In: Llewellyn, G. C.; Dashek, W. V.; O’Rear, C. E., eds.
Biodeterioration research 4: Mycotoxins, wood decay,
plant stress, biocorrosion, and general biodeterioration:
Proceedings of 4th meeting of the Pan American Biodeterioration
Society; 1991 August 20-25; as an electronic symposium.
New York: Plenum Press: 257-293; 1994.
CHEMICAL CHANGES IN WOOD COMPONENTS AND COTTON
CELLULOSE AS A RESULT OF BROWN ROT:
IS FENTON
CHEMISTRY INVOLVED?
DOUGLAS S. FLOURNOY, USDA Forest Service, Forest Products Laboratory,
Madison, WI 53705-2398 USA
INTRODUCTION
The most destructive form of wood decay, brown rot, is caused by a
relatively small number of species of Hymenomycetous basidiomycetes. These
basidiomycetes are unique among cellulose destroyers because they are the only
known microbes that can degrade the cellulose in wood without first removing
the Iignin (Cowling, 1961; Liese, 1970). Brown-rot fungi leave a brown
residue–hence their name–that has been partially o-demethylated (Kirk, 1975).
Furthermore, brown-rot fungi degrade cellulose in an unusual manner that differs
from that of other cellulolytic organisms. Hyphae of these ubiquitous fungi
invade wood cells and bring about a rapid depolymerization of the cellulose with
low losses in total wood substance (Cowling, 1961; Kayama, 1962b). The
average number of glucosyl residues per cellulose molecule (degree of
polymerization, DP) is thereby reduced from about 10 4 (Goring and Timell, 1962)
to about 200 (Cowling, 1961). The resulting fragments correspond to the size
of the cellulose “crystallites.” This effect is thought to be brought about by
cleavages within the amorphous regions of the cellulose that separate the
crystallites (Cowling, 1961). Similar depolymerization of cellulose to the “limit
DP” (to the crystallites) is effected by acid hydrolysis (Battista, 1950) and by
chemical oxidants (Koenigs, 1972a, 1974a,b, 1975; Highley, 1977; Kirk et al.,
1991). As a result of the initial attack by brown-rot fungi and the
depolymerization of the cellulose, wood strength collapses. How this rapid
Biodeterioration Research 4, Edited by G.C. Llewellyn
et al., Plenum Press, New York, 1994
257
depolymerization occurs is a perplexing biochemical question: as Cowling and
Brown (1969) recognized over two decades ago, even the smallest cellulases
(approximate diameter 25 _, length 140 _) are too large to penetrate the pores
of wood (median pore diameter approximately 10 _; maximum 35–100 _). AIso,
cellulases do not mimic the action of brown-rot fungi in generating cellulose
crystallites (Chang et al., 1981; Phillip et al., 1981). Our own examination of
the change in pore structure of wood as it is decayed by a brown-rot fungus
suggests that the depolymerizing agent is between 12 and 38 _ in diameter
(Flournoy, 1991; Flournoy et al., 1991).
Thus, brown-rot fungi seem to utilize an entirely different mechanism to
degrade cellulose than do other cellulolytic organisms. The biochemistry and
physiology of the degradative system employed by brown-rot fungi to
accomplish this feat are far from clear. The prevailing hypothesis implicates low
molecular weight transition metal (usually iron) chelates, which act in two
possible ways: (1) through biochemical conversion into potent oxidizing species,
which diffuse into the wood pores and oxidatively cleave the cellulose, or (2)
through participation in the biochemical generation of an activated oxygen
species (hydroxyl radical or equally potent metallo-oxygen species), in close
juxtaposition to the cellulose, which oxidizes the cellulose leaving the Iignin
substantially unchanged, This review is a critical examination of this hypothesis.
We begin by examining what is known about the chemical changes in wood
components and cotton cellulose as a result of brown-rot decay. The literature
concerning the production of reduced oxygen species by brown-rot species is
then reviewed. We conclude by considering the action of Fenton’s reagent on
wood, Iignin, cotton cellulose, and related systems.
BACKGROUND
In discussing the problem of brown-rot depolymerization of cellulose,
Cowling and Brown (1969) noted that Halliwell (1965) had described the
degradation of cotton cellulose by Fenton’s reagent (H 2O 2/Fe 2+), which generates
hydroxyl radical or a similar oxidant (Halliwell and Gutteridge, 1988). Based on
these observations, Halliwell was the first to propose the possibility of the
existence of a nonenzymatic cellulolytic system involving peroxide and iron.
Subsequently, Koenigs (1972a,b; 1974a,b, 1975) demonstrated that cellulose
in wood can be depolymerized by Fenton’s reagent, that brown-rot fungi produce
258
extracellular hydrogen peroxide, and that wood contains enough iron to make
Halliwell’s hypothesis reasonable. In commenting on this hypothesis, Koenigs
(1974a) stated that “the hypothesis is reasonable pending proof that brown-rot
fungi oxidize cellulose. ” Support for an oxidative system was later provided by
Highley (1977), who obtained evidence that brown-rotted cellulose contains
carbonyl and carboxyl groups. Later, Schmidt and co-workers (Schmidt, 1980;
Schmidt et al., 1981) suggested that oxalic acid, which is secreted by brown-rot
fungi in liquid cultures (Takao, 1965), can reduce Fe 3+ to Fe2+ under certain
conditions, and proposed that oxalic acid acts to generate Fe2+ for the Fenton
system. Some authors have suggested a more direct role for oxalic acid in
depolymerization of cellulose. Shimada and co-workers (Shimada et al. 1991;
Akamatsu et al., 1991) reported that oxalic acid decreased the viscosity of kraft
pulp and proposed that oxalic acid may depolymerize cellulose during brown-rot
decay.
In an attempt to gain direct evidence for a nonenzymatic decay
mechanism, Cobb (1981) grew liquid cultures of Gloeophyllum
trabeum in an
apparatus in which uniformly labeled [ 14C]cellulose was separated from the
fungus by an ultrafiltration membrane. The membrane was reported to prevent
the passage of enzymes between the two chambers. Under these conditions,
[14C]CO 2 was detected and radioactive water-soluble products accumulated in
the growth chamber. These observations have been taken as proof of a
nonenzymatic cellulose decay mechanism being employed by Gl. trabeum (Cobb,
1981; Eriksson et al., 1990).
The existence of a low molecular weight enzyme responsible for
depolymerization, a cellulase, cannot yet be ruled out. Little is known about the
cellulase system of brown-rot fungi. The most striking feature is the apparent
lack of C 1 (ß-1,4-glucan cellobiohydrolase) activity (Nilsson, 1974). A few
cellulases of brown-rot fungi have been isolated and characterized, but little is
known about their chemical and physical properties (Highley, 1975). An
endo-1,4-ß-glucanase of L e n z i t e s trabea has been purified and partially
characterized (Herr et al., 1978). It has a M r of ~29,000, shows maximal
activity at pH 4.4 and 70C, and appears to be a typical endocellulase. Welter
and co-workers (1980) isolated a polysaccharide-degrading complex from Postia
placenta with an apparent Mr of 185,000, which showed activity against eight
substrates. Similar results were obtained from solid substrate cultures of P .
