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International Biodeterioration & Biodegradation 57 (2006) 99– 106
On the changes of pinewood (Pinus sylvestris L.) Chemical
composition and ultrastructure during the attack by brown-rot
fungi Postia placenta and Coniophora puteana
I. Irbe
a,
, B. Andersons
a
, J. Chirkova
a
, U. Kallavus
b
, I. Andersone
a
, O. Faix
c
a
Laboratory of Wood Protection and Emission from Wood Based Products, Latvian State Institute of Wood Chemistry,
27 Dzerbenes Str., LV-1006 Riga, Latvia
b
Centre for Materials Research, Tallinn University of Technology, Estonia
c
Institute of Wood Chemistry and Chemical Technology of Wood, Federal Research Centre for Forestry and Forest Products (BFH), Hamburg, Germany
Received 14 April 2005; accepted 14 December 2005
Available online 15 February 2006
Abstract
Pine sapwood blocks were exposed to brown-rot fungi Postia placenta and Coniophora puteana for 1, 2, 3, and 4 months. Ion exchange
chromatography method was used to determine the changes in sugar content during brown-rot decay. The most remarkable feature was
the preferred degradation of mannose both by P. placenta (80.9% weight loss) and C. puteana (77.5% weight loss) in comparison to the
control. It can be interpreted as a result of the preferred degradation of the backbone chains of O-acetyl-galacto-glucomannans.
For scanning electron microscopy, the wood degradation sequences obtained by the fracture method were used. The fractured surface
of cell wall reflected the pattern of degradation of cellulose microfibrils. The surface of secondary wall decayed by P. placenta revealed
both smooth and uneven regions throughout the test. C. puteana retained uneven cell wall surface only after the first month, while later
the surface became smooth. This suggests that P. placenta tended to degrade cellulose amorphous regions more readily, while C. puteana,
possessing full enzyme complement, was able to degrade both amorphous and crystalline regions more readily.
The water vapor sorption method was applied for microstructural characteristics of wood. The formation of new pores of size
2.1–9.9 nm occurred during brown-rot decay, whose sizes and volume depend on the fungus culture and exposure time. It is assumed that
the appearance of these pores is caused by the destruction of carbohydrates.
r2006 Elsevier Ltd. All rights reserved.
1. Introduction
Brown-rot fungi cause the most considerable decay of
wood in service. These fungi depolymerize and metabolize
wood polysaccharides, leaving behind a brown lignin
residue. Wood polysaccharides are extensively degraded
(Blanchette and Abad, 1988), while lignin is modified or
slightly depolymerized (Jin et al., 1990). Incipient decay of
wood by brown-rot fungi caused measurable strength losses
in wood before measurable weight loss occurs (Wilcox, 1978;
Curling et al., 2002).
The role of small diffusible agent in brown-rot decay is
widely discussed. Several authors (Koenigs, 1974;Illman
et al., 1989;Backa et al., 1992;Goodell et al., 1997) support
the hypothesis that the free radicals produced in the Fenton
reaction (Fe
2+
–H
2
O
2
) are involved in wood degradation
by brown-rot fungi. Extracellular H
2
O
2
is detected in
hyphae of brown-rot fungi, and in the wood cell walls in
the early stages of decay (Kim et al., 2002). This suggests
that H
2
O
2
plays an important role in the early degradation
of cellulose by brown-rot fungi.
This study characterized brown-rotted wood at the
increasing stages of biodeterioration. A better under-
standing of lignocellulose decomposition by brown-rot
fungi could contribute to the application of biochemical
processes in biotechnology and wood decay control.
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0964-8305/$ - see front matter r2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2005.12.002
Corresponding author. Tel.: +371 7545137; fax: 371 7550635.
E-mail address: ilzeirbe@edi.lv (I. Irbe).
2. Materials and methods
2.1. Fungal strains
Two fungal strains were used in this study: Postia placenta (Fr.) Lars
et Lomb. (FPRL 280) and Coniophora puteana (Schum.:Fr.) Karst. (BAM
Ebw.15). Isolates were maintained on malt agar slants at 6 1C. Mycelium was
transferred aseptically to Petri dishes containing 5% malt extract and 3%
Bacto Agar (Ferak, Berlin), pH 6.4, and incubated at 22 1C and 70% RH.
