Controlled ageing of wooden test pieces by Fentons reagent, mimicking decay of brown-rot fungi

D.Tsipotas1 ; Petrou M2 ; and P.K. Kavvouras3

1. Faculty of Creativity & Culture, Buckinghamshire Chilterns University College (BCUC) UK 2. Division of archaeological, Geographical and Environmental Sciences, University of Bradford, UK 3. Division of Wood Technology, National Agricultural Research Foundation (N.AG.RE.F.), Forest Research Institute of Athens, Greece

Abstract

The present work demonstrates a procedure for the artificial degradation of fresh wood samples. The target set, is the development of a tool for the evaluation of the suitability of a conservation method to be applied in wooden artefacts of varying degrees of deterioration. It is well established that the efficiency of a conservation method applied to deteriorated archaeological wood depends largely on its degree of deterioration. So it appears that for carrying out the crucial laboratory tests, a set of wood test pieces of prescribed degrees of deterioration is required. This work investigated the application of Fentons reagent to fresh wood as a means of artificial degradation. Test pieces of dry poplar sapwood were subjected to treatment in order to obtain information on their chemical and physical modification. The test pieces were treated repeatedly until final stages of degradation. After the integration of every treatment cycle a number of specimens were removed and analysed. On sample recovery, maximum moisture content, density, hardness and solubility in 1%NaOH were measured for each cycle of Fentons treatment. The ultrastructure and the modification of test pieces were studied using Scanning Electron Microscopy and FT-Raman, respectively. 1

Treatment of fresh wood with Fentons reagent was quite efficient in preparing test pieces of controlled degree of deterioration. Keywords: Fentons reagent, artificial ageing, poplar wood, brown-rot fungi, degradation of carbohydrates

1. Introduction

It has been well established that the efficiency of a conservation method applied to deteriorated archaeological wood depends largely on its degree of deterioration (Brunning 1995). For carrying out comparative studies for the evaluation of conservation treatments, a set of wood test pieces of prescribed degrees of deterioration is required. The objective of the present work is to develop a laboratory process for producing test pieces of controlled deterioration degree. When biodeterioration of wood occurs there are distinct morphological and chemical changes that are signatures of the casual organism (Blanchette 1995). The past few years thorough investigations on the patterns of wood attacking decay have been reported (Singh et al. 1994, Blanchette 1995, Bjordal and Nilsson 2002). Growth characteristics of the microorganisms in wood and the type of the degradative system, results in different decay patterns (Blanchette 1998). Fungi can cause rapid structural failure and for that they are mentioned as the most serious kind of microbiological degraders (Green and Highley 1997). Brown-rot and soft-rot are the two types of fungal degradation observed repeatedly in objects from archaeological and art collections. Specifically brown-rot fungi depolymerise cellulose rapidly during incipient stages of wood colonization. Considerable losses in wood strength occur very early in the decay process, often before decay characteristics are visually evident even before detection of significant weight loss. Cell wall carbohydrates are degraded extensively during decay leaving a modified, lignin-rich substrate. The residual wood is brown and often cracks into cubical pieces when dry (Green and Highley 1997). The Fentons type oxidation (Fe+2 + H2O2 = OH + Fe+3 + OH- and Fe+3 + H2O2 = Fe+2 + OOH + H+) was proposed to be involved in the brown-rot decay

