, S.K., Richter, E.R., 1990. Effects of electricity on microorganisms - a review. J. Food Process. Preserv. 14 (5), 393e414. Plaschkies, K., Jacobs, K., Scheiding, W., Melcher, E., 2014. Investigations on natural durability of important European wood species against wood decay fungi. Part 1: laboratory tests. Int. Biodeterior. Biodegrad. 90 (0), 52e56. Read, D., 2003. Report on copper, chromium and arsenic (CCA) treated timber. Environmental Risk Management Authority, New Zealand. Richter, K., Gugerli, H., 1996. Wood and timber derived products in comparative life cycle assessment studies. Holz Als Roh-Und Werkst. 54 (4), 225e231. Schmidt, O., 2006. Wood and Tree Fungi - Biology, Damage, Protection and Use. Springer Verlag, Berlin Heidelberg. Simons, P.J., Spiro, M., Levy, J.F., 1998. Electrical properties of wood- Determination of ionic transference numbers and electroosmotic water flow in Pinus sylvestris L. (Scots pine). Journal of the Chemical Society. Faraday Trans. 94 (2), 223e226. Skoog, D.A., Holler, F.J., Nieman, T.A., 1998. Principles of Instrumental Analysis. Thomson Learning London. Stienen, T., Schmidt, O., Huckfeldt, T., 2014. Wood decay by indoor basidiomycetes at different moisture and temperature. Holzforschung 68 (1), 9e15. Tanikawa, M., Furuno, T., Jodai, S., Sonobe, H., 1994. Liquid permeation into refractroy wood species. 2. Permeability and anatomical characteristics in the radial direction. Mokuzai Gakkaishi 40 (3), 328e335. Torgovnikov, G. (Ed.), 1993. Dielectric Properties of Wood and Wood-based Materials. Springer Series in Wood Science. Springer-Verlag, Berlin, Heidelberg, New York. Treu, A., Bardage, S., Johansson, M., Trey, S., 2014. Fungal durability of polyaniline modified wood and the impact of a low pulsed electric field. Int. Biodeterior. Biodegrad. 87 (0), 26e33. Treu, A., Larnøy, E., 2010. Wood protection by means of electro osmotic pulsing technology (PLEOT). In: The International Research Group on Wood Protection. IRG/WP 10e40505, Biarritz, France. Treu, A., Nunes, L., Duarte, S., Larnøy, E., 2011. On the use of wood protection by means of electric osmotic pulsing technology against subterranean termites and fungi. In: Caldeira, F. (Ed.), Minimizing the Environmental Impact of the Forest Products Industries. Fernando Pessoa University Editions. Van Acker, J., et al., 2003. Biological durability of wood in relation to end-use. Holz als Roh- Werkst. 61 (1), 35e45. Van Loey, A., Verachtert, B., Hendrickx, M., 2001. Effects of high electric field pulses on enzymes. Trends Food Sci. Technol. 12 (3e4), 94e102. Wazny, J., Kundzewicz, A., 2008. Conditions and possibilities of nanobiocides formulation for wood protection. Drew. Wood 51 (180), 107e115. Xie, Y., et al., 2007. Effect of treatments with 1,3-dimethylol-4,5-dihydroxy-ethyleneurea (DMDHEU) on the tensile properties of wood. Holzforschung 61 (1), 43e50. Yildiz, S., 2007. Retention and penetration evaluation of some softwood species treated with copper azole. Build. Environ. 42 (6), 2305e2310. Zelinka, S., Glass, S., Stone, D., 2008. A percolation model for electrical conduction in wood with implications for wood-water relations. Wood Fiber Sci. 40 (4), 544e552.
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Impact of a low pulsed electric field on the fungal degradation of wood in laboratory trials Andreas Treu*, Erik Larnøy Norwegian Institute of Bioeconomy Research, Department Wood Technology, Pb. 115, 1431, Ås, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2015 Received in revised form 28 June 2016 Accepted 7 July 2016
Wood protection against fungal decay is mainly based on chemical protection. Nontoxic protection methods have become more important in Europe due to environmental concerns. A method using electric fields to inhibit wood decay by fungi has been investigated in laboratory trials and wood mass loss and moisture content after exposure to fungal attack were determined. The results show significantly reduced mass loss for wood samples exposed to a low pulsed electric field (LPEF), while wood samples connected to alternating and direct current displayed higher mass loss compared to LPEF. Changing the electrode material reduced the mass increase due to metal ion transfer into the wood samples for LPEF-exposed samples. The use of conductive polymer instead of metal electrodes and carbon fibers was preferable as no ions were transferred and the integrity of the material persisted. Decay of pre-exposed wood samples to white rot could be stopped or slowed down by means of LPEF. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Low pulsed electric field Wood decay Wood protection Fungal attack Conductive polymer
1. Introduction Extending service life of wooden products used in adverse environments is still a partially unsolved and highly topical issue for a sustainable future of wood protection. There are limitations to using untreated wood when high durability and long service life are needed. Many European wood species have low durability and need to be protected when used in outdoor applications (Van Acker et al., 2003; Plaschkies et al., 2014). Protection of wood by means of liquid chemical impregnation is dependent on time- and energyconsuming processes, and the use of non-refractory wood species (Tanikawa et al., 1994). Wood protection measures are linked to several restrictions and directives concerning biocides (Aston, 2001). Alternatives to conventional preservatives have become more important in European and North American markets, leading to the development of organic and inorganic chromium-free wood preservatives (Murphy, 1998; Hingston et al., 2001; Read, 2003). Alternative technologies that change the chemical structure of wood, such as wood modification with furfuryl alcohol (Lande, 2008), acetylation with acetic anhydride (Hill et al., 1998),
* Corresponding author. E-mail addresses: [email protected] (E. Larnøy).
