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Appl Environ Microbiol, June 1998, p. 2117-2125, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Fate of Residual Lignin during Delignification
of Kraft Pulp by Trametes versicolor
Ian D.
Reid*
Pulp and Paper Research Institute of Canada,
Pointe-Claire, Quebec, Canada H9R 3J9
Received 18 November 1997/Accepted 11 March 1998
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ABSTRACT |
The fungus Trametes versicolor can delignify and
brighten kraft pulps. To better understand the mechanism of this
biological bleaching and the by-products formed, I traced the
transformation of pulp lignin during treatment with the fungus.
Hardwood and softwood kraft pulps containing 14C-labelled
residual lignin were prepared by laboratory pulping of lignin-labelled
aspen and spruce wood and then incubated with T. versicolor. After initially polymerizing the lignin, the fungus depolymerized it to alkali-extractable forms and then to soluble forms.
Most of the labelled carbon accumulated in the water-soluble pool. The
extractable and soluble products were oligomeric; single-ring aromatic
products were not detected. The mineralization of the lignin carbon to
CO2 varied between experiments, up to 22% in the most
vigorous cultures. The activities of the known enzymes laccase and
manganese peroxidase did not account for all of the lignin degradation
that took place in the T. versicolor cultures. This
fungus may produce additional enzymes that could be useful in enzyme
bleaching systems.
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INTRODUCTION |
Trametes versicolor is a
white rot fungus that can delignify kraft pulps (28). The
lower lignin contents of pulps treated with this fungus lead to higher
brightness and higher bleachability of the pulps. Although the
application of this biological delignification process to pulp
bleaching has been studied for several years, its mechanism
the
chemical changes produced in the lignin and the enzymes that catalyze
themis not yet known in detail.
Biodegradation of native lignin, the model polymer dehydrogenative
polymerizate, and lower-molecular-weight model compounds by
Phanerochaete chrysosporium and other white rot fungi has
been studied extensively (17). Kraft lignin, the lignin
which is solubilized during kraft pulping, has also received some
attention (21). White rot fungi are able to extensively
mineralize (convert to CO2) and solubilize these lignins
(8, 11, 25, 26, 33). These fungi produce various
combinations of the extracellular oxidative enzymes lignin peroxidase,
manganese peroxidase (MnP), and laccase (13), which are
thought to play important roles in lignin degradation (12,
35). Participation of MnP has been implicated in kraft pulp
delignification by T. versicolor (22), and it has
been determined that lignin peroxidase is not needed (2).
MnP and laccase can cause substantial decreases in the kappa numbers
and improvements in the bleachability of kraft pulps under appropriate
conditions, but their activities do not fully explain the delignifying
effects of T. versicolor cultures (3).
The structure of the residual lignin in kraft pulps is significantly
different from the structure of the lignin in wood and even from the
structure of dissolved kraft lignin. During kraft pulping, aryl ether
bonds are depleted, condensed bonds are enriched, and links to
polysaccharides are formed (3). Kraft pulps have much lower
lignin contents than wood has, and the porosity of the polysaccharide
matrix enclosing them is increased (31). Consequently, the
mechanism of pulp delignification may differ from the mechanism of wood
delignification.
This study traced the fate of 14C-labelled lignin removed
from hardwood kraft pulp (HWKP) and softwood kraft pulp (SWKP) during delignification, and the molecular sizes of intermediates and products
were determined. Preliminary results showing that solubilization and
mineralization of labelled lignin from HWKP occur have been published
previously (27).
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MATERIALS AND METHODS |
The labelled pulps used in this study were prepared on two
occasions, once in 1991 and once in 1994. These pulps are referred to
below as HWKP*1, SWKP*1, and SWKP*2.
14C labelling of wood.