(Koenigs 1974, Shimada et al. 1997). The Fenton oxidation of cellulose mimics the brown-rot decay in many respects (Jellison et al. 1997, Shimada et al. 1997). Halliwell (1965) described the degradation of cotton cellulose by Fentons reagent (H2O2/Fe+2) which generates hydroxyl radical or a similar oxidant reagent, proposing the possible existence of a nonenzymatic cellulolytic system involving peroxide and iron. Extracellular enzymes through reduction of Fe+3 to Fe+2 and O2 to H2O2 produce hydroxyl radicals, the strongest oxidants in biological systems, which depolymerise cellulose (Henriksson et al. 2000). Koenings (1972, 1974, 1975) demonstrated that brown-rot fungi produce extracellular hydrogen peroxide that can depolymerise wood cellulose through the Fentons reagent. His proposal has been strengthened with time and direct evidence for hydroxyl radicals in brown-rot degradation has been obtained (Wood 1994). Goodell et al. (1997) give a detailed description on the Fentons reactions, along with the role of iron in the fungal environment, the ferric reduction etc. Much work on the chemistry of Fentons reagent and the process of nonenzymatic decay mechanism by brown-rot fungi has also been described in much detail by Green and Highley (1997). Kohdzuma et al. (1990, 1991), trying to prepare artificial models of waterlogged wood, degraded wood (Cryptomeria japonica and Aesculus turbinata) by acid, fungi and Fentons reagent. They concluded that it was impossible to attain the desired degradation level by the fungi treatment. The physical properties of wood treated with Fentons reagent were comparable to those of moderately degraded waterlogged softwood, but it was also found that a considerable amount of lignin together with polysaccharides, has been lost during treatment (Kohdzuma et al. 1990, 1991). To our knowledge, the use of Fentons reagent for mimicking brown-rot decay has been the only means of artificial degradation of wood. Other current research on laboratory degradation includes only accelerated weathering.

2. Materials and methods

2.1 Artificial degradation process

Figure 1: Laboratory set up for the experimental procedure of artificial ageing. From left to right, the cooling device controlling the temperature of the vessel in the middle, containing the flask with the wood test pieces submerged in Fentons reagent. Next to it, the pump and the thermometer monitoring the temperature around the flask.

Ninety wooden test pieces measuring 20x20x10 mm were cut from air dried black poplar (Populus nigra) sapwood. The test pieces were pre-treated according to Kohdzuma et al. (1991) for the enhancement of the accessibility of wood to the Fentons reagent. They were kept immersed into hydrochloric acid solution (5.5 mol/l) for four days at 40oC and three more days at 65oC. The hydrochloric acid was then removed by washing the test pieces under running water for seven days. For the treatment of 265 mg of oven dry wood with the Fentons reagent solution, 160 mg of FeSO4.7H2O were dissolved into 1000ml of 0.1M acetic acid buffer solution (pH:4.2). The FeSO4.7H2O solution was poured into the flask containing the test pieces and quantity of H2O2 measuring five times the quantity of FeSO4.7H2O, was added. The reaction flask was immersed into methanol bath kept at 0oC. The impregnation of test pieces was carried out for 72h, under on-off vacuum. After the termination of impregnation phase, the flask was removed from the methanol bath and placed on a reciprocating shaker for the enhancement of cellulose

oxidation. The reaction phase was carried out at 24oC and lasted also 72h. At the end of first Fentons treatment cycle, 15 test pieces were removed from the reaction flask and were washed in running water for three days. The remaining test pieces were subjected to a series of five subsequent Fentons treatment cycles according to the procedure mentioned above. The quantity of fresh H2O2
Figure 2: The reciprocating shaker device with the flask including the Fentons solution and the test pieces

added at the beginning of each new cycle was in accordance with the number of the test pieces left into the reaction flask.

2.2 Evaluation of treated test pieces The treated test pieces were photographed and macroscopically compared for dimensional changes (excessive shrinkage, deformation etc.) and color variation. For the determination of density and maximum moisture content, all test pieces immersed in water were placed under vacuum for 5 hours to exclude trapped air and ensure full waterlogging. After weight and volume measurement, the test pieces were oven dried at 105oC for 24 hours, reweighed and the dry density and maximum moisture content were calculated. Hardness (Janka test) was measured using an Amsler universal testing machine. The test pieces were examined using an FEI Quanta 400 scanning electron microscope (Oxford Instruments, Abingdon, Oxfordshire) to determine their

ultrastructure and state of preservation. Micrographs were taken using 20 kV with the secondary electron detector (SED). Sections were cut from each test piece in the transverse, radial longitudinal and tangential longitudinal directions, using a double sided razor blade or a scalpel. The sections were then placed on double-sided carbon disks on aluminium SEM stubs. The sections were analysed for both the internal and external structures of each test piece. No preparation was considered necessary for the test pieces,