(A.
http://dx.doi.org/10.1016/j.ibiod.2016.07.007 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
Treu),
[email protected]
modification with 1,3-dimethylol-4,5-dihydroxy-ethyleneurea (Xie et al., 2007) and heat treatment (Kamdem et al., 2002), are commercially available in Europe and are characterized by different non-toxic modes of action compared to traditional biocide-based wood preservatives. However, these wood modification systems are costly and the majority of today’s commercial wood protection treatments still rely on conventional wood preservatives with toxicity as the action principle (Cheremisinoff et al., 2008). The increased durability of protected wooden products is a major contributor to increasing the use of wood and wooden products, which again results in a positive contribution to the climate (Richter and Gugerli, 1996; Pajchrowski et al., 2014). The search for alternatives to existing wood protection treatments is motivated by the reduction of process costs, less interaction with dangerous substances, reduction of hazardous waste production, reduction of wood preservative leaching, and minimizing the negative influence of the treatment process on the wood material, such as reduction of strength properties. Furthermore, the possibility of using wood protection treatments that do not depend on wood species is desirable, since product performance is dependent on uptake and distribution of wood protection agents (Yildiz, 2007). A variety of electric and electromagnetic treatments have been evaluated with respect to their interaction with microorganisms in the field of food research (Aronsson et al., 2001; Ho and Mittal,
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2001; Hermawan et al., 2004; Huang et al., 2012). The efficacy of these treatments is based on thermal or non-thermal effects (Palaniappan et al., 1990). A pulsed electric field (PEF) is an effective method for killing bacteria in liquid while avoiding thermal damage. The efficacy of these treatments depends on an electricallyconductive medium. Wood, however, has a low specific conductivity and is considered a dielectric material (Torgovnikov, 1993). Water, therefore, plays a major role in the dielectric properties of wood. Dry wood has a specific resistance ranging from 1013 to 1015 Ohm-m. Electrical conduction in wood is described by the percolation theory and the existence of a continuous path of loosely bound or capillary water in wood (Zelinka et al., 2008). Applying an electric field through a natural material, such as wood, can lead to electrical transport of water and ions. The ionic transference and the electro-osmotic water flow were shown to be two independent processes (Simons et al., 1998). However, it is unclear whether the movement of ions and water can be accounted for the mode of action in preventing fungal decay. Alternative wood protection technologies, such as the use of electric fields against wood decay, were evaluated in an early study and showed that direct current had a greater effect against wooddestroying fungi compared to alternating current and highfrequency current (Hattori and Tamura, 1939). The growth of blue stain fungi in different electric fields showed that a direct current inhibits the growth and germination of the fungi. Polarity switching, however, decreased the inhibiting effect, whereas short pulses of high voltage could inhibit the growth of surface fungi (Bjurman, 1996). While high-voltage electric fields are not feasible for the area of wood protection close to human accessibility, a low-current electric system is needed in order to influence hyphal growth or wood degradation by fungi. The application of a specific low-pulsing pattern of a low voltage (LPEF) has been shown to protect Scots pine (Pinus sylvestris) samples from Coniophora puteana fungi and molds (Treu and Larnøy, 2010; Treu et al., 2011, 2014). LPEF used in these studies has a low frequency and has been proven not to be harmful to humans (Berget, 2012). Little is known in terms of the mechanism of protection, the effect on material moisture content, or the degree of protection provided when applied to larger samples. The aim of the study was to analyze the influence of a slow, low pulsed electric field (LPEF) on wood samples exposed to basidiomycetes in fungal trials in dependence on different test setups. The specific objectives were. 1) To evaluate the influence of different electric fields on the mass loss of Scots pine sapwood when exposed to the brown rot fungus Coniophora puteana. 2) To evaluate the influence of electrode material, wood species, fungal species and exposure time on the efficacy of LPEF.