Mycelial plugs were transferred to Kolle flasks for the wood decay test.
2.2. Decay test
Scots pine (Pinus sylvestris L.) sapwood blocks (50 25 15 mm)
were exposed to the fungi for 1, 2, 3 and 4 months at 22 1C and 70% RH.
Test procedures were done according to the European standard EN 113
(1996). Three replicates for the fungus and each test period were taken.
Subsequent to cultivation, blocks were removed from the culture vessels,
brushed free of mycelium and oven dried at 103 721C. The percentage of
weight loss was calculated from the dry weight before and after the test.
2.3. Ion exchange chromatography (IEC)
The carbohydrate composition in brown-rotted wood was determined
by total acidic hydrolysis, followed by the analysis of monomeric sugars
(Uremovic et al., 1994). Samples for quantitative borate IEC were taken
from wood blocks decayed by the fungi for 2 and 4 months. The samples
were taken from the middle part of the wood block after its cutting in a
cross-direction.
Wood material was ground and then subjected to acid hydrolysis. In
the primary hydrolysis, 200 mg of dried sawdust was hydrolyzed with 72%
sulfuric acid at 30 1C for 1 h. The secondary hydrolysis was performed
after dilution with water to a 4% sulfuric acid concentration by
autoclaving at 120 1C for 40 min. Then the hydrolysate was filtered on a
glass filter to determine its lignin part. The acidic filtrate containing sugars
was directly applied to a column, filled with anion-exchanger resin. The
mobile phase was 0.475 M potassium-borate buffer, pH 9.2. 2,2-
bicinchoninate reagent for reducing sugars was used for detection of
sugars in the column eluate.
2.4. Scanning electron microscopy (SEM)
Two sequences of wood degraded by P. placenta and C. puteana for 4
months were obtained from the wood block lower surface close to the
substrate.
Wood blocks were sawn into two parts (1/3 and 2/3), then the larger
piece was split with a knife and broken to get the fractured surface. After
getting a proper sample, it was attached to the specimen stub with
conductive glue, coated with gold and examined with a JEOL scanning
electron microscope JSM 840A at 15 kV. All micrographs taken at the
same magnification. In this study, only latewood cells were used, as thicker
cell walls showed more distinct results.
2.5. Water vapor sorption (WVS)
Wood samples after fungal degradation were dried at 103 1C, milled,
and a fraction of size 0.5–1.0 mm was taken for measurement of the
isotherms. Water vapor sorption–desorption isotherms were measured on
a vacuum balance with quartz spirals as the sensible element (the
sensitivity was 1.5–2.0 mm/mg) at the temperature 22 70.1 1C.
The elongation of the spirals was measured by a horizontal microscope
with the accuracy 0.005 mm. The time of reaching the equilibrium in each
point of the isotherm was 20–24 h.
The isotherms were analyzed by the comparative method in combina-
tion with the BET method (Chirkova et al., 2004). The following
characteristics of the samples’ microstructure were determined: accessible
specific surface (A,m
2
/g), mass and surface concentrations of hydrophilic
centers (a
m
, mmol/g and a, groups/nm
2
, respectively), volume of the
sorption space (W,cm
3
/g) and average pore size ðDaver ¼ð4WÞ=AÞ.
The distributions of the pores’ volume in terms of their sizes
½dW=dD¼fðDÞwere calculated from the desorption branch of the
isotherms. The D-values were calculated from the Kelvin equation:
lnðP=P0Þ¼gv=DRT,
where gis the surface tension of the sorbate; vthe molar volume of the
liquid sorbate; Rthe gas constant; and Tis the temperature.
3. Results and discussion
3.1. Ion exchange chromatography
The composition of control specimens truly reflected the
composition of softwood polysaccarides. They consist of
approx. 43% cellulose, 10–15% arabino-4-O-methyl-
glucurono-xylan (Xyl/GluA/L-Ara ¼10/2/1.3), 5–10% O-
acetyl-galacto-glucomannan (Man/Glu/Gal ¼3/1/1), and
27% lignin (Timell, 1967).
Glucose content in the control sample after hydrolysis
was the highest (49.1 abs.%) followed by mannose, xylose,
galactose, and arabinose (12.9, 5.7, 1.7, and 1.2 abs.%,
respectively) (Table 1). The number calculated by differ-
ence to 100 (29.4% for the control) was mostly due to the
lignin and resin content. Experimental hydrolysis losses
could also contribute to this figure.
