Fentons Final Article11

7/31/2019 Fentons Final Article11

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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

freshwood 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 integrationof 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.


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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


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(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.


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2. Materials and methods

2.1 Artificial degradation process

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

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.


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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

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,

Figure 2: The reciprocating shaker device with the

flask including the Fentons solution and the testpieces


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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 5 th 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.


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

treatment series.


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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 (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

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.


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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 ofFentons 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 oftest piece recovered after one treatment with

Fentons reagent (magnification x130).

Figure 6: SEM micrograph of test piecerecovered after two treatments with Fentons

reagent (magnification x150).

Figure 7: SEM micrograph of test piece

recovered after three treatments with Fentons


Figure 8: SEM micrograph of test piece recovered

after four treatments with Fentons reagent

(magnification x150).


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ofFentons 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 ofFentons 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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Treatment

cycle

M.C. max(%)

Density dry(gr/cm3) Hardness

(Janka test)(kg)

Solubility in

1% NaOH(%)

Firstseries

Secondseries

Firstseries

Secondseries

1 325 ---- 0.373 ---- 6.90 24.40

2 343 ---- 0.312 ---- 4.44 39.00

3 284 ---- 0.326 ---- 3.12 43.38

4 434 ---- 0.218 ---- 1.42 52.39

5 445 336 0.213 0.289 2.46 52.45

6 539 356 0.163 0.246 ---- 56.61

7 ---- 443 ---- 0.134 ---- ----

8 ---- 613 ---- 0.100 ---- ----

9 ---- 753 ---- 0.063 ---- ----

10 ---- 1036 ---- ---- ---- ----

Table 1: Analysis of the artificially degraded test pieces


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

treatment series.

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

1 2 3 4 5 6 7 8 9 10

Treatment ser. 1

Treatment ser. 2

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

Density(g/cm3)

Cycles

1 2 3 45 6 7

8 9 10

Treatment ser . 1

Treatment ser . 2

0

200

400

600

800

1000

1200

Maximum

moisturecontent(%)

Cycles


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13

-.025

-.02

-.015

-.01

-.005

0

.005

1700 1600 1500 1400 1300 1200 1100

(1)

(2)

(3)

(4)

(5)

(6)

Lignin Cellulose

Cellulose and hemicellulose

Lignin

Lignin Hemicellulose

Holocellulose

Lignin

.005

.01

.015

.02

.025

3050 3000 2950 2900 2850

(1)

(2)

(3)

(4)

(5)

(6)

Extractives and hemicellulose

Cellulose

Cellulose and hemicellulose

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.

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.

References

ASTM 1984. Standard test method for 1% sodium hydroxide solubility of wood. ASTM

D 1109-84.

Bjordal C., and T. Nilsson, (2001), Decomposition of waterlogged archaeological wood,

in Hoffmann P., Spriggs J. A., Grant T., Cook C. and A. Recht (editors) Proceedings ofthe 8th ICOM Group on Wet Organic Archaeological Materials Conference, Stockholm,

pp 235-244.

Bjordal C., and T. Nilsson, (2002), Waterlogged archaeological wood a substrate forwhite rot fungi during drainage of wetlands, International Biodeterioration and

Biodegradation, 50, 2002, pp 17-23.

Blanchette R.A., (1995), Biodeterioration of archaeological wood, Biodeterioration

Abstracts, 9, 2, 1995, pp 113-127.

Blanchette R.A., (1998), A guide to wood deterioration caused by microorganisms and

insects, in: Dardes K., and A. Rotne (editors), The structural conservation of panel

paintings, Los Angeles, Getty Conversion Institute, pp 55-68.

Edwards H.G.M. and D.W. Farwell, (1994), FT-Raman spectrum of cotton: a polymericbiomolecular analysis, Spectrochimica Acta, 50A, 4, 1994, pp 807-811.

Edwards H.G.M., Farwell D.W., and D. Webster, (1997), FT Raman microscopy ofuntreated natural plant fibres, Spectrochimica Acta Part A, 53, 1997, pp 2383-2392.

Goodell B., Jellison J., Liu J., Daniel G., Pazsczynski A., Fekete F., Krishnamurthy S.,Jun L., and G. Xu, (1997), Low molecular weight chelators and phenolic compounds


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15

isolated from wood decay fungi and their role in the fungal biodegradation of wood,

Journal of Biotechnology, 53, 1997, pp 133-162.

Green F., and T.L. Highley, (1997), Mechanism of brown-rot decay: paradigm or

paradox, International Biodeterioration and Biodegradation 39, 1997, pp 113-124.

Halliwell G., (1965), Catalytic composition of cellulosic substrates, Biochemistry

Journal, 95, 1965, pp 35-40.

Henriksson G., Zhang L., Li J., Ljungquist P., Reitberger T., Pettersson G., and G.

Johansson, (2000), Is cellobiose dehydrogenase from Phanerochaete chrysosporium a

lignin degrading enzyme?, Biochimica et Biophysica Acta, 1480, 2000, pp 83-91.

Jellison M., Connoly J., Goodel B., Doyle B., Illman B., Fekete F., and A. Ostrofsky,

(1997), The role of cations in the biodegradation of wood by the brown-rot fungi,

International Biodeterioration and Biodegradation, 39, 2-3, 1997, pp 165-179.

Jensen P., and D. Gregory, (2006), Selected physical parameters to characterize the state

of preservation of waterlogged archaeological wood: a practical guide for theirdetermination, Journal of Archaeological Science, 33, 4, 2006, pp 551-559.

Koenigs J.W., (1972), Production of extracellular hydrogen peroxide by wood-rotting

fungi, Phytopathology, 62, 1972, pp 100-110.

Koenigs J.W., (1974), Hydrogen peroxide and iron: a proposed system for decomposition

of wood by brown-rot Basidiomycetes, Wood and Fiber, 6, 1, 1974, pp 66-80.

Koenigs J.W., (1975), Hydrogen peroxide and iron: a microbial cellulolytic system?,

Biotechnology Bioengineering Symposium, 5, 1975, pp 151-159.

Kohdzuma Y., Itakura S., Minato K., Katayama Y. and K. Okamura, (1990), A trial for

preparation of artificial waterlogged wood I. Comparison of some characteristics of acid

hydrolyzed and decayed wood with those of waterlogged wood, Mokuzai Gakkaishi, 36,5, 1990, pp 389-397.

Kohdzuma Y., Minato K., Katayama Y. and K. Okamura, (1991), Preparation of artificial

waterlogged wood II. Comparison of some characteristics of wood degraded by Fentonsreagent with those of waterlogged wood, Mokuzai Gakkaishi, 37, 5, 1991, pp 473-480.

Shimada M., Akamatsu Y., Tokimatsu T., Mii K. and K. Okamura, (1997), Possiblebiochemical roles of oxalic acid as a low molecular weight compound involved in brown-

rot and white-rot wood decays, Journal of Biotechnology, 53, 1997, pp 103-113.

Singh A.P., Nilsson T. and G.F. Daniel, (1994), Microbial decay of an archaeologicalwood, The international research group on wood preservation, IRG/WP 94-10053.

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