2. Materials and methods Different experimental configurations (test A-C) were used to investigate the effect of electric current on wood decay. For all trials (test A-C), unleached Scots pine (Pinus sylvestris) sapwood samples from eastern Norway were used. Additionally, beech wood (Fagus sylvatica) samples were used in tests B and C. An overview of samples and test setup is given in Table 1. All wood samples were sterilized including the already installed electric cabling by exposure to 25e50 kGy g-radiation using a 60Co source prior to fungal exposure. Other sterilization measures such as steam sterilization influenced negatively the cable- and electrode material and were therefore not used in this study. The wood samples were dried at
245
103 C after fungal exposure, and mass loss and wood moisture content were calculated. Mass loss of each sample caused by fungi is given by Eq. (1):
mass lossð%Þ ¼
ðm0 md Þ 100 m0
where m0 is dry mass of wood prior to test, and md is dry mass of wood after the test. Wood moisture content (MC) after fungal exposure was calculated by Eq. (2):
MCð%Þ ¼
ðmu md Þ 100 md
where mu is the mass of wood samples after fungal exposure and md is dry mass after test. 2.1. Fungal trials The brown rot fungus Coniophora puteana (BAM Ebw. 15) and the white rot fungus Trametes versicolor (CIB 863A) were used as test organisms, since basidiomycetes are the major hazard for above-ground applications and these fungal species are obligatory fungi when testing according to the European standard (CEN, 1996). The fungi were grown and maintained in agar plates (87 mm diameter and 14 mm high for test A, 87 mm diameter and 20 mm high for test C) or Kolle flasks (test B). Petri dishes contained 25 ml 4% (w/v) malt agar. A plastic mesh was used to avoid direct contact between the samples and the medium. Sterile cables (EKKX copper signal cable, Ø ¼ 0.5 mm) were introduced into the Petri dishes (tests A and C) through small holes in the lid, which were waxsealed afterwards (see Fig. 1). Kolle flasks in test B were sealed by cotton with cables passing through the cotton. Contamination of Petri dishes and Kolle flasks by mold and bacteria was reduced by disinfection of the working area and equipment between each set of samples, and by using sterile gloves. Samples that showed visible contamination after the test (less than 5% of the samples) were removed and not used for the evaluation. Four replicates per exposure time were used in test A, and eight replicates per exposure time were used in tests B and C. For both fungi, virulence samples were included to measure the vitality of the fungal strains. Virulence samples in this study were untreated wood samples exposed to fungi without the influence of an electric field. The incubation time of the fungi was between 4 and 16 weeks (see Table 1) at 22 C and 70% relative humidity. After the incubation period, fungal mycelium was removed from the samples which were dried at 103 C, and mass loss [%] was calculated (see Eq. (1)). 2.2. Electric fields The electric fields used in this study were connected to the wood material permanently or for a certain period of time. A lowintensity and low-frequency pulsed electrical current was used in all tests. The pulsed electric field described by Kristiansen (1998) was selected and was generated by a pulse generator provided by EPT AS. A pulsed electric field (0.806 Hz) with 13 V was used in test A while 40 V was used for tests B and C using the same pulse pattern. The pulse consisted of a longer positive pulse (tþ) followed by a short negative pulse (t) and a pause (t0). Amplitude and the different pulse lengths are described in Fig. 2. In addition to a pulsed electric field with 13 V in test A, an alternating and direct current was included. A TG2000 - 20 MHz DDS Function Generator (Aim TTi) was used in combination with an EL302RT Triple power supply in order to generate an alternating electric field (AC) with 13 V. A battery (Sønnak Nautilus, 56,000, 12 V, 60 Ah, 400 A) was
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Table 1 Overview of test setup used in test A, B and C. Wood species
Wood dimension (r t l) [mm]
Fungal species
Test Exposure time
Scots pine sapwood (Pinus sylvestris) 5 10 30
Coniophora puteana
Scots pine sapwood (Pinus sylvestris), 15 25 50 Beech (Fagus sylvatica) 5 10 30
Coniophora puteana, Trametes versicolor Coniophora puteana, Trametes versicolor
test A test B test C
Electrode material
4 weeks, 8 weeks, 12 weeks metal screws 6 weeks, 16 weeks, 6 weeks þ 10 weeks LPEF 4 weeks, 8 weeks, 4 weeks þ 4 weeks LPEF
carbon fibers
Electric field
Number of replicates
DC, AC, LPEF 13V LPEF 40V
4
conductive polymer, LPEF 40V metal screws
8 8
DC ¼ direct current, AC ¼ alternating current, LPEF ¼ low pulsed electric field.
Fig. 1. Examples of electrodes attached in different ways to test blocks in decay test A, B and C.
50 40 30
pulse amplitude (V)
20 10 0 LPEF -10 -20 -30 -40 -50 0
500
1000 1500 pulse length (ms)
2000
2500
Fig. 2. Pulsing pattern of LPEF used in this study; tþ ¼ 856 ms, t ¼ 128 ms, t0 ¼ 242 ms.
used as a source for direct current (DC). Wood samples in all tests were connected in parallel to the electric source. 2.3. Element analysis by IPC-AES The determination of metal components in wood samples from test A was performed by a simultaneous ICP-AES technique with axial or radial viewing of plasma (Skoog et al., 1998) on a Thermo Jarell Ash ICP-IRIS HR Duo. For sample preservation 0.05 ml of HCL were added to 7 ml sample. The following elements were determined: Chromium (Cr), Copper (Cu), Iron (Fe), Manganese (Mn), Molybdenum (Mo), Nickel (Ni) and Lead (Pb). Wood samples were divided into three equally sized parts in axial direction prior to analysis.