259
placenta (Highley et al., 1981;
Highley and Wolter, 1982). None of these
isolated proteins showed activity against highly ordered substrates such as
microcrystalline cellulose (avicel) or was able to depolymerize cotton cellulose.
Enoki et al. (1989) reported the ability of brown-rot fungi to oxidize
2-keto-4-thiomethylbutyric acid (KTBA) to ethylene; KTBA is converted to
ethylene by one-electron oxidants such as hydroxyl radical. Ethylene production
was correlated with weight loss but not cellulose depolymerization. Recently,
Enoki et al. (1990) reported the isolation of an extracellular protein from cultures
of Gl. trabeum requiring H2O 2 and capable of KTBA oxidation. These authors
partially purified the protein and reported it to be an iron-containing glycoprotein
of Mr ~ 1600-2000, Based on their work, Enoki and co-workers have suggested
the existence of “a unique wood-component degrading system that participates
directly or indirectly in the fragmentation of cellulose as well as of Iignin in wood
and oxidizes KTBA to give ethylene.”
However, it is yet to be established
whether the H2O 2-dependent KTBA-oxidizing ability of this protein is related to
cellulose depolymerization. Similar glycoproteins were isolated from white-rot
and soft-rot fungi (Enoki et al., 1991), raising questions about their role in wood
decay.
If the brown-rot fungi do employ a Fenton-type system, it seems likely
that the Fe (or other metal) is complexed and some control exerted over its
action. This control may be expressed through chelation of the metal ion
involved. Chandhoke and co-workers (1991) purified siderophores from the
brown-rot fungus Gl. trabeum. They demonstrated that purified siderophores
were able to carry out the one-electron oxidation of KTBA and the Fenton
reaction. Reaction rate was influenced by siderophore, iron, manganese, and
oxalate concentration and pH. Cleavage of cellulose azure in the presence of Fe
was demonstrated to be directly correlated with siderophore concentration.
Rapid progress is being made in the isolation and characterization of these
extracellular proteins, siderophores, and chelators (Fekete et al., 1989; Enoki et
al., 1991; Jellison et al., 1990, 1991). Yet, no one has demonstrated a cell-free
system capable of depolymerizing cotton cellulose in the manner characteristic
of brown-rot fungi.
A solid substrate system for brown-rot fungal
depolymerization of isolated cellulose has been described (Highley, 1977). Using
this system, some progress has been made in understanding the chemical nature
260
of cellulose depolymerization by brown-rot fungi (Highley, 1977; Highley et al.,
1988, 1989; Kirk et al., 1989, 1991).
CHANGES IN CHEMICAL PROPERTIES OF WOOD COMPONENTS AND COTTON
CELLULOSE
Lignin Properties
Cowling’s (1961) comparative study of wood decay by white- and
brown-rot fungi showed that the Iignin content of brown-rotted wood remains
relatively constant when expressed on the basis of original sound wood. Thus,
brown-rot fungi do not utilize Iignin to an appreciable extent. However, several
lines of evidence indicate that the Iignin remaining after consumption of the
carbohydrate fraction of the wood is altered. Early work (Cowling, 1961, and
references therein) suggested that the methoxyl content of brown-rotted Iignin
is decreased compared to that of sound wood. In addition, the volubility
properties of the Iignin have been altered (Cowling, 1961; Brown et al., 1968;
Kirk, 1975). Lignin from decayed samples possesses greater volubility in water
and 1% sodium hydroxide than does Iignin from sound wood (Cowling, 1961),
These results indicate that brown-rot fungi at least modify the properties of Iignin
during wood decay. No major structural changes have been demonstrated to
occur in the Iignin, such as depolymerization, and several points of evidence
argue against depolymerization (Brown et al., 1968): (1) acid-soluble Iignin does
not increase with carbohydrate removal, (2) the molecular weight of
formamide-soluble Iignin increases rather than decreases with increasing extent
of carbohydrate removal, and (3) structural integrity of the wood is maintained
even at high weight loss.
Wood that has been extensively degraded by brown-rot fungi consists
almost entirely of Iignin residue. Several structural studies have been made of
this residue. In the earliest of these, Kirk and Adler (1969) detected the presence
of catechol moieties in brown-rotted Iignin. Using L . trabea (= Gl. trabeum),
“enzymatically liberated Iignin” was prepared by extensively degrading the
sapwood of sweetgum. This Iignin residue was ethylated with diethyl sulfate and
then oxidized with permanganate and hydrogen peroxide, Gas chromatography
of the methyl esters of the resulting acids revealed that four compounds were
prominent.
These compounds were identified as the methyl esters of
3-methoxy-4-ethoxybenzoic acid (l), 3,4-diethyoxybenzoic acid (II),
3,5-dimethoxy-4-ethoxybenzoic acid (Ill), and 3,4-diethoxy-5-methoxybenzoic
acid (IV).
Figure 1. 3-methoxy-4-ethoxybenaoic acid
Figure 2. 3, 4-diethyoxybenzoic acid
Figure 3. 3, 5-dinethooxy-4-ethoxybenzoic acid
Figure 4. 3, 4-diethoxy-5-methoxybenzoic acid
Compounds I and Ill are formed from phenolic guaiacyl- and syringyl-type
substructures of sound Iignin, as expected. But the presence of compounds II
and IV indicates that catechol groups are formed in the degraded Iignin by action
of the brown-rot fungus L. trabea, (= Gl. trabeum).
Kirk and Adler found that
nearly 30% of the phenolic guaiacyl and syringyl unit had undergone net
demethylation. Similar results were found with Poria monticola (= Postia
placenta).
Further studies (Kirk and Adler, 1970) showed that demethylation was not
limited to phenolic substructures and that a hydroxyl group adjacent (ortho) to
262
Figure 5. Methyl esters of 2, 3, 4-trimethoxybenzoic acid
Figure 6. 2, 3, 4, 5-tetramethoxybenzoic acid
the methoxyl group was not required for demethylation, The extent of
demethylation of phenolic units of both guaiacyl and syringyl types was found
to be greater than that in corresponding nonphenolic units. Also phenolic and
nonphenolic syringyl-type subunits were more extensively demethylated than
were guaiacyl subunits.
Kirk et al. (1970) characterized Iignin isolated from sweetgum wood
decayed by L. trabea (= Gl. trabeum) and compared it to milled wood Iignin of
sound sweetgum wood. The Iignins were methylated, oxidized with
permanganate and hydrogen peroxide, and finally esterified. The resulting
mixtures of methyl esters were examined by gas chromatography mass
spectroscopy (GC-MS). Two compounds were produced from Iignin of
brown-rotted wood that were not produced from Iignin recovered from sound
wood. These compounds were identified by GC-MS as the methy esters of
2,3,4-trimethoxybenzoic acid (V) and 2,3,4,5-tetramethoxybenzoic acid (VI). Kirk
and co-workers concluded that the Iignin substructures had been hydroxylated
as a result of fungal action and that this hydroxylation had occurred ortho to the
side chains of phenylpropane units of Iignin.