L-[U-14C]phenylalanine (39 MBq; 17.6 GBq/mmol; catalog no. CFB.70; Amersham) was converted to
[U-14C]cinnamic acid with phenylalanine ammonia lyase
(catalog no. P-1016; Sigma) (23). The labelled cinnamic acid
was dissolved in 10 mM potassium phosphate buffer (pH 7) and was fed to
cut stems of aspen (Populus tremuloides Michx.; 16 MBq; June
1991) and white spruce (Picea mariana L.; 15 MBq; July
1991). The labelled stems were kept in a fume hood with their cut ends
immersed in water and with illumination for 10 h per day for 2 weeks, until the aspen leaves wilted and the white spruce needles began
to fall off. The leaves or needles and small branches were removed and
discarded, and the bark was peeled off the main branches and discarded.
The wood from the main branches was cut into matchstick-sized pieces
and sequentially extracted in a Soxhlet apparatus with water,
benzene-ethanol (1:1), ethanol, and water again and then air dried.
In a separate experiment (July 1994), 32 MBq of
[U-14C]cinnamic acid was fed to a white spruce stem as
described above. After debarking, the labelled wood was ground in a
Wiley mill until it passed through a 10-mesh screen, and then it was
extracted as described above.
Pulping of labelled wood.
The extracted labelled wood pieces
were placed, along with kraft white liquor (15% active alkali, 27%
sulfidity) (5), inside Teflon-lined stainless steel bombs
and heated in a computer-controlled oil bath to H-numbers
(34) of 1,400 (aspen) and 1,600 (spruce). After cooling, the
bombs were opened, and the pulps were separated from the black liquor
by filtration, thoroughly washed with water, and freeze-dried. Portions
(0.4 g) of the pulps were then sequentially extracted with 0.05% Tween
80 (2% consistency, 20 min at 120°C) and 0.01 M NaOH (2%
consistency, 15 min at 120°C) and again freeze-dried. The specific
radioactivity of the extracted aspen pulp (HWKP*1) was 57 kBq/g, and
the specific radioactivity of the extracted spruce pulp (SWKP*1) was 72 kBq/g.
The ground labelled spruce wood was pulped by mixing it at a ratio of 4 ml/g with white liquor (16% active alkali, 14% sulfidity)
in
stainless steel bombs, heating the preparations in an oil bath
from
room temperature to 170°C over a 90-min period, and then
keeping them
at 170°C for an additional 70 min (H-number, 1,100).
After cooling in
cold water, the bombs were opened, and the pulp
was recovered by
filtration on a glass frit and washed with hot
distilled water. This
pulp (SWKP*2) had a specific radioactivity
of 73 kBq/g.
Cellulase isolation of residual lignin.
Crude cellulase
(product code BR-1101; 154 filter paper units/ml) was purchased from
Iogen Corporation, Ottawa, Canada, and was used without purification.
Pulp (up to 2.5 g [oven dry weight]) was weighed into a 250-ml
polypropylene centrifuge bottle with a conical bottom (catalog no.
25350; Corning) and suspended in 200 ml of 0.02% NaN3. The
pH of the suspension was adjusted to 4.7 with acetic acid. Concentrated
Iogen cellulase solution (0.5 ml) was added, and after thorough mixing,
the suspension was incubated at 37°C for 24 h with occasional
shaking. Then the pH was adjusted to 2.0 with 2 M
H2SO4, and the suspension was centrifuged for 10 min at 2,550 × g in a swinging bucket rotor. The
supernatant was discarded, and the pellet was resuspended in 200 ml of
0.02% NaN3, mixed with 0.5 ml of cellulase concentrate,
adjusted to pH 4.7, and incubated at 37°C for another 48 h. Then
4 ml of 5 M NaOH was added to dissolve the lignin, and the mixture was
incubated at 37°C for 1 h. After centrifugation in a swinging
bucket rotor at 2,550 × g for 10 min, the supernatant
was carefully decanted into a clean 250-ml centrifuge bottle. The
pellet was discarded. The pH of the supernatant was adjusted to 2.0 with 2 M H2SO4. After another centrifugation,
the supernatant was discarded, and the pellet was used as the lignin
preparation.
Incubation with the fungus.