The weight loss of the samples was an important
measure of the fungi’s bioactivity being after 4 month of
exposure 47% (P. placenta) and 40% (C. puteana).
Conclusive is the change of the sugar composition,
expressed both in abs.% and rel.%, in the hydrolysates
of degraded samples. Table 1 reveals that the sum of the
detectable sugars (Sabs.%) is lower than the residual
amount of solid wood left after degradation (100—weight
loss). After 4 month of exposure to P. placenta, for
example, 29.9 g sugar was detectable in 53 g of residual
wood. In the case of C. puteana 40.6 g sugar was found in
60 g of degraded wood (Fig. 1).
In this context, the difference between the corresponding
values can be explained by the presence of non-hydro-
lysable wood moieties. These consist mostly of lignin. In
the case of pine the presence of resins cannot be excluded.
Carbohydrate conversion products, as a result of metabo-
lism, may also be difficult to determine by routine IEC
sugar analysis. Moreover, hydrolysis losses may occur,
though to a small extent.
Table 1 also give more details of sugar composition; the
most remarkable feature is the preferred degradation of
mannose both by P. placenta (80.9% weight loss) and C.
puteana (77.5% weight loss) in comparison to the control.
By contrast, in the case of the glucose, enrichment can be
observed. For example, after 4 month 78.5 rel.% (P.
placenta) and 81.8% (C. puteana) glucose was found in the
hydrolysate, while in the control 69.5 rel.% glucose was
present. In absolute values, the amount of glucose changed
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I. Irbe et al. / International Biodeterioration & Biodegradation 57 (2006) 99–106100
dramatically compared to other sugars, i.e. totally glucose
was consumed in the highest amount. In 4 months, P.
placenta degraded glucose from 49.1% (initial) to 23.5%
and C. puteana to 33.2%.
These observations can safely be interpreted as a result
of the preferred degradation of the backbone chains of O-
acetyl-galacto-glucomannans. This polymer is probably
associated with less crystalline parts of the microfibril.
Highley (1987) had demonstrated that glucomannans of
Pinus monticola are more easily metabolized by brown-rot
than xylans. In a study of southern pine degradation by
brown-rot was shown that initial strength loss (up to 40%)
without measurable weight loss corresponded to loss in the
galactan and arabinan components (Curling et al., 2002).
Subsequent strength loss (greater than 40%) and significant
weight loss (5–20%) were associated with the loss of the
mannan and xylan components. Major loss of glucan
component (representing cellulose) was achieved at 475%
strength loss and 420% weight loss. Our results also
showed (Table 1) a slight loss of glucose at the initial stage
of wood decay. 12.6% (P. placenta) and 8.9% (C. puteana)
of glucose were lost in 2 months at 20% weight loss, while
hemicellulose sugars in this period were lost at significantly
higher amounts. This can be attributed to the low
accessibility of (crystalline) cellulose to the fungal enzyme
system.
3.2. Scanning electron microscopy (SEM)
In this study the wood degradation sequences obtained
by the fracture method were included as they provided
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70.6
29.4
55.2
44.8
20
29.9
70.1
47
56.5
43.5
21
40.6
59.4
40
0%
20%
40%
60%
80%
100%
control P. placenta
2 months
P. placenta
4 months
C. puteana
2 months
C. puteana
4 months
carbohydrates lignin weight losses
Fig. 1. Relation among weight losses, non-hydrolysable (ligin) and hydrolysable (carbohydrates) wood components after decay by P. placenta and
C. puteana.
Table 1
Weight losses and changes in wood components after pinewood degradation by brown-rot fungi P. placenta and C. puteana
Fungus Exposure
(month)
Weight
loss (%)
a
Glucose (%) Mannose (%) Xylose (%) Galactose (%) Arabinose (%) SS100 minus
Abs. Rel. Loss Abs. Rel. Loss Abs. Rel. Loss Abs. Rel. Loss Abs. Rel. Loss Abs.
(%)
Rel.