Brown-rotted Iignin has similar but not the same volubility properties as
that of milled wood Iignin (Kirk, 1975). Also, the sulfuric acid Iignin value of
brown-rotted Iignin is lower than that of sound Iignins; carbohydrate content is
similar to that of sound Iignin. Elemental analysis of brown-rotted Iignin indicated
that the Iignin was slightly enriched in oxygen and somewhat depleted in carbon
and hydrogen compared to sound Iignin. On a C9-unit basis, the brown-rotted
Iignin was about one oxygen atom rich in nonmethoxyl oxygen. Functional group
analysis indicated the carboxyl content of brown-rotted Iignin was twice that of
milled wood Iignin. In addition, the total hydroxyl content was somewhat higher.
This increase in hydroxyl content is associated with the aromatic residues rather
than the side chains. Thus, the phenolic hydroxyl content of brown-rotted Iignin
was more than twice that of milled wood Iignin. Aliphatic hydroxyl content was
depressed. This result is consistent with the observed increase in conjugated
carbonyl content of brown-rotted Iignin and with earlier findings that brown-rot
fungi hydroxylate and demethylate Iignin.
The ability of brown-rot fungi to mineralize synthetic [14C]lignin was first
reported by Kirk and co-workers (Kirk et al., 1975). These fungi mineralize
predominately methoxyl carbon but also some side-chain and aromatic-ring
carbon. These results are in accord with results of demethylation studies (Kirk
and Adler, 1969; Kirk and Adler, 1970). Haider and Trojanowski (1980)
examined the ability of several brown-rot fungi to mineralize synthetic [14C]lignin
(DHP). In liquid culture medium, all the carbons were mineralized to varying
extents but the dominant reaction was demethylation. Mineralization of the
methoxyl carbon was repressed by cellulose and hemicellulose, and more so by
glucose, cellobiose, and xylose. It is difficult to say with certainty what these
results indicate because the molecular weight distribution of the DHP was not
reported and mineralization may have resulted from intracellular oxidation of a
low molecular weight component of the [14C]DHP. Haider and Trojanowski also
reported the extensive mineralization of phenolcarboxylic and cinnamic acids and
coniferyl alcohol by brown-rot fungi. Ring cleavage was observed in these latter
compounds, and it is likely that these reactions occur intracellularly.
Jin and co-workers (1990b) isolated brown-rotted Iignin and methylated
it with
14
C H3l, producing [3, 4-O- 1 4 C H3]lignin. Incubation of this Iignin with
brown-rot fungi resulted in generation of
14
C O2 when added to solid substrate
cultures. Mineralization was stimulated in an atmosphere of oxygen. Cultures
supplemented with nutrient nitrogen suppressed degradation of methoxylcarbon,
and supplemental carbon (glucose or glycerol) had little or no effect in the
presence of nutrient nitrogen. However, addition of only these carbon sources
to the cultures sharply suppressed mineralization of the methoxyl carbon.
Extraction and molecular
weight
analysis
of
undegraded
[14C]methoxyl-lignin from the active cultures revealed that on incubation with Gl.
trabeum, the molecular weight of the [ 1 4 C]lignin actually increased during
incubation. Because no [ 1 4 C]-labeled low molecular weight compounds
accumulated in the cultures, Jin and co-workers (1990b) concluded that the
demethylating activity was extracellular.
264
Ander et al. (1984) and other authors (Enoki et al., 1985, 1988) showed
that some brown-rot species are capable of degrading Iignin model compounds
when grown on agar plates and somewhat less capable in liquid cultures. The
nature and meaning of this degradation are unclear because the products have
not been characterized. Mineralization was not demonstrated. Since these are
low molecular weight uncharged molecules, it is unclear whether this
degradation is intra- or extra-cellular.
Although a great deal has been learned about the chemical modification
of the Iignin polymer by one brown-rot fungus, Gl. trabeum, caution should be
exercised in extending these results to brown-rot fungi in general. Brown-rot
fungi, like white-rot fungi, show great variability in their behavior, which can
depend on culture conditions and the nature of the substrate. Further studies
concerning the action of other brown-rot fungi on Iignin are needed before any
broad generalizations can be made.
Cotton Cellulose Properties
To our knowledge, only two reports have addressed the chemical
properties of degraded cellulose from brown-rotted wood. Bray and Andrews
(1924) observed a decrease in both the quantity and quality of wood cellulose
as a function of weight loss. These conclusions were based on the observation
of decreasing percentage of a-cellulose content and increasing percentage of
alkali volubility with increasing weight loss. Bray and Andrews also observed that
reducing power increased as cellulose content decreased. Kayama (1961,
1962a) also found an increase in the copper number of pulp recovered from
wood decayed by Poria vaporaria relative to the copper number of pulp from
sound wood.
Although little information is available on the chemical properties of
cellulose decayed by brown-rot fungus, substantial information is available on
chemical characterization of brown-rotted cotton cellulose. Highley (1977)
compared the oxidative properties of brown-rotted cellulose and cellulose treated
with oxalic acid or H2O 2/FeSO 4. In conducting these studies, Highley compared
the copper number, weight loss in alkaline boil, pH in 10% NaCl, and ion
exchange capacity of the various cellulose. These results are presented in
Table 1.
As the data in Table 1 show, brown-rotted cellulose has increased copper
number, increased weight loss on boiling in sodium hydroxide, decreased pH in
10% NaCl, and increased ion-exchange capacity relative to native cellulose.
265
Brown-rotted cellulose also gave a positive test with ferrous sulfate–potassium
ferricyanide, indicating the presence of oxycellulose. No increase in uronic acid
content of the brown-rotted cellulose was detected. Similar results were
obtained from cellulose treated with 1% H2O 2 and ferrous sulfate.
Highley found that mycelium alone (P. placenta) possessed ion-exchange
capacity, but he did not quantify this capacity. Although efforts were made to
remove the mycelium from the samples prior to analysis, microscopic
examination revealed that not all the mycelium could be removed; therefore the
values in Table 1 may be slightly high.
Infrared spectra showed that carbonyl groups had been introduced into
brown-rotted cellulose. Spectra of mycelium also showed the presence of
carbonyl absorbance, but they did not correspond to spectra of brown-rotted
cellulose. This indicated that the carbonyl groups present in the mycelium did not
contribute to the carbonyl absorbance observed in the brown-rotted cellulose.
The spectra of brown-rotted cellulose were similar to the spectra of cellulose
treated with H2O 2/FeSO 4.
In following up this work, Highley and co-workers (1988, 1989) produced
infrared spectra (obtained by diffuse reflectance) of undegraded cotton cellulose
that showed a low intensity broad carbonyl band centered at ~ 1740 cm-1. This
finding is consistent with the presence of a reducing-end group at the end of
each cellulose molecule. In brown-rotted cellulose, this band has greatly
increased intensity and is centered at a slightly lower wavenumber. Reduction
of the brown-rotted cellulose sample with sodium borohydride resulted in a
decrease in the intensity of the carbonyl band but did not eliminate it completely.
This led the authors to conclude that part of the carbonyl absorbance was due
to the presence of carboxyl groups.
Complete acid hydrolysis of the
brown-rotted cellulose sample led to the observation of several unidentified acids
on HPLC profiles. No uronic acid was reported, and it was suggested that based
on the methylene blue determination of ~ 0.5% mole carboxyls, there was
approximately one carboxyl moiety per cellulose chain in the depolymerized
sample.