Portions (ca. 100 mg) of the
labelled pulps HWKP*1 and SWKP*1 were accurately weighed into 500-ml
Erlenmeyer flasks (three flasks for the aspen pulp and three flasks for
the spruce pulp). To each flask containing aspen pulp 3.9 g (dry
weight) of a commercial HWKP (kappa number, 12.7) was added. To each
flask containing spruce pulp 3.9 g (dry weight) of a white spruce
kraft pulp (kappa number, 27.6) prepared in the Paprican pilot plant
was added. One 125-ml Erlenmeyer flask containing ca. 25 mg of labelled
pulp and 975 mg (dry weight) of unlabelled pulp was also prepared for each pulp type. The pulp in each flask was suspended at 2% consistency in a defined medium containing 0.5 g of Tween 80 per liter
(29), and the flasks were fitted with rubber stoppers
traversed by stainless steel tubes for air inlet and outlet. Latex
tubing stuffed with cotton wool was attached to the external ends of
the aeration tubes, and the flasks were autoclaved for 20 min. After
cooling, the flasks were placed overnight on a gyratory shaker
operating at 225 rpm to thoroughly mix their contents. Then a 10-ml
sample of the pulp suspension was aseptically pipetted from each flask, and the 500-ml flasks were each inoculated with 30 ml of a shake culture of T. versicolor 52J (1) that had been
grown for 5 days in the defined medium; the 125-ml flasks served as
noninoculated controls. The air inlet tube of each flask was connected
to a source of humidified air, and the air outlets were connected to Pasteur pipettes with their tips immersed in 5 ml of 0.1 M NaOH in test
tubes to trap CO2. The airflow through each culture flask was adjusted to give vigorous bubbling but no splashing in the CO2 traps. The flasks were incubated on the shaker with
continuous airflow in a room with the temperature controlled at
27.5°C. Samples (10 ml) were aseptically removed from the inoculated
flasks after 1, 2, 3, 4, and 5 days of incubation for the aspen pulp
and after 1, 2, 4, 8, and 12 days for the spruce pulp. The
CO2 traps were replaced daily. At the end of incubation,
the contents of the 125-ml flasks and the remaining contents of the
500-ml flasks were harvested and fractionated.
Pulp SWKP*2 was incubated with
T. versicolor 52J for 14 days
in a similar way, with these differences: the initial glucose
concentration in the medium was increased to 15 g/liter, and the
sulfate concentration was lowered to 0.2 mM; the cultures were
incubated in 2-liter flasks containing 800 ml of pulp suspension;
the
unlabelled pulp was prepared from black spruce in the Paprican
pilot
plant and was washed with 0.1 M NaOH and water at room temperature
before use; the labelled pulp SWKP*2 was added to each flask as
1.96 ml
of a suspension containing 53.3 kBq; the flask headspaces
were
continuously flushed with O
2 during incubation; and the
effluent
gases were bubbled through 20 ml of 2 M NaOH, which was
replaced
every 2 days, to trap CO
2. Samples (25 ml) were
aseptically removed
every 2 days from each flask, and 40-ml samples
were removed on
days 0, 7, and 14 to measure the kappa number and
brightness.
The pulp was recovered by filtration on Whatman no. 1 filter paper
in a Buchner funnel, washed with distilled water, and
lyophilized.
Fractionation of labelled products.
The contents of the
CO2 traps were washed into 20-ml scintillation vials with
three 5-ml portions of EcoLite(+) scintillation fluid (ICN) and were
counted with a Beckman model LS6800 liquid scintillation counter, after
chemiluminescence was allowed to decay for 24 h in the dark. The
14CO2 yields were corrected for the removal of
radioactivity from the cultures by sampling. The pulp suspension
samples were filtered on a stainless steel mesh with fines recycle.
Subsamples (0.5 ml) of the filtrates were mixed with 5 ml of EcoLite(+)
for counting. The pulp mats were thoroughly washed with water and then
suspended at 2% consistency in 0.01 M NaOH and heated for 15 min at
120°C in an autoclave. After cooling, the pulp was again filtered on the stainless steel mesh with fines recycle, and 0.5 ml of the filtrate
was mixed with 5 ml of EcoLite(+) for counting. The extracted pulp mat
was thoroughly washed with deionized water and freeze-dried. The dried
pulp samples were combusted in a model OX400 Biological Oxidizer
(R. J. Harvey Instrument Corp.); the CO2 produced was collected and counted in 15 ml of Carbon 14 Cocktail (R. J. Harvey Instrument Corp.)