which were waterlogged and they were examined under low vacuum. The solubility in 1% NaOH was measured according to ASTM D 1109-84, using test pieces from each Fentons treatment cycle. Fourier-Transform Raman spectra were obtained using a Bruker IFS66 instrument with an FRA 106 Raman module attachment and Nd / YAG laser excitation at 1064 nm. Spectra were recorded at the range 50-3500 cm-1 at 1 cm-1 spectral resolution with 1000 scans accumulation. The laser power varied from 30 Mw to 120 Mw. The wave number positions were at 1 cm-1. The Opus software, provided by Bruker, was used to collect spectral data and find peak positions. To investigate the differences in peak components between the deteriorated test pieces, a peak component analysis based on the Gauss / Lorentz curve-fit was attempted using Galactic GRAMS / 386 software. The software data were exported to Microsoft Office PowerPoint for graphical display. Test piece preparation was kept to a minimum and included only air drying. Provided that the surface of the wood was flat, the test piece was inserted into the analyser; otherwise a flat surface was produced using a razor. The reproducibility and the degradation efficiency of the method were checked after the completion of the above mentioned first series of Fentons treatment cycles. For this, a second set of 90 test pieces was subjected to 10 successive Fentons treatment cycles. During this second series of Fentons treatment cycles, test pieces were recovered only after the completion of the 5th cycle onwards, to overlap the first series. This second series ended after the completion of the 10th cycle following the complete destruction of the test pieces which lost major quantities of their mass (see Figure 4). The after treatment evaluation of test pieces of the second series included exclusively the maximum moisture content and density while the supplementary evaluation methods mentioned above are still being assessed. The maximum moisture content and density of the first and second series of Fentons treatments were compared and the results are provided below.

Figure 3: Dimension and form diversity of the dried test pieces recovered from both Fentons treatment series.

3. Results The test pieces dimensions reduce in accordance to the number of Fentons cycles as shown in Figure 3. The first cycle appears to have minimal effect on the dimensions of the test pieces, while the samples subjected to six cycle treatment, completely lost their shape. The dimensional changes could be attributed to the degradation of the cell wall carbohydrates leaving a modified, lignin-rich substrate which cannot maintain the shape of the test piece and results in shrinkage and cracks after drying (see Figure 3). The colour of the treated test pieces appears to darken respectively in accordance to the number of Fentons cycles (see Figure 3). The maximum moisture content and density are generally used for a first estimation of the degradation degree of wooden test pieces (Jensen & Gregory 2006). The maximum moisture content of the treated test pieces generally increases with the number of treatment cycles. There is a considerable gradual decrease in the density of the test pieces as the Fentons treatment cycles progress, reaching 0.163 gr/cm3 after the last one
Figure 4: The test pieces subjected to ten Fentons cycles to confirm the reproducibility of the method, have lost major quantities of their mass as well as their shape.

(see Table 1),

demonstrating the progressive degradation of the test pieces and the depletion of carbohydrates. For the evaluation of

process reproducibility the maximum moisture content and density of test pieces recovered from the 5th and 6th cycle of the first and second series of treatments is of great importance. The values appear to overlap each other (see Figures 11 and 12). As it has been already mentioned, hardness, ultrastructure, solubility in 1% NaOH and FT-Raman are assessed in the test pieces recovered from the first series of Fentons treatment only. There seems to be an almost linear decrease in the hardness of the test pieces with the progress of the process. The results coincide with what was expected from the

maximum moisture content and density values. The test pieces recovered from the sixth cycle are fractured thus not subjected to hardness test. Observing the SEM micrographs of radial sections of selected test pieces it is noticeable that there is a progressive deterioration of wood ultrastructure (see Figures 5 to 10). The test pieces recovered after one cycle of Fentons reagent treatment present minor degradation of cell walls not clearly distinguished (see Figure 5). After two cycles of Fentons reagent treatment, the first signs of ultrastructure degradation are obvious in both vessel members and fibers. The dome of several pits in the cross-fields appear degraded (see Figure 6). The test pieces recovered after the third, fourth and fifth cycles

Figure 5: SEM micrograph of radial sections of test piece recovered after one treatment with Fentons reagent (magnification x130).

Figure 6: SEM micrograph of test piece recovered after two treatments with Fentons reagent (magnification x150).

Figure 7: SEM micrograph of test piece recovered after three treatments with Fentons reagent (magnification x160).