Brown-rotted cellulose deploymerization products were further chemically
characterized and compared with other depolymerized cellulose samples (Kirk et
al., 1989, 1991). In this study, the following four depolymerized cellulose
samples were prepared from pure cotton cellulose: (1) acid-hydrolyzed (HCI) to
266
267
the limit DP, (2) H2O 2/FeSO 4-oxidized (Fenton-oxidized), (3) HIO 4/Br 2-oxidized,
and (4) brown-rotted (Postia placenta). These four samples were characterized
as to molecular size distribution, yield of glucose on complete acid hydrolysis,
carboxyl content, uronic acid content, carbonyl content, and sugar acids released
on acid hydrolysis (Table 2). Consistent with earlier results, the Fenton system,
but not the other oxidation system, mimicked the brown-rot system in nearly all
measured characteristics. The acid-hydrolyses sample also possessed similar
characteristics. The following sugar acids were identified by GC/MS in the
hydrolysates of the brown-rotted and Fenton-oxidized samples: glyceric,
erythronic, arabonic, and gluconic. These results were consistent with the
depolymerizing agent being related to the Fenton system, but they did not
establish that the fungi employ such a system.
These studies (Highley, 1977; Kirk et al., 1991) did not establish a
correlation between the oxidation of cellulose and depolymerization. We do not
know,
for instance, whether oxidation
depolymerization,
of the cellulose precedes
whether oxidation is a result of post-de polymerization
modification, or whether oxidation and depolymerization are coupled. The
cellulose used in these studies was highly degraded. Samples from early decay
were not examined; these will be the samples of most interest in understanding
the depolymerization mechanism. In addition, the fungus-decay ensamples in one
study (Kirk et al., 1991) were washed with 0.1 N NaOH at 50C for 3 hr prior to
analysis, which resulted in only 70% recovery of the original sample.
In light of the alkaline lability of oxidized cellulose (Davidson, 1934, 1936,
1940) and the high weight losses observed, we think it prudent to re-examine
the carboxyl content of brown-rotted cellulose with particular attention to
analysis of samples of low weight loss (early decay). Also, the cellulose should
not be exposed to alkali prior to analysis. Brown-rotted cellulose should be
further characterized to determine whether formation of carboxyl groups is
correlated with depolymerization of cellulose. A shortcoming of techniques
currently employed for carboxyl group analysis work is their lack of sensitivity.
At low weight loss, the expected yield is very low (<0.5%), and such a small
number of carboxyls may easily escape detection. Definitive analysis will depend
on the development of more sensitive techniques based on radiochemical
labeling.
268
PRODUCTION OF REDUCED OXYGEN SPECIES BY BROWN-ROT FUNGI
The extracellular formation of reduced oxygen species by brown-rot fungi
remains a controversial topic. This topic has attracted a great deal of research
interest over the past 20 years because early models of cellulose
depolymerization incorporated this reduced oxygen species. Very little
information has been gained since these early studies, and the role of reduced
oxygen species in cellulose depolymerization, if any, remains speculative.
Production of Hydrogen Peroxide
Koenigs (1972b) reported the first observation of extracellular hydrogen
peroxide (H2O 2) production by brown-rot basidiomycetes. In these experiments,
extracellular peroxide was inferred from the green discoloration of sheep’s blood
medium or bovine hemoglobin agar under and around a colony. After 13 days of
incubation, all brown-rot fungi tested produced green discoloration. Koenigs
concluded that H2O 2 had diffused away from the hyphae in these plates. In liquid
cultures containing buffered hemoglobin, all brown-rot fungi examined caused
a decrease in the absorbance of the methemoglobin peak at 630 nm and of the
Soret band at 405 nm, consistent with H 2O 2 formation. Based on these kinds of
experiments, Koenigs concluded that generation of extracellular H 2O 2 was a
general characteristic of wood-rotting fungi. Later, Koenigs (1974a,b) reported
H 2O 2 production by brown-rot fungi grown on wood and correlated its production
with cellulose depolymerization. Unfortunately, the assay for H2O 2 production
employed in these studies (catalase-aminotriazole) was later established by
Highley (1981) to be an invalid method for measuring H 2O 2 by wood decay
fungi. Therefore, the results of Koenigs’ later studies (1974a,b) must be
discounted.
Highley (1982) found only one brown-rot fungus (L. olivascens) capable
of H2O 2 production in liquid culture even though he used many of the same
species as did Koenigs (1972b). Highley (1982) also failed to detect H2O 2 in
sawdust or cellulose decayed by several brown-rot fungi (Gl. trabeum, P .
placenta, L. lepideus). An important control of Highley’s was the incubation of
known concentrations of H 2O 2 with culture filtrates. He failed to observe
lowering of H2O 2 concentrations even after 24 hr of incubation, ruling out the
presence of H2O 2-degrading components.
In these studies, Highley (1982) utilized an o -dianisidine and a titanium
270
tetrachloride reagent for H 2O 2 detection. He examined white-rot fungal cultures
and found H2O 2 when assayed with o -dianisidine, but the same cultures were
negative when assayed with titanium tetrachloride. Efforts to detect H2O 2 with
the titanium reagent had also failed in Koenigs’ hands (Koenigs, 1972). The
explanation for this discrepancy is unknown. One possibility is that the technique
employing o -dianisidine is not selective for H2O 2 and that some other metabolize
is responsible for the positive results attained with this method.
In his thesis, Cobb (1981) correlated growth and H2O 2 accumulation in
stationary liquid cultures of Gl. trabeum. Using the peroxytitanic method for
peroxide quantitation, Cobb examined stationary liquid cultures using a basal
salts medium supplemented with ballmilled southern yellow pine, holocellulose
extracted
from
ballmilled
southern
yellow
pine,
a-cellulose,
carboxymethylcellulose (CMC), or a hemicellulose monosaccharide mixture.
Flasks showing poor or no growth (a-cellulose, hollocellulose, ballmilled southern
yellow pine, and CMC) showed no peroxide accumulation. Those cultures
showing good growth (hemicellulose monosaccharide mixture) gave good
accumulation of peroxide. Cobb concluded that Gl. trabeum does not produce
peroxide except from hemicelluloses. Growth and production of peroxide on a
strictly glucose or cellobiose supplemented medium was not examined.
In cytochemical studies (Highley and Murmanis, 1985a,b), P. placenta was
grown on hemlock sawdust for 6 weeks. Samples were examined for localization
of H2O 2 production by staining with 3,3’-diaminobenzidine tetrahydrochloride
(DAB). On oxidation, DAB binds osmium tetroxide and can subsequently be
visualized by electron microscopy. In control experiments, Coriolus versicolor, a
white-rot fungus, produced large amounts of dark deposits, presumably oxidized
DAB. Hyphae of P. placenta failed to produce a similar deposition. Extracts from
sonicated mycelium of both fungi were positive for catalase activity. Electron
micrographs of the white-rot fungus treated with aminotriazole, a catalase
inhibitor, failed to produce oxidized DAB deposits. Therefore, the electron-dense
regions observed were ascribed to peroxidative oxidation of DAB by catalase. An
important control, omitted in this study, was the treatment of the brown-rot
fungus with H2O 2 and DAB to ensure the catalytic competence of the sample.
In any case, it is apparent that H2O 2 does not accumulate in the mycelium of
brown-rot fungi as it apparently does in white-rot fungi.