The samples removed from the cultures containing SWKP*2 were
fractionated as outlined in Fig.
1. The
pulp suspensions were
filtered on Whatman type GF/C filters with
0.65-µm pores. The
radioactivity in the filtrate was measured by
liquid scintillation
counting of 5-ml samples mixed with 15 ml of
Optiphase 3 (Wallac).
One milliliter of the filtrate was used to
measure laccase and
MnP activities, and the remainder of the filtrate
was freeze-dried.
The pulp on the filter was washed with 100 ml of
water, freeze-dried,
and weighed. The pulp was extracted with 5 ml of
1.6% NaOH for
60 min at 60°C and then filtered with suction on a
type GF/C filter.
One milliliter of the alkali extract was mixed with 5 ml of OptiPhase
3 for counting, and the remainder was stored at 4°C.
The extracted
pulp was washed with 100 ml of water and then transferred
to a
50-ml polypropylene centrifuge tube for cellulase digestion. The
cellulase treatment was carried out as described above, except
that the
procedure was scaled down to a liquid volume of 40 ml.
At the end of
the second incubation with cellulase, the digest
was acidified to pH 2 and centrifuged. The pellet was suspended
in 5 ml of 0.1 M NaOH and
incubated at 37°C for 1 h. After centrifugation,
the supernatant
was decanted and used as the cellulase lignin
preparation. One
milliliter of this solution was counted, and
the remainder was saved
for gel filtration analysis. Five-milliliter
samples of the
supernatants obtained from centrifugation of the
acidified first- and
second-stage cellulase digests were also
counted. The pellet remaining
after extraction of the cellulase
lignin was washed with 10 ml of water
and then transferred to
a combustion boat lined with filter paper. The
radioactivity in
this residue was determined by burning the residue in
a Harvey
Biological Oxidizer and trapping and counting the
14CO
2 produced.
Size exclusion chromatography.
Samples (450 µl) of culture
filtrates were mixed with 50 µl of 1 M NaOH; freeze-dried culture
filtrates were redissolved in a minimum volume (0.5 to 1.5 ml) of 0.1 M
NaOH; and alkali extracts and cellulase lignin solutions were used
directly. All samples (500 µl) were mixed with 500 µl of ethanol in
1.5-ml microcentrifuge tubes and centrifuged for 2 min at full speed in
a Beckman Microfuge 12 centrifuge. The molecular size distributions
were analyzed by chromatography on Superose 12 (Pharmacia) as
previously described (24). One-milliliter effluent fractions
were collected in scintillation vials and counted after mixing with 5 ml of Optiphase 3. The radioactivity applied to the column was
recovered quantitatively in the eluted fractions. The amount of
radioactivity in each 1-ml fraction was converted to a percentage of
the total radioactivity supplied to the culture by dividing by the
amount of radioactivity applied to the column and multiplying by the
percentage of the total radioactivity in the extract which the sample
represented. Cytochrome c and veratryl alcohol were used as
molecular size standards. The recorder tracings of the effluent
A280 were digitized with the program Un-Scan-It
(Silk Scientific) after scanning with a Hewlett-Packard ScanJet 4C
scanner.
Analytical methods.
Kappa numbers were determined by the
micro kappa method (4). Pulp brightness (International
Standards Organization) was measured on handsheets with a Gretag model
SPM 50 spectral photometer at 457 nm. Laccase activity was measured
with 2,2'-azinobis(3-ethylbenzthiazoline-5-sulfonate) (7),
and MnP activity was measured by determining the rate of appearance of
the Mn(III)-malonate complex (22). Klason lignin was
measured by the method of Effland (10) with estimation of acid-soluble lignin from the A205 of the
hydrolysis filtrates.
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RESULTS |
Lignin solubilization.
When the labelled hardwood pulp HWKP*1
was incubated with T. versicolor for 5 days, a small
fraction (1%) of the radioactivity was given off as
14CO2 (Fig. 2).
