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Appl Environ Microbiol, July 1998, p. 2409-2417, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Role of Organic Acids in the Manganese-Independent
Biobleaching System of Bjerkandera sp. Strain
BOS55
María Teresa
Moreira,1,2
Gumersindo
Feijoo,2,3
Tünde
Mester,3
Pablo
Mayorga,1
Reyes
Sierra-Alvarez,1 and
Jim A.
Field3,*
Division of Wood Science, Department of
Forestry,1 and
Division of Industrial
Microbiology, Department of Food Technology and Nutritional
Sciences,3 Wageningen Agricultural University,
Wageningen, The Netherlands, and
Department of Chemical
Engineering, University of Santiago de Compostela, Santiago de
Compostela, Spain2
Received 5 February 1998/Accepted 16 April 1998
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ABSTRACT |
Bjerkandera sp. strain BOS55 is a white rot fungus that
can bleach EDTA-extracted eucalyptus oxygen-delignified kraft pulp (OKP) without any requirement for manganese. Under manganese-free conditions, additions of simple physiological organic acids (e.g., glycolate, glyoxylate, oxalate, and others) at 1 to 5 mM stimulated brightness gains and pulp delignification two- to threefold compared to
results for control cultures not receiving acids. The role of the
organic acids in improving the manganese-independent biobleaching was
shown not to be due to pH-buffering effects. Instead, the stimulation
was attributed to enhanced production of manganese peroxidase (MnP) and
lignin peroxidase (LiP) as well as increased physiological
concentrations of veratryl alcohol and oxalate. These factors
contributed to greatly improved production of superoxide anion
radicals, which may have accounted for the more extensive biobleaching.
Optimum biobleaching corresponded most to the production of MnP. These
results suggest that MnP from Bjerkandera is purposefully produced in the absence of manganese and can possibly function independently of manganese in OKP delignification. LiP probably also
contributed to OKP delignification when it was present.
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INTRODUCTION |
White rot fungi produce
extracellular oxidative enzymes which initiate the oxidation of lignin
(10, 27). Due to their lignin-degrading capacity, whole
cultures of various white rot fungi cause extensive brightness gains
and delignification of kraft pulp (21, 29, 40, 42). Of all
the extracellular oxidative enzymes produced by white rot fungi,
manganese peroxidase (MnP), lignin peroxidase (LiP),
manganese-independent peroxidase (MIP), and laccase, MnP is considered
to be the most important enzyme involved in kraft biobleaching. The
hypothesis is supported by several observations. A partial correlation
between MnP activity and biobleaching has been found in several
screening programs (2, 21, 40). In time course experiments,
the occurrence of MnP coincides with the period of maximum brightness
gains and delignification (23, 43). MnP-deficient mutants of
the best-characterized biobleaching species, Trametes
versicolor, are not able to cause bleaching, and the bleaching
activity can be restored by the exogenous addition of MnP
(3). Semipurified preparations of MnP have been shown to
cause kraft pulp delignification in vitro (19, 24, 28, 43,
45). Furthermore, prior extraction of manganese (Mn) from kraft
pulp results in the complete loss of biobleaching activity by white rot
fungi (22, 44).
Recently, we demonstrated that the biobleaching activity of the white
rot fungus Bjerkandera sp. strain BOS55 toward
oxygen-delignified kraft pulp (OKP) was not dependent on the
presence of Mn (39). Even when kraft pulp was extracted free
of Mn by EDTA, it was bleached as extensively as pulp supplemented with
Mn. In spite of the lack of Mn, MnP was found to be the major oxidative
enzyme present in fast protein liquid chromatograms; LiP and MIP were also present. The production of MnP by Bjerkandera sp.
strain BOS55 in the absence of Mn nutrients is remarkable because most white rot fungi require Mn for mnp gene expression and
protein production (7, 17). The results suggested that under
Mn-deficient conditions, MnP from Bjerkandera may have roles
in pulp biobleaching.
The addition of physiological organic acids to cultures of
Bjerkandera sp. strain BOS55 was shown to stimulate the
production of MnP (37). Here we report on the stimulatory
effects of simple organic acids on the Mn-independent biobleaching of
eucalyptus OKP. The role of the organic acids in oxidative enzyme
production and secondary metabolite concentrations was evaluated and
compared to pulp brightness gains and decreases in kappa number.