White-rot fungi have been found to produce H 2O 2 and enzymes that
271
produce and utilize hydrogen peroxide under conditions of carbon and/or nitrogen
starvation. Highley (1987) proposed that this might be the case for brown-rot
fungi as well; i.e., failure to detect H2O 2 might be due to excess nitrogen or
carbohydrate in the growth medium. Therefore, he examined cultures of whiteand brown-rot fungi grown on agar under varying conditions of nitrogen and
carbohydrate supply. H 2O 2 was detected as coloration of the medium in the
presence of either o -dianisidine or 2,2’-azino-di-(3-ethylbenzthiazole-6-sulphonic
acid) (ABTS) and horseradish peroxidase, which were included in the medium.
Highley found that under conditions of high carbohydrate and high nitrogen, 2
of 13 brown-rot fungi and 5 of 7 white-rot fungi gave positive results with
ABTS. All brown-rot fungi were negative in plates containing o -dianisidine,
whereas the white-rot fungi gave similar results with both reagents.
By varying the nitrogen source, Highley (1987) was able to detect H2O 2
production in 6 of 13 brown-rot fungi tested; 7 fungi failed to give a positive
reaction regardless of the conditions employed. Of those brown-rot fungi that
gave positive results, H2O 2 production was stimulated somewhat at low nitrogen
concentrations. All seven white-rot fungi employed gave positive results. By
varying the carbohydrate source and concentration (0.02% NH 4N O3), Highley
detected H 2O 2 production in all but 2 of the 13 brown-rot fungi tested. Peroxide
production was stimulated by low concentrations of carbohydrate and supported
by cellobiose, glucose, mannose, xylose, and malt extract.
Highley and co-workers (1988) were unable to detect H 2O 2 in liquid
cultures of P. placenta under various oxygen concentrations (0-1.0 atm O 2) using
the titanium reagent or ABTS linked with peroxidase. Similar negative results
were obtained with C. puteana (Highley et al., 1989).
Illman et al. (1989) were able to detect H2O 2 production with ABTS and
horseradish peroxidase in stationary liquid cultures of Postia placenta supported
by several sugars under conditions of nitrogen limitation. The authors did not
report how much time was required for these cultures to develop green
discoloration, which was taken as a positive indicator of peroxide, nor was the
temporal nature of color development explored. Therefore, we do not know
whether the H2O 2 was produced transiently as the result of the presence of a
carbohydrate oxidase for instance or whether H 2O 2 was exported by the
mycelium and accumulated in the liquid medium. Hydrogen peroxide production
was not strictly dependent on nitrogen limitation.
272
Using the horseradish peroxidase and ABTS method for detecting
peroxide, Micales and Highley (1989) found that H 2 O 2 was produced by
degradative and nondegradative isolates of P. placenta under a variety of carbon
and nitrogen conditions. No correlation was found between H2O 2 production and
the ability of the isolates to degrade wood. A nondegradative isolate (ME-20)
was reported to produce large quantities of H 2O 2 but the production was not
quantified.
Recently, Veness and Evans (1989) examined liquid cultures of both
white-rot and brown-rot basidiomycetes, grown under a variety of conditions, for
H 2O 2 production by using an oxygen electrode and catalase. They detected no
H 2O 2 in culture fluids of 14- and 28-day-old carboxymethylcellulose and
7-day-old glucose cultures. Similar results were obtained with the titanium(IV)
assay. However, Ritschoff et al. (1990) detected H2O 2 in two of four brown-rot
fungi when ABTS-peroxidase was added to a solid, wood-based culture medium.
The first attempt to quantify H 2O 2 production by brown-rot fungi was
recently made by Ritschkoff and Viikari (1991a,b). These workers grew liquid
cultures of Serpula Iacrymans (Sl 1), P . placenta (FPRL 208), and Gl. trabeum
(BAM Ebw 109) in liquid culture
with 1% crystalline cellulose (Avicel) or
amorphous cellulose (prepared from Avicel by phosphoric acid treatment). Using
ABTS and horseradish peroxidase, in vivo, the authors measured the intensity of
the formed color at 560 nm during the cultivation. They found that when grown
on crystalline cellulose as the sole carbon source, S. lacrymans and P. placenta
produced peroxide beginning at 4 and 8 days incubation, respectively. Gl.
trabeum did not produce significant amounts of peroxide; the slight color that did
develop in the cultures after 8 days soon disappeared. In the flasks containing
amorphous cellulose, no peroxide was detectable.
The results of these various studies do not lead us to conclude that
production of extracellular H2O 2 is a general characteristic of brown-rot fungi.
Several problems persist, the most serious of which is the lack of a selective and
reliable assay for H 2O 2. The most serious attack of this problem was made by
Veness and Evans (1989), who examined liquid cultures of basidiomycetes with
an oxygen electrode and catalase. Previous studies have employed a chromagen
such as ABTS or o -dianisidine in combination with horseradish peroxidase, and
as noted previously, this system may not be selective for H2O 2.
This approach allows qualitative assessments, but results have been
273
mixed. White-rot fungi, which are generally conceded to be producers of
extracellular H 2O 2, do not always give uniform results (Highley, 1982, 1987).
Thus, it is difficult to interpret data from brown-rot fungi in experiments in which
only one-half of the fungi tested gives a positive result. A critical examination of
Koenigs’ data (1972b) was suggested by Veness and Evans (1989). The
correlations between three tests for H 2O 2 made by Koenigs (1972b) with sheep’s
blood agar, hemoglobin agar, and hemoglobin liquid medium are very poor. We
agree with Veness and Evans (1989) that it is difficult to assign a meaning to
these data.
It has also been noted that some fungi, two white-rot fungi in this
instance, may lose the ability to produce H2O 2 (Highley, 1987) when cultured for
long periods. Similarly, loss of phenol oxidase production by the white-rot fungus
C. versicolor was found to occur when the fungus was kept in culture for long
periods (Szklarz et al., 1989).
It may be that brown-rot fungi produce H 2 O 2 transiently or in an
environment in which it is sequestered or rapidly utilized, and thus it has escaped
detection in cell-free extracts. Only one research group (Ritschkoff and Viikari,
1991a,b) has carefully examined H2O 2 production as a function of culture age.
Most of the results, reported herein, are those obtained at 7 and 14 to 28 days
growth (Veness and Evans, 1989) after 6 weeks growth on sawdust (Highley,
1987) or at 7, 14, and 21 days growth in stationary liquid cultures (Highley
1982) or 7 days still-culture (Koenigs, 1972b), Transient production of H2O 2 may
not be detected in such studies.
Expression of the cellulose-depolymerizing activity of brown-rot fungi has
not yet been accomplished in a defined liquid medium. Production of H 2O 2 may
be tightly coupled to expression of this activity and may not be observed in the
absence of cellulose depolymerization.
At this time, whether brown-rot
basidiomycetes produce extracellular H 2O 2 is not definitively established. The
answer to this question awaits the isolation and characterization of enzymes that
produce or utilize H2O 2.
Production of Radical Oxygen Species
Highley (1982) found that filtrates of 21-day-old cultures of brown-rot
fungi bleached the spectrum of p -nitrosodimethylaniline when cultured on
glucose, suggesting the presence of hydroxyl radical. However, this assay is not
a very selective assay for hydroxyl radical. Organic radicals and easily
auto-oxidizable compounds can produce the same results.