(%)
S
Abs. (%)
Control — — 49.1 69.5 — 12.9 18.3 — 5.7 8.0 — 1.7 2.4 — 1.2 1.8 — 70.6 100 29.4
P. placenta 1672
22071 42.9 77.7 12.6 6.6 12.0 48.7 3.9 7.1 31.2 0.9 1.6 48.5 0.9 1.7 25.0 55.2 100 44.8
33671
44774 23.5 78.5 52.2 2.5 8.3 80.9 2.7 8.9 53.1 0.7 2.3 59.2 0.6 2.0 51.6 29.9 100 70.1
C. puteana 11272
22171 44.7 78.9 8.9 6.1 10.8 53.1 4.0 7.1 23.0 0.9 1.5 49.7 1.0 1.7 23.4 56.5 100 43.5
32872
44071 33.2 81.8 32.3 2.9 7.2 77.5 3.0 7.3 47.4 0.7 1.8 56.2 0.8 1.9 38.7 40.6 100 59.4
a
Mean (3) 7standard deviation.
I. Irbe et al. / International Biodeterioration & Biodegradation 57 (2006) 99–106 101
more detailed view on wood cell structure in comparison
with the cutting method.
Degradation sequence of P. placenta attacked wood is
shown in Fig. 2. The fractured surface of cell wall gradually
turned smoother but the differences between fractures after
1 and 3 months were almost unnoticed. As the degradation
of cellulose turned deeper (the mass loss increased) the
surface became smoother, with glass-like appearance. After
4 months, the surface was less uneven than after 1–3
months. By the end of test, a visible boundary between the
cell wall and the compound middle lamellae (CML) was
still observed.
Fig. 3 shows the sequence of wood attacked by C.
puteana. After 1 month the fracture surface was rough,
with deep helical cracks and robust sliced surface. Starting
from the 2nd to the 4th month the cell wall broke evenly
showing smooth structure. The S
3
layer of the secondary
wall had already cracked after 2 months of attack leaving
deep canyons along the lumen surface. Morphological
differences between the secondary wall and the CML were
well distinguished after 1 month of degradation. The cell
wall surface revealed a sliced structure across to the fiber
axis, while the CML showed smooth structure. The
difference between both layers disappeared in the later
degradation periods, when the secondary wall looked like
the CML. The cell wall thinned in a similar way when
degraded by white-rot fungi, which remove all cell wall
components causing a widespread thinning of the wall
(Eriksson et al., 1990).
The micrographs showed difference in wood degradation
patterns between brown-rot species under this study.
According to the IEC (Table 1), C. puteana could be
regarded as less active in consuming cellulose, while SEM
showed opposite results i.e., smooth breaking surface
already in early decay stages. The smooth feature observed
in the fractured S
2
layer can be explained (Encinas et al.,
1998) as a consequence of the effects on the degree of
depolymerization of cellulose chains by enzymes. It is
supposed that C. puteana caused rapid depolymerization of
cellulose chains throughout the cell wall without a
considerable loss of cellulose.
Weight loss in the case of both fungi after 4 months
fluctuated in the range of 40–47% with the higher value of
P. placenta, while SEM micrographs illustrated uneven
wood breakdown by the fungus throughout the test.
According to Highley (1977),P. placenta contains en-
doglucanases and glycosidases, but not exoglucanases,
while Coniophoroid brown-rots produce the full enzyme
complement (Highley, 1988). Endoglucanases are supposed
to attack in the middle of the more disordered regions of
cellulose, and exoglucanases the crystalline areas at the
opposite chain ends. A typical endoglucanase cleaves the
cellulose chains, resulting in a rapid decrease in the degree
of polymerization (DP), while exoglucanases decrease the
substrate DP very slowly. The endo–exo synergy enhances
the activity of the individual enzymes (Teeri, 1997). It is
supposed that P. placenta tended to degrade cellulose
amorphous regions more readily, while the crystalline ones
remained less damaged. This could affect the wood fracture
and reveal the cell wall surface as uneven. C. puteana with
the endo–exo enzymes was able to degrade both amor-
phous and crystalline regions more readily, that appeared
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Fig. 2. Degradation sequence of wood blocks decayed by P. placenta in 4 months.
Fig. 3. Degradation sequence of wood blocks decayed by C. puteana in 4 months.
I. Irbe et al. / International Biodeterioration & Biodegradation 57 (2006) 99–106102
in micrographs as a smooth fractured surface. The
fractured surface of wood cell reflected the pattern of
degradation of cellulose microfibrils.