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MATERIALS AND METHODS |
Microorganisms.
Bjerkandera sp. strain BOS55 (ATCC
90940) was maintained and an inoculum was prepared as described before
(36).
Culture conditions.
The standard medium used contained
10 g of glucose per liter, 2.2 mM NH4+-N
as ammonium tartrate, and BIII mineral medium (including a trace
element solution) (54). The trace element solution was modified to provide an initial added Mn concentration of 0, 33, or 330 µM Mn(II) in the medium as MnSO4. The addition of
2,2-dimethylsuccinate (DMS) buffer to the medium was omitted, unless
otherwise stated. In some experiments, organic acids were added to the
medium at 1 to 50 mM. The pH of the organic acid-supplemented medium
was adjusted to 4.5. Control cultures not receiving organic acids were
adjusted to either pH 4.5 or pH 6.0 with NaOH. The medium was
autoclaved (120°C for 20 min), and then a filter-sterilized thiamine
solution (400 mg liter
1) was added (5 ml
liter1). Aliquots of 25 ml of medium were placed in a
presterilized cultivation system as described below and were incubated
statically in air at 27°C for 14 days.
The glassware for experiments conducted under Mn-free culture
conditions was washed thoroughly with HNO3 (5 M) and then
with double-distilled demineralized water to prevent Mn contamination.
The maximal soluble Mn concentration of the Mn-free trace element
solution was 0.423 µM and, when diluted into the final biobleaching assay medium, accounted for approximately 0.004 µM. Direct
measurements of soluble Mn in Mn-free assay medium containing up to 5 mM organic acid supplementation showed a maximal concentration of 0.010 µM. The concentrations of soluble Mn in assay medium supplied with 50 mM organic acid ranged from 0.041 to 0.072 µM.
Biobleaching assays.
OKP from Eucalyptus globulus
was obtained as handsheets (60 g m2) from a kraft mill in
Pontevedra, Spain (ENCE). The OKP had a kappa number of 8.6 and a pulp
brightness of 62 ISO (International Organization for Standardization)
units. In experiments conducted with Mn-free medium, the handsheets
were extracted with EDTA prior to fungal treatment to remove Mn present
in the pulp (39). The residual Mn contamination in the pulp
after EDTA extraction was determined previously to be 0.38 mg
kg1 (39), which would contribute 0.024 µM Mn
in the biobleaching medium.
Fungal biobleaching of 3.8- by 3.8-cm
2 pulp handsheets was
assayed as described previously (
39). All assays were
carried
out in quadruplicate, with the exception of the time course
experiment,
which was conducted with six replicates. Enzyme activities
were
monitored on days 6, 9, and 14 of incubation, unless otherwise
indicated. Day-zero abiotic controls were obtained after 12 h
of
incubation of the handsheets in sterile medium. Before pulp
brightness
and kappa numbers were measured, the handsheets were
extracted with
oxalic acid for 1 h in order to remove precipitated
MnO
2 (
38).
Enzyme assays.
MnP and MIP activities were measured by the
oxidation of 2,6-dimethoxyphenol to coerulignone
(E469, 49,600 M1
cm1) as described by De Jong et al. (11). LiP
activity was measured by the oxidation of veratryl alcohol to
veratraldehyde (E310, 9,300 M1
cm1) (54) and was corrected for veratryl
alcohol oxidase activity. Aryl alcohol oxidase (AAO) activity was
monitored spectrophotometrically as described by Muheim et al.
(41) by monitoring the oxidation of p-anisyl
alcohol to p-anisaldehyde (E290,
15,000 M1 cm1).
TNM reduction.
The formation of superoxide anion radical was
measured by a modified method based on the reduction of
tetranitromethane (TNM) to trinitromethane
(E350, 14,600 M1
cm1) by extracellular fluids (56). The
extracellular fluids were recovered from biobleaching cultures at
selected times over 14 days and were centrifuged (12,000 × g, 10 min). Aliquots (1 ml) of extracellular fluids were
incubated at 30°C in a quartz cuvette together with 1 mM TNM. The
increase in absorbance at 350 nm was monitored for several minutes and
is reported as nanomoles of superoxide formed per milliliter per minute
(e.g., units liter1). No absorbance increase was detected
in boiled extracellular fluids.