2.4. Test A Unleached Scots pine sapwood samples with dimensions 5 10 30 mm were connected to different electric fields by drilling holes that were 5 mm deep and 2.1 mm in diameter in the center of each cross section of the samples. Acid-resistant and rustfree nickel-chromium plated screws, 15 mm in length, were inserted on both sides of the samples. Isolated copper cables (0.5 mm) were connected to the screws to transfer the different electric fields to the wood samples. The samples were exposed to C. puteana and harvested after 4, 8 and 12 weeks, in accordance with Bravery (Bravery, 1978). Each plate in test A contained one test sample connected to an electric field and one untreated control sample (see Fig. 1).
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Additionally, virulence samples were used, which were exposed to fungal attack without LPEF or a neighboring LPEF- connected wood sample. Additionally, wood samples connected to screws and cables, but without LPEF, were used as reference. Petri dishes in test A consisted of compartments that divided the dishes into two separate sections, in order to avoid neighbor effects. The higher mass loss of AC- and DC-connected samples after fungal exposure compared to LPEF samples resulted in the exclusion of these fields and the use of LPEF for further trials (tests B and C). 2.5. Test B Scots pine sapwood (P. sylvestris) and beech wood (F. sylvatica) samples with dimensions 15 25 50 mm exposed to a low pulsed electric field were examined according to the European standard CEN/TS 15083-1 (CEN, 2005) for 6 or 16 weeks using the brown rot fungus C. puteana and the white rot fungus T. versicolor as test fungi. Additionally, samples were exposed to fungal attack for 6 weeks without LPEF and connected to LPEF for the remaining 10 weeks. Kolle flasks were used for test B, with two LPEF-connected samples per Kolle flask. Two untreated wood samples per Kolle flask were used as virulence. Additionally, Kolle flasks with one LPEF-connected and one untreated control sample were used per Kolle flask and exposed to decay for 16 weeks according to EN 113 (CEN, 1996) (see Table 4, marked with *) in order to analyze any neighbor effects. Carbon fibers provided by Miljøteknologi AS were used as electrodes and introduced into the samples by gluing them in a 10 mm deep and 2.1 mm wide hole in both cross sections of the sample, using a two-component resin (S&P resin 220 epoksilim, S&P Clever Reinforcement Company AG). The 50 mm long carbon fibers were connected to splices and further connected to insulated copper cables. 2.6. Test C Scots pine sapwood (P. sylvestris) and beech wood (F. sylvatica) samples with dimensions 5 10 30 mm were exposed to C. puteana and T. versicolor for 4 or 8 weeks. Additionally, wood samples were exposed to fungal attack for 4 weeks without LPEF and connected to LPEF for the remaining 4 weeks. Petri dishes in test C contained two LPEF-connected samples or two untreated virulence samples. Additionally, for wood samples exposed for 8 weeks, one LPEF-connected and one untreated control sample per Petri dish were used, (see Table 5, marked with *). Petri dishes without compartments were used in test C. Metal-free conductive solid polymer (Elektroplast AS), 45 mm in length and 2 2 mm in lateral dimensions, was used in order to connect LPEF to the wood samples. The conductive polymer was analyzed for its antifungal behavior prior to test C and did not inhibit fungal growth. Additionally, acid-resistant and rust-free screws plated with nickel chromium, 15 mm in length, were used for the exposure of P. sylvestris to C. puteana in order to study the influence of metal ion transfer. Electrodes were inserted into wood samples by drilling holes that were 5 mm deep and 2.1 mm diameter in the center of each cross section of the wood samples. Conductive polymer was pressed into the each hole in the cross sections and connected to splices and insulated copper cables, which led to the LPEF generator. 2.7. Statistical analysis Analysis of variance and Tukey-Kramer HSD tests were performed by JMP Pro software version 10.0 in order to analyze the differences in mass loss and wood moisture content after fungal
247
exposure. Mean mass loss values without common letters are significantly different according to Tukey Kramer HSD test on a significance level of a ¼ 0.05. 