Figure 8: SEM micrograph of test piece recovered after four treatments with Fentons reagent (magnification x150).

Figure 9: SEM micrograph of test piece recovered after three treatments with Fentons reagent (magnification x150).

Figure 10: SEM micrograph of test piece recovered after three treatments with Fentons reagent (magnification x160).

of Fentons reagent treatment present intense sings of cell wall degradation, in the form of heavy cracks (see marking circles in Figures 7, 8 and 9) and collapsing, which lead to their deformation. The fibers are heavily degraded as well as the domes (border) of the pits in the cross sections (see arrow in Figure 8). The radial parenchyma completely looses its form after five cycles of Fentons treatment. The six cycles of Fentons reagent treatment result to extended alteration of the wood structure. The anatomical characteristics are hardly recognizable (see Figure 10). The alkali solubility in 1 % NaOH of the test pieces from each treatment cycle appears increasing with the cycle number. That is, as the degradation of the wood substances progresses (according to the repetitive Fentons cycles), the percentage of the material soluble in 1 % NaOH increases (see Table 1). The FT-Raman spectroscopy of the artificially degraded test pieces identifies the progressive degradation of holocelluloses with the progress of the Fentons reagent treatment cycles. The strongest bands of the holocelluloses reported in the literature (Edwards & Farwell 1994; Edwards et al. 1999) appear to progressively lose their intensity. The treatment seems to have less effect on the degradation of lignin, however a slight degradation is observed (see Figures 13 and 14).

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Table 1: Analysis of the artificially degraded test pieces M.C. max (%) First series 325 343 284 434 445 539 ------------Second series ------------336 356 443 613 753 1036 Density dry (gr/cm3) First series 0.373 0.312 0.326 0.218 0.213 0.163 ------------Second series ------------0.289 0.246 0.134 0.100 0.063 ---Hardness (Janka test) (kg) 6.90 4.44 3.12 1.42 2.46 ---------------Solubility in 1% NaOH (%) 24.40 39.00 43.38 52.39 52.45 56.61 -------------

Treatment cycle

1 2 3 4 5 6 7 8 9 10

11

1200

1000
Maximum moisture content (%)

800 600 400 200 Treatment ser. 2 0 1 2 3 4 Treatment ser. 1 5

Cyc le s

10

Figure 11: Maximum moisture content of test pieces recovered from the first and second Fentons treatment series.

0,4 0,35 0,3

Density (g/cm3)

0,25 0,2 0,15 0,1 0,05 0 1 2 3 4 5

Cyc le s

Treatment ser. 2 Treatment ser. 1 6 7 8 9 10

Figure 12: Density of test pieces recovered from the first and second Fentons treatment series.

12

.025

(6)

.02

(5)

.015

(4)

(3)
.01

(2) Extractives and hemicellulose

.005

Cellulose Cellulose and hemicellulose

(1)
3050 3000 2950 2900 2850

Figure 13: FT-Raman spectrum of test pieces recovered after the six cycles with Fentons reagent treatment between 3050 and 2700 cm-1, where the major influence of the treatment on peak positions is observed.

.005

(6)

(5)

-.005

(4)
-.01

(3)
-.015

-.02

(2) Lignin Holocellulose Lignin Hemicellulose Lignin

Cellulose and hemicellulose Cellulose

-.025

Lignin (1)
1700 1600 1500

1400

1300

1200

1100

Figure 14: FT-Raman spectrum of test pieces recovered after the six cycles with Fentons reagent treatment between 1750 and 1050 cm-1 where the major influence of the treatment on peak positions is observed. .

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4. Conclusions The test pieces treated were appreciably degraded by the Fentons reagent. The artificial degradation resulted in considerable modification of physical, mechanical and chemical properties as well as ultrastructure. It appears that these alterations could be controlled, in an acceptable level, by the number of Fentons treatment cycles. The selective degradation of cell walls emulates the wood biodegradation by brown-rot fungi in some aspects. FT-Raman spectroscopy highlighted that lignin did not disintegrate considerably, specifically at the initial cycles of the procedure. The experimental data from the second series of Fentons treatment will facilitate the assessment of the Fentons reagent effect on lignin decomposition in the near future.

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