Determination of H2O2 concentration.
Centrifuged extracellular fluids were incubated for 20 min at 80°C in
order to inactivate the enzymes present. The
H2O2 concentration was determined by a
modification of the method described by Pick and Keisari
(46). The reaction mixture (1 ml) contained 150 µl of
culture fluid, 200 µl of H2O, 200 µl of 0.5 M sodium
phosphate buffer (pH 6), and 200 µl of 1.41 mM diammonium
2,2'-azinobis(3-ethyl-6-benzothiazoline sulfonate) (ABTS) as a
substrate. The reaction was started by the addition of 200 µl of 5 U
of horseradish peroxidase (Boehringer GmbH, Mannheim, Germany)
ml1. The maximal absorbance at 420 nm was measured after
the peroxidase addition. As a blank, the same reaction mixture was used
after preincubation of the sample with 6 U of catalase from
Aspergillus niger (Sigma, St. Louis, Mo.). A calibration
curve was established with known concentrations of
H2O2.
FPLC.
Concentration of the extracellular culture fluid and
fast protein liquid chromatography (FPLC) of the concentrated filtrate were done as described previously (39), with the exception
that the proteins were eluted with a linear gradient of sodium acetate (pH 6.0) up to 450 mM over 45 min.
Analytical techniques.
Handsheet brightness was determined
with a CM-508i spectrophotometer (Minolta Camera Benelux,
Maarssenbroek, The Netherlands) by method T452 om-92 of the Technical
Association of the Pulp and Paper Industry, Atlanta, Ga. The brightness
is reported as the biologically mediated brightness gain (BMBG), which
is the brightness of the fungus-treated OKP handsheet minus the
brightness of the OKP handsheet incubated in parallel in sterile
culture medium of the same composition. The change in pulp brightness with the sterile incubation was less than 2% (ISO units).
The kappa number of pulp samples was determined with potassium
permanganate according to the micro method (
6).
The Mn concentration was determined with membrane-filtered
(0.2-µm-pore-size filter; Millipore) liquid samples that were
acidified
to pH 2 with HNO
3 by inductively coupled plasma
mass spectrometry
at 257.6 nm (Elan 6000; The Perkin-Elmer
Corp.-Meyvis en Co.,
Bergen op Zoom, The Netherlands). The detection
limit of this
technique for Mn was 0.002 µM.
Organic acids were analyzed by high-performance liquid chromatography
with an Aminex HPX-87H column (Bio-Rad, Veenendaal,
The Netherlands) at
40°C (thermostat controlled). The eluent was
H
2SO
4 (5 mM) at a flow rate of 0.6 ml
min
1. Organic acids were monitored in a UV detector at
210 nm. Compound
identification was carried out by matching retention
times of
the observed products with their standards. The identity of
oxalate
was confirmed by the disappearance of the corresponding peak in
the high-performance liquid chromatograms of selected samples
preincubated with oxalate oxidase (Sigma).
Veratryl alcohol was analyzed by high-performance liquid chromatography
by use of a previously described method (
35).
Chemicals.
All chemicals were commercially available and
were used without further purification.
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RESULTS |
Role of organic acid supplementation.
Biobleaching of OKP and
MnP production by Bjerkandera sp. strain BOS55 were compared
by use of culture medium without and with supplementation with
glycolate (5 mM) and with four different Mn nutrient regimens. These
included two Mn-free media with EDTA-extracted OKP, normal OKP, and OKP
supplemented with 33 and 330 µM MnSO4. The measured
soluble Mn concentration in the Mn-free media with EDTA-extracted OKP
and normal OKP was less than 0.010 µM. Theoretically, the insoluble
Mn content of the pulp contributed 0.024 and 1.480 µM Mn when
expressed as a concentration in the media, respectively. The results in
Table 1 demonstrate that the Mn-free
media lacking glycolate supported limited biobleaching and only traces
of MnP activity. On the other hand, with glycolate supplementation, the brightness gains were comparable to those in media containing Mn
nutrients. Also, the titers of MnP were highly stimulated both in the
presence and in the absence of Mn. The presence of Mn supported limited
oxalate production, whereas oxalate was not detectable at all in the
basal media without Mn or organic acid supplementation (Table 1). The
inclusion of glycolate in the culture medium greatly stimulated oxalate
production in both the absence and the presence of Mn nutrients.