3. Results 3.1. The influence of different electric field sources on fungal attack (test A) No significant differences in mass loss were observed after the exposure to C. puteana in any of the wood samples connected to alternating or direct current in test A (see Table 2). Virulence samples experienced more than 26% mass loss after 8 weeks of fungal exposure. However, LPEF-exposed samples showed low mass loss (<1%) during the entire exposure. Moreover, the LPEF samples showed a mass increase from 8 weeks onward due to the migration of metals from the screws. The analysis of wood samples confirmed the uptake of metals during the exposure (see Table 3). Especially the high amount of copper and chromium in LPEFconnected samples could explain the anti-fungal properties of the wood samples and the mass increase after 12 weeks of exposure. Metals which were analyzed in wood were most likely transported from the screws into the wood samples. Some of the samples protected by LPEF showed some discoloration on the negative pole side. Comparable to this was the accumulation of a substantially higher amount of chromium and copper on the negative side of the sample (see Table 3). DC-connected samples experienced significantly lower mass loss compared to untreated control samples and virulence samples after 8 weeks. The analysis of DC-connected wood samples revealed the uptake of a large amount of chromium and copper aswell. AC-connected samples showed no significant mass loss differences compared to their untreated control samples and virulence samples after 8 weeks and did not show a high uptake of metals. The non-connected, untreated control neighbor samples of alternating current (AC), direct current (DC) and low pulsed electric field (LPEF) in test A showed no significant difference in mass loss compared to mass loss of virulence samples after 12 weeks of exposure. A neighbor effect can therefore be excluded. The wood moisture content (MC) of samples in test A exceeded 40% and should therefore have created a favorable environment for fungal growth. However, MC exceeded 80% after 12 weeks. ACconnected samples had significantly higher moisture content compared to virulence and LPEF-connected samples. Wood moisture content after 8 weeks in test A was above literature value (80%) of optimal fungal growth according to the standard EN 113 (CEN, 1996). AC showed significantly lower MC compared to virulence and untreated controls (LPEF-neighbor samples). Significant differences in MC could not be observed in any samples after 12 weeks of exposure to fungi. 3.2. The influence of different wood- and fungal species in LPEF experiments on fungal attack (test B) Virulence samples in test B showed a mass loss above the threshold values described in EN 113 (CEN, 1996), making the test results valid. All LPEF-connected pine and beech wood samples showed significantly lower mass loss after 6 and 16 weeks of exposure compared to untreated controls and virulence samples (see Table 4). The influence on wood mass loss was higher for the exposure of white rot fungus on hardwood and for brown rot fungus on softwood. Mass loss after six weeks of non-LPEF connected wood samples to fungi could not be significantly inhibited by subsequent 10
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Table 2 Mass loss and wood moisture content of Scots pine miniblock sapwood samples in test A, after 4, 8 and 12 weeks of exposure to C. puteana. Samples were connected to different electric fields and tested with non-connected reference and virulence samples. Metal screws were used as connection material. Values in parenthesis represent one standard deviation. Values followed by the same letter do not differ significantly at a ¼ 0.05 for a given incubation time. Exposure time (weeks)
Mass loss (%)
4
AC untreated controls (AC) DC untreated controls (DC) Cable with no LPEF Virulence LPEF untreated controls (LPEF) AC untreated controls (AC) DC untreated controls (DC) Virulence LPEF untreated controls (LPEF) AC untreated controls (AC) DC untreated controls (DC) Cable with no LPEF Virulence LPEF untreated controls (LPEF)
8
12
MC (%)
5.6 7.5 12.3 6.5 13.1 6.1 0.2 4.8 12.8 17.5 9.9 27.8 26.4 1.2 35.9 24.0 28.6 21.4 31.5 23.9 22.5 3.0 23.9
(3.9) (8.2) (9.8) (2.8) (3.2) (5.4) (0.7) (4.2) (5.4) (9.9) (6.2) (6.6) (2.8) (0.3) (4.6) (11.2) (4.3) (17.4) (8.7) (10.7) (8.1) (1.1) (5.8)
A A A A A A A A CD BC CD AB ABC D A A A A A A A B A
93.7 57.9 67.9 60.4 69.4 57.0 50.6 59.2 64.9 84.1 93.3 85.7 131.7 73.6 130.9 110.2 106.0 93.7 125.7 132.9 120.1 108.3 165.5
(33.7) (12.6) (12.9) (8.8) (3.5) (13.3) (7.5) (20.1) (19.54) (11.10) (28.71) (20.51) (11.96) (23.42) (22.08) (50.35) (7.24) (40.65) (7.79) (17.69) (15.94) (49.25) (69.5)
A AB AB AB AB B B AB C ABC ABC ABC AB BC A A A A A A A A A
Table 3 Amount of metal analyzed on three different positions (negative and positive electrode side and center of the sample), in wood samples after 12 weeks of exposure to brown rot decay and different electric fields; AC ¼ alternating current; cable ¼ wood samples connected to screws and cables, but without electric field; DC ¼ direct current; LPEF ¼ low pulsed electric field. Electric field
Position
N
Mean Cr (mg/kg)
Mean Cu (mg/kg)
Mean Fe (mg/kg)
Mean Mn (mg/kg)
Mean Mo (mg/kg)
Mean Ni (mg/kg)
Mean Pb (mg/kg)
AC
center negative positive center negative positive center negative positive center negative positive negative positive
4 4 4 1 1 1 4 4 4 4 4 4 4 4
10.56 (8.63) 22.48 (29.33) 59.31 (105.45) 2.72 (0) 3.59 (0) 2.69 (0) 3054.4 (3030.9) 5163.6 (6795.5) 4855.9 (6188.3) 540.1 (178.3) 2852.2 (2793.1) 809.8 (1242.6) 2.29 (0.51) 1.95 (0.36)