274
In soil-block cultures, wood blocks or cellulose impregnated with a number
of different chelators and radical and peroxide quenching agents had little effect
on weight loss produced by P. placenta, Gl. trabeum or L. olivascens (Highley,
1982; Highley and Murmanis,
1985a). Even blocks impregnated with
chloroamphenicols had no effect on wood decay. This result indicates that
brown-rot fungi may very well circumvent any toxin that may be present in wood
on colonization. Two notable exceptions to this result were thiourea (1.0 M)
(<2% weight loss compared to 64% for control) and fluorouracil (0.001 M)
(~5% weight loss) (Highley, 1982).
Illman et al. (1989) observed the electron spin resonance signal of the
dimethyl-1-pyrroline N-oxide (DMPO) derivative of the hydroxyl radical in liquid
cultures of P. placenta supported by cellobiose or glucose. The appearance of
this signal was time dependent and did not appear until the tenth day of growth.
This signal was also detected in wood slivers of Douglas-fir and white fir. The
exact chemical source of the hydroxyl radical was not determined in this study.
To our knowledge, no report of the involvement of peroxy-radical (•OOH)
in cellulose depolymerization exists in the literature. This radical is easily
generated by protonation of the superoxide anion (O2•) whose pKa is ~4.8. The
superoxide anion has been shown to be an efficient degrader of cotton cellulose
(Thompson and Corbett, 1985). The DP of 1 g of cotton cellulose in 100 mL of
dimethyl sulfoxide (DMSO) containing 15 mg KO 2 is reduced from 6,700 to 313
in 5 hr. The degraded cellulose was not characterized, and how its properties
relate to those of brown-rotted cellulose are unknown.
In a general test for the production of free radicals, Veness and Evans
(1989) grew white- and brown-rot fungi in liquid culture in flasks whose interior
surface had been silvered. Free radicals were detected by the desilvering of these
mirrors in the culture flasks. Several white-rot fungi were able to completely
desilver the flask below the level of the growth medium completely. Several
brown-rot fungi showed little deslivering (Fibroporia vaillantii) or no deslivering
(Lentinus Iepideus, Serpula Iacrymans). Lundborg (1988) suggests that hydroxyl
radicals are formed by brown-rot fungi o the basis of depression formation in
cellulose agar plates by Fomitopsis pinicola. Depression formation was inhibited
by radical scavengers.
As most of these reports concerning free radicals are phenomenological
in nature, it is difficult to assign any meaning to them and little can be concluded
regarding their significance to brown-rot decay.
275
Detection of Peroxidases and Oxidases in Brown-Rot Fungi
Koenigs (1972b) reported the detection of peroxidases using o -dianisidine
as a substrate in two species of brown-rot fungi, Coniophora puteana and L.
lepideus, but the activities reported were very low. Koenigs also reported that
production of H2O 2 by 2- to 3-week-old mycelial mats was supported by glucose.
He used the C-AT-G system to detect peroxide, a method that has since been
discredited (Highley, 1981). Koenigs suggested that brown-rot fungi support
peroxide production from a number of other sugars (xylose, mannose, galatose,
etc.), but no report has appeared in the literature detailing these experiments.
Koenigs’ paper (1972b) summarizes the relevant literature concerning
wood-rotting fungal carbohydrate oxidases up through the late 1960s.
Ferm and Cowling (1972) examined three brown-rot fungi for intracellular
and extracellular activities of Iaccase, tyrosinase, and peroxidase. All these
activities were detected intracellularly at some point in liquid culture growth.
Poria monticola ( = Postia placenta) was negative for all three extracellular
activities. Very low activities of Iaccase and tyrosinase activities were detected
in extracellular filtrate of Poria cocos ( = Wolfiporia cocos) after 3-12 and 3-25
days of growth; peroxidase was detected at day 25. With L . trabea ( = G l .
trabeum), no extracellular Iaccase was detected; low levels of tyrosinase were
detected after 17-25 days of growth and peroxidase after 14-25 days.
Highley (1981, 1982) reported that L . olivascens filtrates produced
peroxide from glucose. However, he could not detect peroxidase activity in that
organism (Highley, 1982) or in other brown-rot fungi. Likewise, Szklarz et al.
(1989) did not find any extracellular oxidase or peroxidase in cultures of L .
trabea ( = Gl. trabeum). Again, these studies were not concerned with a detailed
temporal approach, so it can only be concluded that peroxidase activity does not
accumulate. Highley and Murmanis (1985b) were unable to find alcohol oxidase
activity in sonicated mycelium extracts of P . placenta recovered from liquid
culture.
Enoki et al. (1989) reported the ability of brown-rot fungi to oxidize KTBA;
KTBA is converted to ethylene by one-electron oxidants such as hydroxyl radical
or ferric ammonium sulfate. It is also oxidized by horseradish peroxidase or Iignin
peroxidase in the presence of H 2O 2. Of 10 brown-rot fungi tested, ethylene
production from KTBA was correlated with weight loss from Japanese cedar
sapwood and Japanese beech sapwood. Some cultures reached peaks of
276
ethylene production, which subsequently declined; most showed an increase in
ethylene production in the first 15 to 30 days, which then reached a plateau.
The authors did not correlate ethylene production with cellulose
depolymerization.
These workers (Enoki et al., 1989) also showed that degradation of Iignin
model compounds correlated with ethylene production from KTBA, albeit 8 of
the 10 brown-rot fungi showed no degradative activity against Iignin model
compounds. Cellulose (filter paper) degradation was also correlated with ethylene
production. Again, most of the brown-rot fungi showed little activity against
cellulose. Based on these results, Enoki and co-workers (1989, 1990) have
suggested the existence of “a unique wood-component-degrading system that
participates directly or indirectly in the fragmentation of cellulose as well as of
Iignin in wood and oxidizes KTBA to give ethylene.”
Enoki and co-workers (1989) isolated the extracellular proteins from
wood-containing cultures of brown-rot fungi and examined KTBA oxidation by
these proteins. Under anaerobic conditions, these extracellular proteins produced
ethylene in the presence of KTBA and H2O 2. In the presence of O2, a reductant
such as nicotinamide adenine dinucleotide (NADH) (reduced form) was needed
to stimulate ethylene production. This reaction was inhibited by catalase. Enoki
and co-workers proposed the existence of an extracellular H2O 2-requiring protein
capable of oxidizing KTBA and of oxidizing NADH in the presence of O2 to
generate H 2O 2. These extracellular proteins were incapable of polymerizing
phenols to colored products, in the presence of H2O 2, ruling out phenoloxidase
activity.
Recently, the work of these authors (Enoki et al., 1990) with Gl. trabeum
and Tyromyces palustris has led to a partially purified compound reported to be
an iron-containing glycoprotein of Mr ~ 1600-2000. It is yet to be established
whether the H 2O 2-dependent KTBA-oxidizing ability of these compounds is
related to cellulose depolymerization.
REACTION OF FENTON’S REAGENT WITH LIGNIN AND CELLULOSE
A number of studies have concerned themselves with the reaction of
wood compounds with the Fenton reagent. It would be of interest to examine
the results of these studies and compare these with the known chemistry of
brown-rotted Iignin and cellulose. Such a comparison will allow us to critically
277
evaluate whether a role for the Fenton reagent is plausible in brown-rot decay.