However, the oxalate concentration was higher when Mn was present.
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TABLE 1.
Effect of glycolate on the manganese dependency of
biobleaching of eucalyptus OKP by the white rot fungus
Bjerkandera sp. strain BOS55a
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Aside from glycolate, a large number of other physiological organic
acids supplied at 5 mM supported the stimulation of brightness
gains
and decreases in kappa number (Fig.
1A)
as well as the stimulation
of MnP activity (Fig.
1B) in the absence of
Mn. Some of the acids,
such as succinate, glycolate, oxalate,
glyoxylate, and citrate,
also markedly induced LiP activity. A
nonphysiological acid, DMS,
had only a marginal stimulatory effect on
biobleaching and had
no effect on MnP titers. However, DMS at 20 mM
supported limited
biobleaching stimulation, accounting for a BMBG of
approximately
8.5 ISO units in various experiments (results not shown).
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FIG. 1.
Effect of different organic acids supplied at 5 mM on
BMBG and percent kappa number (KN) decrease (A) and on the peak titers
of MnP and LiP (B) in manganese-free biobleaching cultures with
EDTA-extracted pulp. Error bars indicate standard deviations.
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Many of the physiological organic acids were metabolized during the
course of the experiment (Table
2).
Acetate, glycolate,
glyoxylate, citrate, and succinate supplementation
resulted in
the formation of 0.10 to 0.76 mM oxalate. Media with
acetate,
glyoxylate, malonate, and succinate also resulted in the
formation
of 0.25 to 0.42 mM citrate. Furthermore, it was observed that
succinate was converted stoichiometrically to acetate prior to
being
metabolized to citrate and oxalate.
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TABLE 2.
Effect of adding different organic acids to biobleaching
cultures of Bjerkandera sp. strain BOS55 on the formation of
other organic acid metabolites under culture conditions
lacking Mna
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Effect of organic acid concentration.
The effect of organic
acid concentration on brightness gains and decreases in kappa number in
the absence of Mn was studied for glycolate and oxalate. The results
are shown in Fig. 2. The optimum
glycolate concentration range for brightness gains and decreases in
kappa number was 1 to 5 mM. At higher glycolate concentrations, the
percent decrease in kappa number (delignification) was reduced, while
the brightness gains were affected less. The trend for MnP activity
followed that for biobleaching. LiP activity was present in media
supplied with 3 to 20 mM glycolate. However, there was no measurable
LiP activity at 1 mM glycolate, which supported extensive brightness
gains and delignification. MIP was produced to a lesser extent than the
other enzymes, with optimal activities in media with 5 to 10 mM
glycolate added.
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FIG. 2.
Effect of the initial glycolate concentration (A and B)
and oxalate concentration (C and D) on biobleaching and oxidative
enzyme activities in manganese-free biobleaching cultures with
EDTA-extracted pulp. (A and C) BMBG ( ) and percent kappa number
decrease ( ). (B and D) Peak titers of MnP ( ), LiP ( ), and MIP
( ). Error bars indicate standard deviations.
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Oxalic acid supplementation from 1 to 50 mM supported extensive
brightness gains; however, there was a sharp optimum at 1
mM oxalate
for delignification. Beyond 1 mM, delignification steadily
decreased
with increasing oxalate concentrations. MnP, LiP, and
MIP activities
were present in media supplemented with 1 mM oxalate;
however, optimal
activities were observed with 3 to 5 mM oxalate.
These enzymes were
present only at trace levels or were nondetectable
with 20 to 50 mM
oxalate. At these high oxalate concentrations,
brightness gains were
clearly not due to delignification.
Optimal physiological pH for biobleaching.