11.48 (13.90) 30.78 (41.15) 25.32 (15.66) 7.47 (0) 17.45 (0) 10.40 (0) 178.6 (177.4) 996.6 (913.1) 2774.9 (1844.7) 1852.4 (1730.9) 5169.9 (8285.9) 533.7 (367.4) 8.53 (5.55) 7.63 (7.19)
0.58 (0.29) 2.73 (3.48) 5.84 (10.60) 0.19 (0) 0.28 (0) 0.19 (0) 292.9 (279.4) 439.4 (297.8) 457.5 (440.5) 60.03 (94.57) 271.0 (191.4) 82.43 (150.2) 0.163 (0.04) 0.14 (0.02)
1.19 1.36 1.25 0.52 0.53 0.60 0.85 3.67 9.16 5.45 2.32 5.68 0.66 0.65
16.89 (6.33) 43.85 (60.26) 87.06 (146.29) 11.33 (0) 14.99 (0) 11.21 (0) 3459.6 (3825.6) 9804.8 (13409.1) 5558.8 (11086.9) 56.15 (70.0) 3952.1 (3515.0) 948.7 (1819.8) 9.47 (2.26) 8.06 (1.41)
0.17 (0.191) 0.31 (0.50) 0.66 (1.22) 0.046 (0) 0.061 (0) 0.05 (0) 14.84 (18.22) 43.76 (67.23) 105.5 (96.6) 52.99 (32.62) 15.43 (2.68) 23.28 (5.39) 0.04 (0.01) 0.03 (0.01)
5.68 (1.45) 698.32 (1384.0) 784.12 (1556.57) 1411.16 (0) 6.94 (0) 5.19 (0) 709.6 (1370.9) 39.56 (26.56) 783.5 (1395.5) 22.47 (19.59) 44.37 (46.79) 11.91 (11.89) 4.38 (1.05) 3.73 (0.65)
cable
DC
LPEF
untreated
weeks of LPEF connection in test B compared to virulence samples. Some of the carbon fibers in test B were destroyed during the exposure period, especially after 16 weeks of exposure. It cannot be determined at which point in time the failures occurred. High standard deviation of average mass loss values was observed for 6 and 16 weeks as well as 6 þ 10 weeks LPEF connected samples. All wood samples displayed wood moisture content between 40 and 120%.
(0.35) (0.33) (0.17) (0) (0) (0) (0.85) (5.28) (8.33) (5.11) (1.63) (2.7) (0.21) (0.21)
subsequent connection to LPEF after 4 weeks for the white rot fungus T. versicolor compared to mass loss of virulence samples after 8 weeks (see Table 5). However, this inhibition effect cannot be seen when wood samples are exposed to the brown rot C. puteana. Conductive polymer electrodes performed without any visible failure. Electrode material from metal screws in test C led to a mass increase as observed in test A. All wood samples displayed a wood moisture content between 40 and 120%.
3.3. The influence of electrode material in LPEF on fungal attack (test C)
4. Discussion
All virulence samples showed sufficient mass loss after 8 weeks to validate the test, except beech samples exposed to C. puteana, which showed very low mass loss. All LPEF samples showed significantly lower mass losses after 4 and 8 weeks of exposure compared to untreated controls and virulence samples. In contrast to the results of test B, mass loss of non-LPEF connected wood samples in test C was significantly reduced for pine wood samples and slightly reduced for beech wood samples after 8 weeks by
The effect of LPEF on wood decay was more pronounced for samples used in test C (see Table 5), which were smaller in dimension compared to samples used in test B (see Table 4). However, the simplified and more rapid test method in test A and C was deployed because it gave similar results compared with standardized tests, when toxic values were determined (Bravery, 1978). Nevertheless, the influence of sample dimension on wood mass loss cannot be compared directly since different electrode material was
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Table 4 Mass loss of Scots pine sapwood and beech wood samples after 6 and 16 weeks of exposure to C. puteana and T. versicolor connected to a pulsed electric field using carbon fibers as electrodes (test B).* Untreated control sample and LPEF-connected sample (control 16 w* þ LPEF 16 w*) were exposed together in the same Kolle flask, in accordance with EN 113 (1996). Values in parenthesis represent one standard deviation. Values followed by the same letter do not differ significantly at a ¼ 0.05 for a given wood and fungal species combination. Wood species
Fungi species
Fagus sylvatica
Coniophora puteana
Trametes versicolor
Pinus sylvestris
Coniophora puteana
Trametes versicolor
Mass loss (%) 6 weeks þ 10 weeks control 16 weeks* LPEF 16 weeks LPEF 16 weeks* LPEF 6 weeks virulence 16 weeks virulence 6 weeks 6 weeks þ 10 weeks control 16 weeks * LPEF 16 weeks LPEF 16 weeks * LPEF 6 weeks virulence 16 weeks virulence 6 weeks 6 weeks þ 10 weeks control 16 weeks* LPEF 16 weeks LPEF 16 weeks* LPEF 6 weeks virulence 16 weeks virulence 6 weeks 6 weeks þ 10 weeks control 16 weeks* LPEF 16 weeks LPEF 16 weeks* LPEF 6 weeks virulence 16w virulence 6w
LPEF
LPEF
LPEF
LPEF
10.3 14.3 3.1 3.5 1.3 22.8 9.4 31.0 35.6 18.4 10.9 3.8 30.0 13.2 22.8 32.4 9.9 3.8 5.4 32.0 20.0 18.4 26.2 5.4 5.7 0.8 23.3 5.8
MC (%) (5.9) (10.6) (4.4) (4.9) (2.2) (5.1) (2.8) (5.3) (3.6) (8.9) (6.8) (2.4) (1.0) (0.5) (12.7) (3.2) (3.8) (0.4) (3.7) (2.8) (2.5) (3.3) (2.2) (1.8) (2.5) (0.3) (2.2) (0.9)
AB B BC BC C A B A A B BC C A B A AB C C C A B A B C C D A C
49.7 79.3 64.9 59.3 51.6 71.0 46.5 96.9 50.6 68.4 62.8 44.7 48.3 37.8 111.6 87.9 83.5 70.6 58.9 86.6 55.5 92.3 58.6 69.4 91.7 63.1 58.2 43.7