Fractionation of the pulp recovered from the fungal cultures showed
that the radioactivity in the alkali extract increased substantially
during incubation, from 5% initially to 33% by day 4. The increase in
extractable radioactivity was especially marked on the third day of
incubation with the fungus. There was a more gradual increase in the
radioactivity dissolved in the culture medium, from an initial value of
13.4% to 33.3% by day 5. The radioactivity that remained in the pulp
after alkali extraction decreased from 82% before incubation to 38%
after 5 days of fungal treatment.
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FIG. 2.
Distribution of label from residual lignin during
incubation of HWKP*1 with T. versicolor 52J. This is an area
graph; the vertical distances between pairs of lines indicate the
portion of the total radioactivity in each fraction. Data are the means
of values from three replicates ± standard errors.
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When the labelled softwood pulp SWKP*1 was incubated with
T. versicolor for 12 days, the release of
14CO
2 was very small (<0.5%) (Fig.
3). After a 2-day lag, the
alkali-extractable
14C increased from 7 to 42% of the
total by day 8, at the expense
of the insoluble fraction. On day 12, the alkali-extractable fraction
was smaller than the alkali-extractable
fraction at day 8. This
decrease was consistent in all three replicates
and was accompanied
by an increase in the insoluble fraction of the
radioactivity
from 51% at day 8 to 67% at day 12. The radioactivity
dissolved
in the medium increased from 1.4% at day 0 to 8% at day 12.
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FIG. 3.
Distribution of 14C from residual lignin
during incubation of SWKP*1 with T. versicolor 52J. The
vertical distances between pairs of lines indicate the portion of the
total radioactivity in each fraction. The amount of
14CO2 produced was too small to produce a
visible separation between the two top lines. Data are the means of
values from three replicates ± standard errors.
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Molecular size distribution of soluble materials and alkali
extracts.
The molecular sizes of the labelled materials
solubilized and extracted from the pulps before and after fungal
treatment were investigated by gel filtration. The water-soluble
fraction obtained from incubation of the hardwood pulp HWKP*1 with the
fungus produced a single broad peak that eluted before the molecular
size standard veratryl alcohol (molecular mass, 168 Da) but after
cytochrome c (12,384 Da) (Fig.
4). The apparent molecular mass for the
apex of the radioactive peak was 1,550 Da, or 7.3 guaiacylglycerol units. The molecular size distribution of the soluble radioactive material did not change as the amount of soluble material increased during the fungal incubation.
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FIG. 4.
Gel filtration of the labelled water-soluble fraction
obtained from incubation of 14C-lignin-labelled HWKP*1 with
T. versicolor 52J. Symbols:  , day 5;  , day 4;  , day
3;  , day 2;  , day 0;  , control. Dashed lines show elution
volumes of cytochrome c (12,384 Da) and veratryl alcohol
(168 Da).
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The radioactive material in the alkali extracts obtained from the
hardwood pulp HWKP*1 treated with
T. versicolor produced
a
gel filtration profile similar to that of the soluble material
(Fig.
5). The molecular size distribution did
not change with
the length of incubation. The smaller amount of
alkali-extractable
material initially present in the pulp eluted from
the column
at the same volume as the material from the treated pulp.
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FIG. 5.
Gel filtration of the labelled alkali extract obtained
from incubation of 14C-lignin-labelled HWKP*1 with T. versicolor 52J. Symbols: , day 5; , day 4; , day 3; ,
day 2; , day 0; , control. Dashed lines show elution volumes of
cytochrome c (12,384 Da) and veratryl alcohol (168 Da).
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The amount of water-soluble radioactive material obtained from the
softwood pulp SWKP*1 was too small to determine its gel
filtration
profile. The alkali extracts obtained from the softwood
pulps had
molecular size distributions similar to those of the
extracts obtained
from the hardwood pulps, but there was slightly
more radioactivity on
the high-molecular-size flank of the main
peak (eluting at 12 to 16 ml)
(Fig.
6). In these pulps as well,
the
shape of the molecular size distribution profile did not change
during
the fungal incubation, even though the amount of the alkali-extractable
radioactivity increased and then decreased.