The basal medium
used in these studies did not contain buffer. Consequently, the pH in
control cultures dropped during the experiments to values ranging from
3.5 to 3.7. On the other hand, the pH in organic acid-supplemented
cultures ranged from 3.8 to 5.0 during the experiments. Consequently,
the stimulatory effect of the organic acids on biobleaching and
oxidative enzyme production could have been due to their pH-buffering
effect. To exclude this possibility, control cultures were set at an
initial pH of 6.0, which resulted in a drop to pH 4.4 to 4.5. The
results shown in Fig. 3 indicate that the
stimulatory effect of the organic acids was not due to their
pH-buffering effect. The control cultures set at pH 6.0 did not support
the stimulation of brightness gains, even though the physiological pH
was comparable to that of the organic acid treatments. The
physiological pH was defined as the pH on day 6, which was usually the
lowest pH observed during the time period of biobleaching. The pH
optima of biobleaching were studied by supplementing cultures with 5 mM
tartrate and adjusting the initial pH from 3.0 to 6.0. The brightness
gains were plotted as a function of the physiological pH together with
data from other experiments with and without organic acid
supplementation (Fig. 3). The data showed a broad optimal pH range (3.8 to 5.0) for biobleaching by Bjerkandera sp. strain BOS55 in
the Mn-free, organic acid-supplemented cultures.
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FIG. 3.
Effect of physiological pH on BMBG. Results are shown
for control cultures not receiving organic acids and initially set at
pH 4.5 () and at pH 6.0 (). Results are also shown for cultures
receiving from 1 to 5 mM organic acids and initially set at pH 4.5 () and for cultures receiving 5 mM tartrate and initially set at pHs
ranging from 3.0 to 6.0 (). All data are from biobleaching
experiments with manganese-free medium and EDTA-extracted OKP. The
trend for control cultures is indicated by the dotted line, and the
trend for organic acid-supplemented cultures is indicated by the solid
line. Error bars indicate standard deviations.
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Time course of biobleaching.
The time course of biobleaching
was studied in a control culture set at an initial pH of 6.0 and in a
culture treated with 3 mM glycolate and set at an initial pH of 4.5. The brightness gains and percent decrease in kappa numbers during 21 days of incubation are shown in Fig. 4.
The pHs of both cultures were almost equal (pH 4.6 to 4.8) from day 7 onward. The biobleaching occurred in two distinct periods. In the first
period, from days 4 to 9, brightness gains and delignification were
observed in both the control and the glycolate-supplemented cultures.
However, in the second period, from days 9 to 14, brightness gains and delignification continued in the glycolate-supplemented culture but not
in the control culture. Moreover, the delignification was more
extensive in the latter period.
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FIG. 4.
BMBG (A), percent kappa number decrease (B), and pH (C)
during the time course experiment in manganese-free biobleaching
cultures with EDTA-extracted pulp. The control culture not receiving
organic acids was set at an initial pH of 6.0 (). The glycolate (3 mM)-amended culture was set at an initial pH of 4.5 (). Error bars
indicate standard deviations.
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Oxidative enzyme activities of the ligninolytic system of
Bjerkandera sp. strain BOS55 were measured for comparison as
a function
of biobleaching time (Fig.
5).
MnP and LiP were the major oxidative
enzymes present during the two
periods of biobleaching. To a lesser
extent, MIP activities were also
detectable (Table
3). All of
these enzyme
activities were approximately two- to threefold higher
in the
glycolate-supplemented culture than in the control culture.
Fast
protein liquid chromatograms of extracellular heme proteins
in
9-day-old cultures (Fig.
6) clearly
illustrated the stimulated
production of active MnP, LiP, and MIP.
Similar chromatograms
of 6-day-old cultures revealed that at that time,
LiP was not
detectable and an even greater stimulatory effect of the
organic
acids on MnP production was noted (results not shown). In all
chromatograms, most of the MnP activity was associated with the
heme
protein in fractions 31 to 34.
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FIG. 5.
Extracellular activities of MnP (A) and LiP (B) as well
as physiological concentrations of veratryl alcohol (C) and oxalic acid
(D) during the time course experiment in manganese-free biobleaching
cultures with EDTA-extracted pulp. The control culture not receiving
organic acids was set at an initial pH of 6.0 (). The glycolate (3 mM)-amended culture was set at an initial pH of 4.5 (). Error bars
indicate standard deviations.