(7.3) (2.1) (9.8) (3.3) (7.0) (7.7) (4.6) (31.7) (6.4) (41.4) (13.2) (4.7) (6.6) (1.5) (53.2) (4.4) (16.9) (6.5) (19.1) (6.6) (4.9) (35.9) (7.1) (7.4) (15.8) (29.9) (12.0) (3.6)
C A AB BC C AB C A B AB AB B B B A AB AB AB B AB B A ABC ABC AB ABC BC C
Table 5 Mass loss of Scots pine sapwood and beech miniblock samples after 4 and 8 weeks of exposure to C. puteana and T. versicolor connected to a pulsed electric field using conductive polymer electrodes and metal screws (only for P. sylvestris exposed to C. puteana) with non-connected control and virulence samples (test C). * Untreated control samples and LPEF-connected samples (control 8 w* þ LPEF 8 w*) were exposed together in the same Petri dish. Values in parenthesis represent one standard deviation. Values followed by the same letter do not differ significantly at a ¼ 0.05 for a given wood and fungal species combination. Wood species
Fungi species
Fagus sylvatica
Coniophora puteana
Trametes versicolor
Pinus sylvestris
Coniophora puteana
Trametes versicolor
Mass loss (%) 4 weeks þ 4 weeks LPEF control 8 weeks* LPEF 4 weeks LPEF 8 weeks LPEF 8 weeks* virulence 4 weeks virulence 8 weeks 4 weeks þ 4 weeks LPEF control 8 weeks* LPEF 4 weeks LPEF 8 weeks LPEF 8 weeks* virulence 4 weeks virulence 8 weeks 4 weeks þ 4 weeks LPEF control 8 weeks* LPEF 4 weeks LPEF 4 weeks metal LPEF 8 weeks LPEF 8 weeks metal LPEF 8 weeks * virulence 4 weeks virulence 8 weeks 4 weeks þ 4 weeks LPEF control 8 weeks* LPEF 4 weeks LPEF 8 weeks LPEF 8 weeks* virulence 4 weeks virulence 8 weeks
4.6 0.4 0.0 0.4 0.0 1.0 2.0 25.8 38.3 0.2 0.5 0.8 17.2 36.8 25.1 19.1 0.4 0.9 0.2 2.6 0.1 21.1 27.1 6.8 21.1 0.3 0.1 0.6 4.8 19.5
MC (%) (3.4) (0.7) (0.3) (0.5) (0.5) (1.4) (1.3) (4.8) (8.1) (0.4) (1.3) (0.7) (2.6) (3.0) (5.5) (2.9) (0.4) (1.4) (0.2) (3.8) (0.6) (3.2) (4.4) (0.8) (3.9) (0.6) (0.5) (0.3) (1.5) (3.4)
A
44.9 79.3 33.2 53.8 47.6 75.4 96.1 86.7 48.5 46.6 63.7 38.7 70.9 67.9 53.1 115.5 42.5 50.5 50.4 63.0 51.1 110.1 113.1 58.4 103.0 54.7 44.2 54.3 97.0 130.0
(5.8) (3.4) (1.5) (24.1) (19.6) (8.5) (10.7) (39.2) (10.6) (17.2) (31.6) (4.5) (18.1) (8.9) (13.0) (28.3) (19.1) (32.9) (39.5) (33.2) (42.2) (11.2) (19.1) (55.0) (19.1) (32.8) (21.3) (39.9) (22.2) (32.8)
A
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used in the tests and some of the carbon fibers in test B failed. Furthermore, exposure time was different between the tests. Test methods deployed in the field of wood protection are often focused on obtaining toxic values of protective treatments. The protection of wood by means of an electric field, however, is an installation rather than a treatment. Further modification of the test setup could be utilized in the future. As C. puteana mainly attacks softwood, an overall low mass loss was achieved when beech samples were exposed in test C. However, this effect could not be observed in test B (see Table 4). Different pre-exposure times and qualitative analysis of fungal DNA would further explain the colonization pattern of fungi and the mode of action of LPEF. 4.1. The role of wood moisture content Although the mass loss of LPEF-connected samples was significantly lower compared to those samples which had AC or DC applied fields (see Table 2), wood moisture content after 12 weeks of exposure was around 100% for all wood samples exposed to electric fields. Unfavorably low wood moisture content for fungal decay was not reached for any wood sample in test A-C. Elevated wood moisture content in fungal tests is normally due to fungal activity or growth of hyphae (Schmidt, 2006). Optimum wood moisture content for C. puteana is 30e70% (Schmidt, 2006; Stienen et al., 2014). Higher wood moisture content could lead to reduced availability of oxygen and therefore inhibit fungal activity (Boddy, 1983). However, the high wood moisture content in after 12 weeks exposure in test A (see Table 2) cannot explain the efficacy of LPEF on fungal decay, since untreated control and virulence samples showed in general over 20% mass loss and displayed even higher wood moisture content. Due to high variation, wood moisture content of untreated control and virulence samples was not significantly different from other wood moisture contents. Wood held under 20% moisture content can be considered to be protected from basidiomycete decay (Dix and Webster, 1995). However, none of the tested samples from test A-C displayed such low wood moisture content. Wood moisture content differences are therefore not a plausible explanation for the mode of action of LPEF. 4.2. The influence of metal ion transfer and the electrode material The mass increase of LPEF-connected samples during test A is assumed to be a result of ion uptake from the metal electrodes which exceeds a possible mass loss of wood substance (see Tables 2 and 3). It was found that electric fields can be used for in-situ impregnation of wood (Ottosen et al., 2011). A similar effect could explain the weight gain of LPEF-connected samples after exposure in this