One of the difficulties with interpreting these experiments is the fact that, for the
most part, the reactions have been conducted under conditions not typically
found in brown-rot. With this caveat in mind, we believe it still instructive to
examine the findings.
Reaction With Lignin
Gold and co-workers (1983) synthesized [14C]-ring, [14C]-side-chain, and
[14C]-methoxyl labeled dehydropolymerizates
(DHPs), model Iignin polymers.
14
Exposure of these [ C ] D H PS to molar H2O 2 and 10 mM FeSO 4 in dimethyl
formamide (DMF) for 20 hr led to extensive depolymerization of all three Iignins
as demonstrated by gel filtration chromatography on LH-20. This result suggests
that free hydroxyl radical is probably not operative in brown-rot fungi since only
minor modification of the Iignin is observed even after extensive decay by these
fungi (Cowling, 1961; Kayama, 1961). Although this result does not rule out a
role for the Fenton system, it requires us to conclude that the fungus will need
to devise a mechanism that can deliver such a highly reactive species selectively
to the cleavage sites within the cellulose molecule and that will leave the Iignin
polymer virtually intact.
The only other study of Fenton’s reagent with Iignin that this author is
aware of is that of Tatsumi and co-workers (1980). These authors examined the
oxidation of specifically [14C]labeled pine kraft Iignin with hydrogen peroxide and
ferrous salts under alkaline conditions. When small amounts of H2O 2 (0.5 M)
were employed, condensation of monomer to give dimer and oligmer products
was observed. When a large amount of hydrogen peroxide (1.32 M) was
employed, high molecular weight Iignin was considerably degraded to give low
molecular weight products. Ring rupture was also observed. Analysis of gaseous
products released indicated that a large portion of methoxyl carbon was
converted to CO2 and CH4. This study suggests that demethylation of Iignin by
Fenton’s reagent is feasible.
Several studies have addressed the action of Fenton’s reagent on whole
wood. Koenigs (1974a) found that H 2O 2/FeSO 4 treated sweetgum wood meal
resulted in a Iignin that possessed markedly increased volubility during acid
hydrolysis. Up to 74% of the Iignin in sweetgum could be rendered soluble.
Interestingly, Iignin from Ioblolly pine was not depolymerized. These results
indicate that the substrate may have a profound affect on susceptibility to attack
278
under otherwise identical conditions. On the other hand, Kohdzuma et al. (1991)
treated sugi (Cryptomeria japoica D. Don) heartwood and tochinoki (Aesculus
turbinata Blume) in acetic acid buffer solution and Fenton’s reagent and found
that both holocellulose and Klason Iignin were extensively degraded. In addition,
the change in the density of wood substances as a function of weight loss for
brown-rot decay did not match the relationship obtained from wood treated with
Fenton’s reagent.
Reaction With Model Lignin and Aromatic Compounds
The finding of hydroxylation products in brown-rotted Iignin (Kirk et al.,
1970) is consistent with a role of hydroxyl radical in Iignin modification. Ample
evidence exists that the Fenton system under anaerobic conditions and at low
pH will hydroxylate aromatic ring systems ortho- and para- to phenolic moieties
(Maskos et al., 1990, Walling, 1975). Walling and Johnson (1975) concluded
that most, if not all, hydroxyl radical attack on aromatics occurs via addition to
yield hydroxycyclohexadienyl radicals and that in the presence of adequate
oxidant (Fe3+), phenolics can be made the major products. These authors further
concluded that direct side-chain attack cannot be more than a minor reaction
path. Halliwell (1978) found that hydroxylation of aromatic compounds by
Fenton’s reagent was enhanced by the presence of an iron chelator such as
EDTA. Reactions of nonphenolic Iignin model compounds (Kirk et al., 1985) with
Fenton’s reagent result predominately in demethylation of aromatic methoxy
groups and hydroxylation of the aromatic nucelus.
Reaction With Carbohydrates and Cellulose
Studies have addressed the action of Fenton’s reagent on carbohydrates
(Moody, 1963, 1964). Although much work has been done on the chemistry of
Fenton’s reagent with D-glucose, the action of H 2O 2 and ferrous salts on
cellulose has received relatively little attention (Moody, 1963, 1964).
In an acidic medium and in the presence of Fe 2+ , H2O 2 will oxidize a
primary hydroxyl in monohydric alcohols to an aldehyde or carboxyl group
(Ivanov et al., 1953). In polyhydric alcohols, primary hydroxyl groups will react
first, followed by secondary hydroxyl groups. Presumably, if this mechanism
were operative in brown-rot decay, attack would occur in the amorphous region
of the cellulose and the reactivity of the primary hydroxyl group would not be
diminished by hydrogen bonding, as would be expected of the primary hydroxyl
groups in the crystalline region. Thus, uronic acid residues would be expected
279
in the brown-rotted cellulose, but none has been reported (Highkey, 1977; Kirk
et al., 1991). In their examination of the reaction of cotton with H2O 2 and FeSO 4
at pH 4.6, Inanov and co-workers concluded that oxidation proceeds intensively
in acid medium (pH < 4), that primary hydroxyl are oxidized to aldehyde or
carboxyl groups and that secondary hydroxyls groups are oxidized to ketone
groups with eventual rupture of the ring to form two aldehyde groups, which
may be further oxidized to carboxyl groups.
In a study of the oxidation of methyl ß-D-glucospyranoside,
probably the
simplest model for cellulose, by Fenton’s reagent, de Belder and co-workers
(1963) identified as products the four carbonyl compounds resulting from
oxidation at C-2, C-3, C-4, and C-6, although the predominate reaction is
apparently demethylation to form D-glucose. Numerous studies with cellobiose
have also been conducted. Reactions of cellobiose with Fenton’s reagent (Uchida
and Kawakishi, 1988) led to a much more complex product distribution.
However, (1Æ6)-linked disaccharides are apparently more reactive with •OH
than (1Æ4)-linked disaccharides and α− linkages are more reactive than b-linkages.
The earliest report on cellulose as such simply monitored changes in its
viscosity as it is treated with peroxide and various iron salts (Yamafuji and
Urakami, 1950). These workers observed a decrease in cellulose viscosity and
concluded that the cellulose was depolymerized but nothing else.
Halliwell (1965) examined cotton cellulose degradation by peroxide and
ferrous salts under acidic conditions. He found that the cellulose was first broken
down to very short fibers within a few days, with the accumulation of only a
small amount of soluble products. Solubilization was maximum at pH 4.2-4.3
and greatly retarded at higher ( > 5) and lower ( < 3) pH values.
Koenigs (1974a) reported that the holocellulose of sweetgum and Ioblolly
pine meal was depolymerized by the action of H2O 2/ F e S O4. Cotton cellulose
(Koenigs, 1972a, 1975) was also rapidly depolymerized by Fenton’s reagent.
High peroxide levels were required to effect efficient depolymerization (1% or
0.29 M), which was accompanied by high weight loss (DP ~ 180 at 3 2 . 7 %
weight loss). Kohdzuma et al. (1991) also found the holocellulose of wood
extensively degraded by Fenton’s reagent. Unfortunately, the chemical properties
of these holocelluloses were not determined in these studies.