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TABLE 3.
BMBG and kappa number (KN) decrease rates, average enzyme
titers, and average secondary metabolite concentrations for two
different time periods of the biobleaching time course experiment
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FIG. 6.
(A and B) FPLC profiles of heme proteins of 9-day-old
extracellular fluids from the control culture (A) and the 3 mM
glycolate-amended culture (B) from the time course experiment. (C and
D) Peroxidase activities in collected fractions for the control culture
(C) and the glycolate-amended culture (D). Symbols: , MnP;  , LiP;
, MIP.
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The concentrations of two important secondary metabolites implicated in
the ligninolytic system, veratryl alcohol and oxalate,
were also
monitored (Fig.
5). These metabolites were found at
higher
concentrations in the glycolate-supplemented culture than
in the
control culture. Their average concentration was two- to
threefold
higher during the second period of biobleaching in the
glycolate-supplemented culture, which contained 111 µM veratryl
alcohol and 153 µM oxalate (Table
3). The physiological concentration
of H
2O
2 during biobleaching ranged from 1.1 to
2.6 µM and was
not affected much by the organic acid supplementation
(Table
3).
During the periods when biobleaching occurred, only low
levels
of AAO activity were detected (Table
3).
Role of organic acids in superoxide production.
The
extracellular fluids of selected biobleaching cultures were monitored
periodically for the production of superoxide anion by measuring rates
of TNM reduction. The results shown in Table 4 indicate that organic acids
dramatically stimulated the production of superoxide. Rates of up to
approximately 0.5 U liter1 were detected in
oxalate-supplemented cultures. On the other hand, no superoxide
production or otherwise comparatively low rates of superoxide
production were detected in control cultures set at an initial pH of
4.5 or 6.0, respectively.
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TABLE 4.
Effect of adding different organic acids to Mn-free
biobleaching cultures of Bjerkandera sp. strain BOS55 on the
peak rate of superoxide production measured on the basis of
TNM reductiona
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DISCUSSION |
The results of this work demonstrate that simple physiological
organic acids are important components in the Mn-independent biobleaching system of Bjerkandera. The addition of organic
acids to Mn-free biobleaching cultures at concentrations of as low as 1 to 3 mM permitted extensive delignification (50%) and brightness gains
(13 ISO units) for eucalyptus OKP. The brightness gains and
delignification obtained by adding organic acids were comparable to
those obtained by adding Mn to the culture medium (39, 40).
Four possible stimulatory roles of the organic acids in the
Mn-independent pulp biobleaching system can be considered. These include pH-buffering effects of the organic acids and the stimulated production of extracellular peroxidases, secondary metabolites, and
reduced oxygen radicals by the organic acids.
pH-buffering effects of organic acids.
Organic acids provided
better pH buffering of the culture medium than what was found in
control cultures. However, the stimulatory effect of the acids was not
due solely to pH regulation. Biobleaching and the production of all the
vital components of the ligninolytic system (peroxidases and secondary
metabolites) were stimulated in organic acid-supplemented cultures with
an initial pH of 4.5 compared to control cultures with an initial pH of
6.0 (to provide a similar physiological pH). Furthermore, 5 mM DMS, a
nonphysiological acid, had no noteworthy stimulatory effect on
biobleaching or enzyme production.
Role of organic acids in extracellular peroxidase production.
In parallel with biobleaching, the activities and production of
isozymes from three families of extracellular peroxidases (MnP, LiP,
and MIP) were also highly stimulated by the exogenous organic acid
supplements. Although Mn stimulates MnP production by
Bjerkandera sp. strain BOS55, MnP is still produced at
appreciable levels in the absence of Mn (35, 37). This
behavior is distinct from that of many other white rot fungi having an
absolute requirement for Mn for the expression of active MnP production
(7, 17).
Previous work showed that simple organic acids added to Mn-containing
medium stimulated MnP production in
Bjerkandera sp.
strain
BOS55 (
37). In this study, a similar pattern of stimulation
was observed under the Mn-free biobleaching conditions used. The
highest MnP activity (30 U liter
1) was obtained with 5 mM
glycolate. The mechanism of stimulation
does not appear to be unique to
MnP, since LiP production was
also induced by oxalate or oxalate
precursors. A plausible mechanism
is that gene expression occurs in
response to reduced oxygen radicals
derived from organic acids.