SEM micrographs showed that the S
3
layer remained
sound after P. placenta attack, but was altered in some
locations after C. puteana decay (Fig. 3). Endo- and
exoglucanases of C. puteana probably more intensively
affected the S
3
layer causing erosion and thinning of the
cell wall. Degradation of the S
2
layer by brown-rot fungi
without causing noticeable loss of the S
3
has been
previously reported by Highley and Murmanis (1985).It
is because of a more compact layer of residual lignin in the
S
3
layer. The alterations in the S
3
could be explained by the
differences in enzymatic systems between both brown-rot
species.
CML in brown-rotted wood revealed smooth structure
from the beginning to the end of test. In P. placenta-
decayed wood, a visible boundary between the secondary
wall and CML was observed by the end of test. In C.
puteana-attacked wood, the difference between both layers
had disappeared by the second month of degradation,
when the secondary wall looked like the CML. We assume
that the smooth fractured surface of the CML was because
of a high amount of lignin. The cell wall ultrastructure of
brown-rotted wood studied by TEM (Eriksson et al., 1990)
provided evidence of deposits of unhydrolysed lignin
throughout the cell wall, with the greatest amount in the
CML. It is known (Fengel and Wegener, 1989) that the
CML of softwoods contains high amount of lignin, around
60%, in comparison with the secondary wall where it is
around 27%. Cellulose comprises around 14% in CML,
while in secondary wall it is around 60%.
3.3. Water vapor sorption
Fig. 4 shows the isotherms of sorption–desorption of
water vapors of wood samples after a 2-month exposure
with the fungi C. puteana and P. placenta in comparison
with the corresponding isotherms of sound wood control.
For all isotherms, the presence of hysteresis in the whole
range of P/P
0
is typical, which is connected with a partial
inclusion of the sorbate in the structure of non-rigid
sorbent upon desorption.
The hysteresis loop width depends on the strength of
interstructural bonds of the sorbent and grows with their
increase. It narrows on all degraded samples after 1 month
of exposure (weakening of bonds after the removal of
hemicelluloses). With increasing exposure time, the P.
placenta hysteresis loop grows until 4 months of exposure
owing to the formation of new interstructural bonds after
the sample’s biodegradation. In the case of C. puteana, the
widest hysteresis loop occurs after 3 months of exposure.
Fig. 5 shows the change in the structural characteristics
of wood depending on the exposure time. The initial (after
1 month) decrease of specific surface (A) is connected with
the removal of hemicelluloses, which, owing to a low MW,
ensures a higher swelling in the initial wood. In this case,
the concentration of hydrophilic centers (a) grows. These
centers are released upon the removal of low-molecular
carbohydrates and do not manage to compensate at the
expense of re-structuring. The further decrease in the A
value occurs owing to the decrease of MW of carbohy-
drates due to degradation, as well as the enrichment of
samples in lignin, whose swelling in water vapors is lower
in comparison with that of carbohydrates.
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Control
0
50
100
150
200
250
300
0 0.2 0.4 0.6 0.8 1
0 0.2 0.4 0.6 0.8 1
0 0.2 0.4 0.6 0.8 1
a (mg/g)
P. placenta
0
50
100
150
200
250
300
a (mg/g)
C. puteana
-50
0
50
100
150
200
250
300
P/Po
a (mg/g)
Fig. 4. Water vapor sorption–desorption isotherms of sound wood and
wood decayed by P. placenta and C. puteana for 2 months (sorption—
circles; desorption—cubes).
I. Irbe et al. / International Biodeterioration & Biodegradation 57 (2006) 99–106 103
The mass concentration of hydrophilic centers (a
m
) after
1 month of exposure on both cultures grew, since the
increase in surface concentration (a) prevailed over the
decrease in the specific surface. Hereafter (up to 3 months
of exposure), the a
m
value decreased owing to the decrease
of both Aand a. After a 4-month exposure on fungi, the
decrease of the Avalue on P. placenta slowed down, and
even grew somewhat on C. puteana in the case of a
simultaneous increase of the surface concentration of the
hydrophilic centers, which resulted in an increase in a
m
.