DISCUSSION
Although much work has been done on the cellulose depolymerization
mechanism employed by brown-rot fungi, any role for the employment by
brown-rot fungi of the H 2O 2/ F e 2+ system in their depolymerization of wood
cellulose remains speculative. No direct evidence points to a role for iron or any
other transition metal and reduced oxygen species in the cellulose
depolymerization mechanism employed by brown-rot fungi.
Some indirect evidence is consistent with a role for the H 2O 2/Fe 2+ system.
For instance, the finding of demethylation (Kirk and Adler, 1969, 1970) and
hydroxylation (Kirk et al., 1970) of brown-rotted Iignin is consistent with the
presence of hydroxyl radical. Other oxygen species can be responsible for these
findings as well (Hall, 1980).
Much data also argue against the presence of the H 2O 2/Fe 2+ system in
brown-rot decay. Probably the strongest argument against its presence in
brown-rot fungi is the lack of ability of many researchers to demonstrate
convincingly the production of H 2O 2 by cultures of brown-rot fungi in liquid
culture; such production has never been demonstrated in solid substrate cultures
(although, as pointed out by Koenigs (1972a), the absolute concentration of
H 2O 2 at any one time may not be important since effects of H2O 2 are expected
to be cumulative). It is possible that constitutive low levels of H 2O 2 have escaped
detection.
Although much work has been performed on the oxidation of cellulose
over the past 70 years, nearly all has dealt with reaction in the alkaline
environment (Nevell, 1985). Little experimental work has been done under acidic
conditions (Daruwalla et al.,
1960), and there are gaps in the literature
concerning this area of cellulose oxidation. In general, two extreme types of
cellulose are generated on oxidation, depending on the reaction conditions
(Davidson, 1940). The first and most commonly studied is that generated by
oxidants under alkaline conditions. This produces an acidic type of oxycellulose
that is characterized by low copper number, high affinity for methylene blue, and
increased lability toward acid hydrolysis. The second kind of oxycellulose is
generated by oxidation under acidic conditions, which produces a reducing type
of oxycellulose characterized by high copper number, excessive weight loss on
alkaline boiling, low methylene blue absorption, and resistance to acid hydrolysis.
The action of the H 2 O 2 / F e2 + system on cellulose is expected to be
oxidative in nature, and similar changes have yet to be demonstrated in wood
or cotton cellulose. Oxidative changes in brown-rotted Iignin are firmly
established. As Koenigs has observed (1974b), “it seems necessary to settle the
point definitively for cellulose.” Indirect evidence for oxidation of cellulose by
brown-rot fungi include observation of high copper numbers in depolymerized
cellulose (Kayama, 1961; Highley, 1977) and increased alkaline volubility of the
depolymerization product (Cowling, 1961; Kayama, 1961,1962a; Highley,
1977). Based on these observations, brown-rot fungi apparently produce a
reducing type of oxycellulose. Direct evidence includes detection of carboxyl
groups (Highley, 1977) and identification of sugar acids (Kirk et al., 1991) in the
depolymerization products. Again, all of this work was performed on highly
degraded samples. An increase in copper number does not necessarily mean that
the cellulose has been oxidized because acid hydrolysis of cotton cellulose also
leads to an increase in copper number. Moreover, increased alkaline volubility
only establishes the presence of low molecular weight cleavage components in
the sample and may not result from alkaline-sensitive linkages of the kind
expected in certain kinds of oxidized cellulose. Thus, unequivocal demonstration
of oxidation of cellulose by brown-rot fungi has not been accomplished.
Hydroxyl radical reacts with carbohydrates at nearly diffusion-controlled
rates. With cellulose, presumably radicals will be generated that will either
eliminate H2O 2 react with molecular oxygen to form peroxy radicals, or undergo
hydrolysis (von Sonntag, 1980). Elimination of water will generate an
oxycellulose that is expected to be labile in alkali and stable in acid, i.e., a
reducing type of oxycellulose. Reaction with molecular oxygen will produce
peroxycellulose radicals that are expected to readily eliminate HO2• and give the
corresponding carbonyl compound, aldoses and ketoses. Hydrolysis leading to
scission of the glycosidic linkage is expected to be fast and will ultimately lead
to oxidation products as well.
A number of investigators have noted that the course and specificity of
the Fenton reaction may be altered by the presence of substrate, pH, and buffer
ions (Smidsrød, et al., 1965; Larson et al., 1967; Zbaida et al., 1988; Bottu,
1991). It has been suggested that in some iron/peroxide systems, in the
presence of the EDTA or DTPA, hyroxyl radical is not even produced (Rush and
Koppenol, 1986; Rahhal and Richter, 1988; Yamazaki and Piette, 1990) and the
reactive species may be an iron-oxo species such as the ferryl ion. Larsen and
Smidsrød (1967) determined that an iron/peroxide/substrate complex may be
operative in some of their oxidations of polysaccharides by Fenton’s reagent.
Zbaida and co-workers (1988) found that the presence of various iron chelators
282
can promote either seletive hydroxylation, N-demethylation, or sulfoxidation of
cimetidine. Iron chelators with high redox potential, i.e., ethylenediamine,
inhibited both hydroxylation and N-demethylation, whereas iron chelators with
low redox potential, i.e.,
EDTA, promoted both hydroxylation and
N-demethylation of cimetidine. This suggests that it is at least possible that
brown-rot fungi can selectively O-demethylate and hydroxylate Iignin with the
Fenton system if the proper chelator were produced. Another factor that must
be considered is the pH of the medium in which the reaction is conducted, as
this will affect the redox potential of Fe(ll)/Fe(lll), the chelation constants, and
the redox potential of H2O 2. Work on biomimetic systems in an effort to model
Iigninases lent insight into these questions (Habe et al., 1985; Dolphin et al.,
1987), Paszcynski and co-workers (1988) successfully demonstrated that
treatment of wood chips and wood pulps with natural heroes and synthetic
porphyrins in the presence of tert- butyl hydroperoxide resulted in extensive
delignification with very little loss of cellulose. It may be possible, given the
appropriate set of conditions, that preferential depolymerization of cellulose can
be achieved with a Fenton-type system that results in little or only minor
modification of the Iignin.
SUMMARY
The mechanism by which brown-rot fungi degrade wood is not
well-understood. The prevailing hypothesis implicates low molecular weight
transition metal (usually iron) chelates, which act through biochemical conversion
into potent oxidizing species, which diffuse into the wood pores and oxidatively
cleave the cellulose, or through participation in the biochemical generation of an
activated oxygen species (hydroxyl radical or equally Potent metallo-oxygen
species), in close juxtaposition to the cellulose, which oxidizes the cellulose
leaving the Iignin substantially unchanged.
This review has addressed chemical changes in wood components and
cotton cellulose as a result of brown-rot fungal degradation. The literature
indicates that chemical changes in brown-rotted Iignin are oxidative in nature and
result primarily in hydroxylation and demethylation of the Iignin polymer. Results
from studies on the chemical changes in cellulose are less definitive, and
oxidative changes in this substrate as a result of brown-rot decay have not been
unequivocally
demonstrated.
283
A review of the effects of Fenton’s reagent on wood components and
model compounds suggests that the effects of brown-rot decay are not
duplicated by this reagent in many respects and therefore, it does not represent
an adequate model for brown-rot decay.
2s4
285
286
287
288
289
290
291
292
●
293