test. Some of the samples protected by LPEF showed discoloration on the negative pole side, which could be confirmed by the analysis of large amounts of metals on the negative sample side (see Table 3). Even pulse length in AC fields did not lead to substantial accumulation of metals in the wood samples. At this point, the experimental focus was drawn to the possibility that the transfer of ions into the wooden matrix was the sole source of the mode of action. Copper from the wires, as well as nickel and chromium from the screws, were the main concerns (see Table 3). Metal-free electrodes were therefore used in tests B and C. Some of the carbon fibers in test B were destroyed during the exposure period, especially after 16 weeks of exposure. It cannot be determined at which point in time the failures occurred. Despite the failure of some carbon electrode material in test B, the mass loss of all LPEF-connected wood samples is significantly lower than control and virulence samples. However, the mass loss of LPEFconnected samples in test B exceeds the threshold value of 3%
mass loss according to standard EN 113 (CEN, 1996). The carbon fibers as electrode material need to be optimized if used for the installation of LPEF. Conductive polymer electrodes performed without any visible failure during lab trials. Other possible materials could be titanium and platinum; however, a more costefficient material is desired. Earlier trials by the authors on the same wood material showed that already previously LPEF- and fungi exposed samples, which had been shown mass increase after 8 weeks due to the ion transfer, lost 20% of the mass after disconnecting from LPEF and exposure to C. puteana for an additional 8 weeks (Treu and Larnøy, 2010). Movement of metal ions into the wood samples during 8 weeks of exposure in earlier trials was therefore not causing a potentially toxic protective effect in later unprotected (not connected to LPEF) exposure. However, the analysis of the distribution and the amount of introduced metal ions in this study within different exposure time periods has not been part of the investigation, but is assumed to have reached a possible protective level after 12 weeks. Levels of introduced metals need to be compared in relation to toxicity threshold levels for different metals (Wazny and Kundzewicz, 2008). The highest value for copper content in wood samples from test A showed DC and LPEF connected samples with 4e5 g/kg, while literature values describe similar amounts for copper from copper azole treatment (Green and Clausen, 2005). However, the distribution of copper in three different positions in the wood samples was uneven with much lower values on the positive side of the wood samples (Table 3). Ion transport into the wood samples is therefore not the main reason why a protective effect of LPEF was observed, which could be proven by using non-metal electrodes. A long-term effect of LPEF from disconnected samples is not likely and could not be seen in earlier trials. However, it has not been investigated how interrupted pulse dosage would influence the degradation of wood by fungi and if the exposure to LPEF has a short-term effect. The protective effect of pulsed electric fields (PEF) in food protection is mainly based on effects on the intracellular organization of microorganisms or membrane damage. High temperature as the mode of action is unwanted, and non-thermal preservation of food is desired. High voltage is therefore used in small dosage and short exposure times (Aronsson et al., 2001; Van Loey et al., 2001). The LPEF used in this study consists of long repeating pulses and low voltage. Wood samples are exposed to the electric field during the whole test. An influence of LPEF on the wood temperature could not been measured. Differences between the virulence samples (a pair of untreated samples exposed to fungi) and the untreated control samples for both mass loss and MC in test A were not significant, proving that there were no neighbor effects due to electric fields. However, the Petri dishes used in test A consisted of two compartments (see Fig. 1 A) that divided the Petri dish into a compartment with an untreated control and an electric field-exposed wood sample. Tests B and C were therefore designed using non-divided flasks or dishes and proved no influence of an electric field on the neighboring untreated control wood sample (see Fig. 1 B and C). 5. Conclusion Our study analyzed a large influence of a low pulsed electric field (LPEF) on wood samples when exposed to basidiomycetes in laboratory fungal trials. LPEF was able to protect beech and Scots pine sapwood against brown and white rot. Other electric fields showed a significantly lower or no effect against fungal degradation of wood. The test setup, in particular the electrode material, influenced the outcome of the fungal trials. Metal screws and carbon fibers
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