Oxidative stress caused by O
2 and
H
2O
2 has been shown to stimulate
mnp
gene transcription in
Phanerochaete chrysosporium
(
32).
Effect of organic acids on secondary metabolite concentration.
The physiological concentrations of oxalate and veratryl alcohol were
also stimulated by the organic acids. These secondary metabolites have
several roles in lignin degradation (50, 52). Veratryl
alcohol not only protects LiP from H2O2
inactivation (8) but also has cofactor and mediator roles in
LiP catalysis as well (50). Veratryl alcohol was a
requirement for LiP to depolymerize soluble radiolabelled synthetic
lignin during in vitro experiments (18). Also, veratryl
alcohol was required for the biobleaching of OKP by a semipurified
preparation of LiP from P. chrysosporium (4). The
unbound veratryl alcohol cation radical is too short-lived (0.5 ms) to
behave as a diffusible oxidant and thus cannot account for the
oxidation of poorly accessible lignin in pulp fibers (26).
Nonetheless, there is good evidence that lignin in OKP is more highly
exposed and accessible to LiP than is lignin in unbleached kraft pulp
(4).
The roles of veratryl alcohol in LiP catalysis can be extended to
include certain types of MnP isozymes capable of veratryl
alcohol
oxidation. MnP isozymes with this property have been described
for two
white rot fungal genera,
Lentinus edodes and
Pleurotus spp. (
9,
34,
49). While the veratryl
alcohol-oxidizing
capacity of
L. edodes was found to be
dependent on the presence
of Mn, MnP from
Pleurotus spp.
directly oxidizes veratryl alcohol
in the absence of Mn.
Most of the organic acids tested in this study are de novo metabolites
detected in white rot fungi or have been detected in
kraft pulp
biobleaching cultures (
13,
31,
48,
53,
58).
Many of the
organic acids, e.g., acetate, glycolate, glyoxylate,
citrate, and
succinate, served as precursors for the formation
of oxalate in
Bjerkandera sp. strain BOS55. Oxalate is a common
secondary
metabolite of many white rot fungi (
13,
53). Two
enzymes in
basidiomycetes are responsible for the formation of
oxalate:
oxalacetase or glyoxylate oxidase (
12,
53). Oxalacetase
is
an Mn-dependent enzyme, which would account for the stimulated
production of oxalate under culture conditions with 33 µM Mn (Table
1). In media lacking Mn, the formation of oxalate most likely
can be
accounted for by glyoxylate oxidase. Consistent with these
hypotheses
was the finding that glyoxylate and the structurally
related glycolate
were the best precursors for oxalate formation
(Table
2).
Effect of organic acids on reduced oxygen radicals.
Reduced
oxygen species can be formed from the oxidation of oxalate and other
physiological organic acids (12, 52). Mn(III) formed from
the oxidation of Mn(II) by MnP can oxidize oxalate and glyoxylate to
free radicals, ultimately leading to the formation of superoxide
(25, 30). Veratryl alcohol cation radical formed by LiP
mediates the oxidation of oxalate in a similar fashion (5, 47,
52). In this study, the occurrence of superoxide anion radical
was demonstrated in cultures receiving organic acid supplements by
measuring the reduction of a well-known superoxide scavenger, TNM
(56).
The superoxide anion radical may have roles in lignin degradation which
could account for the stimulated delignification of
kraft pulp in
cultures supplemented with organic acids. The superoxide
anion radical
has been suggested to account for the direct oxidation
of recalcitrant
lignin model compounds by Mn(III) oxalate (
20).
The
importance of the superoxide anion radical for kraft pulp
delignification by laccase mediator systems has already been emphasized
in experiments in which superoxide dismutase was used to inhibit
biobleaching (
51). The superoxide anion radical has also
been
implicated in the mechanisms of oxygen delignification and
peroxide
bleaching (
15). Although superoxide itself shows no
reactivity
toward kraft pulp lignin, it can behave as a strong oxidant
of
radicals formed in the lignin molecule, causing ring opening and
cleavage between C
---C
bonds
(
15). Therefore, the stimulating
effect of the superoxide
anion radical on delignification during
white rot fungal biobleaching
can only be rationalized if the
lignin is first oxidized to cation
radicals by the oxidative enzymes.