The average pore size for the swollen wood grew with
increasing biodeterioration time (Fig. 5), reaching the
highest values (4.5 nm) after 3 months of exposure. It
should be mentioned that the samples investigated were
subjected to drying at 103 1C after the exposure. Such a
treatment caused a partial collapse of the structure, which
resulted in a partial loss of information during the analysis
of the sorption branch of the isotherm. However, in the
process of saturating with water vapors, the wood structure
relaxed, and the desorption branch of the isotherm could
be regarded as more equilibrium.
From the desorption branches of the isotherms,the curves
of distribution in volume sizes were plotted for the pores in
size range 3–200 nm (Fig. 6). Table 2 lists the sizes and
volumes of such pores in biodeteriorated wood. These data
show that new pores (D¼2:1–2.5 nm) appeared in the
samples exposed to P. placenta after the first month of the
experiment. With time, the size and volume of these pores
grew. These values were close to those found by Flournoy
et al. (1991), who investigated the porous structure of moist
wood after the decay by P. placenta for 1 month by the
method of sorption from aqueous solutions of substances
with different molecular sizes.
In contrast to P. placenta, the degrading action of C.
puteana manifested itself in a different way. new pores in
the specified size range appeared only after a 3-month
exposure, and their size and volume were less than those of
P. placenta.
It is of interest to elucidate the reasons for the
appearance of these pores and the distinctions in the
behavior of the two fungi. Flournoy et al. (1991) assigned
the newly developed pores to the sizes of the active agent of
the fungus. However, even the cell wall of sound wood in
the swollen state contains a great volume of rather wide
pores. The average pore size for intact pinewood was
3.8 nm. Obviously, such pores, being absent in dry wood,
are accessible not only to low-molecular substances, but
also to rather large molecules. Besides, the actual distribu-
tion of pore volumes in their sizes should be taken into
account, namely, the wood samples had pores, whose size
exceeds the average one.
At the same time, all known enzymes exceed the
micropore size of the intact structure of the wood matrix,
while brown-rot fungi employ a low molecular weight
decay system and rapidly depolymerize cellulose (Goodell
et al., 2003).
The formation of new pores ranged from 2.1 to 9.9 nm
(Table 2) depends on the fungus and exposure time.
It is assumed that the appearance of small pores is
connected with the destruction of cellulose and the
‘‘imperfection’’ of the wall structure. The essential distinc-
tion between the two fungi under study is, as has been
mentioned above, in the different nature of enzymes,
namely, endo- and exoglucanases. Probably, the degrada-
tion of the cell wood attacted by C. puteana occurs firstly
mainly on the wall surface, but in the case of P. placenta
the destruction of cellulose even after 1 month of exposure
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100
150
200
250
300
350
01234
01234
01234
01234
A, m2/ g
1
1.5
2
2.5
3
3.5
am, mM/g
3
4
5
6
7
8
α, groups/nm2
2
2.5
3
3.5
4
4.5
5
Time (months)
Daver, nm
P. placenta C. puteana
Fig. 5. Microstructural characteristics (specific surface A, mass and
surface concentrations of hydrophilic centers a
m
and a, average pore size
D
aver
) of pinewood depending on the exposure time on P. placenta and C.
puteana.
I. Irbe et al. / International Biodeterioration & Biodegradation 57 (2006) 99–106104
proceeds deep inside the wall. This assumption is indirectly
confirmed by microphotographs (Figs. 2 and 3), as well as
the higher activity of P. placenta in comparison with C.
puteana.
Acknowledgement
The authors gratefully acknowledge Dr. J. Puls (Institute
of Wood Chemistry and Chemical Wood Technology,
Federal Research Centre for Forestry and Forest Products,
Hamburg) for the fruitful discussion of the polysaccharides
part of biodegraded wood.
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ARTICLE IN PRESS
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
r, nm
dw/dr
P. placenta, 2 months P. placenta, 4 months
C. puteana, 2 months C. puteana, 4 months
Fig. 6. The distribution of pores by specific size in brown-rotted wood.
Table 2
Characteristics of the porous structure of destructed wood samples
Fungus Time Drange (nm) Volume of pores
(months) in the range (cm
3
/g)
Control — — —
P. placenta 1 2.1–2.5 0.015
2 3.0–83 0.065
3 3.1–9.9 0.055
4 3.1–9.9 0.070
C. puteana 1— —
2— —
3 2.3–3.6 0.020
4 2.7–8.3 0.035
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