Correspondence of biobleaching to ligninolytic enzymes.
MnP
and LiP were the most predominant oxidative enzymes produced during
biobleaching. MnP was consistently present under all culture conditions
which supported extensive biobleaching. The correspondence of
brightness gains with the maximal titer of MnP under variable culture
conditions, with and without Mn as well as with and without
physiological organic acids, is plotted in Fig.
7. From 1 to 10 U liter1,
there was a strong linear correlation between brightness gains and the
MnP titer (R2 = 0.786, P < 0.01); thereafter, an increased MnP titer showed little ability to
improve brightness gains. This trend suggests that MnP from
Bjerkandera might be involved in biobleaching in the absence
of Mn. This trend also indicates that once a minimum level of
biocatalyst has been exceded, other factors, such as poor lignin
bioavailability, appear to become rate limiting.
View larger version (15K):
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|
FIG. 7.
Correlation between peak MnP activity and extent of BMBG
after 14 days of incubation of EDTA-extracted OKP with
Bjerkandera sp. strain BOS55. The open symbols indicate
cultures with manganese-free media. The closed symbols correspond to
cultures with 33 µM manganese. Circles represent no organic acids,
triangles represent 1 mM organic acids, diamonds represent 3 mM organic
acids, and squares represent 5 mM organic acids.
|
|
Provided that Mn is present, MnP is known to delignify kraft pulp in
vitro (
19,
24,
28,
43,
45) as well as to depolymerize
synthetic lignins (
18,
57). Mn was found to be an obligate
mediator substrate of MnP from
P. chrysosporium for the
oxidation
of other substrates (
16). However, it is not yet
fully clear
how MnP can function in the absence of Mn.
Alternative mediators or cofactors besides Mn have to be considered.
Several precedents from the literature give insights
into possible
alternatives. MnP from
Ceriporiopsis subvermispora is known to oxidize several N-substituted aromatics in the absence
of
Mn (
55). Furthermore, the
mnp gene from
C. subvermispora was shown to contain an aromatic binding site
(
33). Naturally
occurring N-substituted aromatics
(3-hydroxyanthranillate and
4-hydroxymethylquinoline) have also been
identified as important
secondary metabolites in some white rot fungi
(
1,
14). MnP
isozymes purified from
Pleurotus
spp. were shown to be capable
of directly oxidizing 2,6-dimethoxyphenol
and veratryl alcohol
in the absence of Mn (
34,
49). These
MnP isozymes might be
able to directly oxidize phenolic moieties in OKP
lignin or otherwise
utilize veratryl alcohol as a nondiffusible
mediator.
Aside from MnP, LiP was detected in biobleaching cultures stimulated by
oxalate or oxalate precursors. When present, LiP could
possibly have
been involved in the biobleaching, since veratryl
alcohol was
available. LiP from
P. chrysosporium in combination
with
veratryl alcohol was previously shown to be effective in
the in vitro
depolymerization of radiolabelled synthetic lignin
(
18) and
in causing brightness gains and delignification of
OKP (
4).
| |
ACKNOWLEDGMENTS |
This study was carried out with financial support from the
Commission of the European Communities Agriculture and Fisheries (FAIR)
specific RTD program CT95-0805, "Oxidative Enzymes for the Pulp and
Paper Industry." Support given to M.T.M. and G.F. from the Wageningen
Agricultural University fellowship program is also greatly appreciated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Industrial Microbiology, Department of Food Technology and Nutritional Sciences, P.O. Box 8129, Wageningen Agricultural University, 6700 EV
Wageningen, The Netherlands. Phone: 31-317-484976. Fax: 31-317-484978. E-mail: JIM.FIELD{at}ALGEMEEN.IM.FTNS.WAU.NL.
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Appl Environ Microbiol, July 1998, p. 2409-2417, Vol. 64, No. 7
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Abstract
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