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FIG. 6.
Gel filtration of the labelled alkali extract obtained
from incubation of 14C-lignin-labelled SWKP*1 with T. versicolor 52J. Symbols: , day 12; , day 8; , day 4; ,
control. Dashed lines show elution volumes of cytochrome c
(12,384 Da) and veratryl alcohol (168 Da).
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Extraction of cellulase lignin.
Polysaccharide hydrolysis with
cellulase has frequently been used to isolate the residual lignin from
kraft pulps (14-16, 36, 37). Typically, the cellulase
solution is replaced one or more times, the solubilized carbohydrates
are removed, and the insoluble lignin-enriched residue is treated with
fresh enzyme. Sometimes the digest is acidified before the soluble and
insoluble fractions are separated (37), and sometimes it is
not (36). Hortling et al. (14) reported that 20 to 60% of the residual lignin in kraft pulps dissolved during the
enzymatic hydrolysis. The residual lignin in the kraft pulps used in
the present study also showed high solubility after cellulase
hydrolysis; the enzyme digests at pH 4.8 had a marked yellow color, and
40 to 80% of the 14C from labelled pulps stayed in the
clear supernatant after centrifugation. Acidification of these digests
precipitated a dark brown material and most of the 14C from
labelled pulps. This brown material readily dissolved in dilute alkali
solutions. The pH dependence of the solubility of the
cellulase-liberated residual lignin is illustrated in Fig. 7. The amount of soluble lignin, as
determined by A280, was large and roughly
constant from pH 4 to 10 and appeared to increase slightly at pH 12. At
pH 2, almost all of the lignin from the softwood pulp and most of the
lignin from the hardwood pulp precipitated.
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FIG. 7.
Effect of pH on the solubility of lignin obtained from
cellulase-digested kraft pulps. Samples of HWKP () and SWKP ( )
were digested with cellulase at pH 4.8 and 40°C for 3 days, and then
subsamples were adjusted to the pH values indicated and centrifuged.
The supernatants were separated from the pellets and diluted 1:10 with
0.1 M NaOH before the absorbance was measured. The
A280 contributed by the cellulase solution was
negligible compared to the A280 contributed by the pulp.
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The standard procedure for cellulase digestion of pulps used in this
study (see Materials and Methods) took this solubility
of the lignin
into account. The digests were acidified to pH 2
before replacement of
the cellulase solution. At the end of the
second digestion, the digests
were made up to 0.1 M NaOH to maximize
the extraction of lignin from
the pulp. The extracted lignin was
then precipitated by acidification
to concentrate it and separate
it from carbohydrates and other
acid-soluble materials in the
digest. After cellulase digestion and
alkaline extraction of the
lignin, a small amount of dark brown
insoluble material always
remained. Further treatment with cellulase
did not dissolve this
residue. The recovery of lignin from the pulp by
the cellulase
digestion-alkaline extraction procedure was determined by
Klason
lignin analysis (Table
1).
Substantial amounts of the lignins
were in the acid-soluble fraction,
as estimated from the
A205 values of the Klason
filtrates. The apparent recovery of lignin
from the hardwood pulp was
100%, and the apparent recovery from
the softwood pulp was 88%. The
Klason lignin contents of the material
extracted by alkali from the
digested hardwood pulp and of the
extract from the softwood pulp were
89 and 63%, respectively.
Distribution of 14C from lignin-labelled SWKP.
Another batch of labelled SWKP (SWKP*2) was prepared and incubated with
T. versicolor for 14 days. After alkali extraction, the pulp
was digested with cellulase, and the cellulase lignin was recovered.
The delignification of this pulp was much more extensive than the
delignification observed in the previous experiment; 22% of the label
was mineralized to 14CO2 and 61% was
solubilized (Fig. 8A). The fraction of
the label removed from the pulp by mineralization and solubilization
agreed well with the decrease in kappa number of unlabelled kraft pulp in the cultures (Fig. 8B). The pulp brightness increased to 53 from an
initial value of 32. The alkali-extractable radioactivity increased
from 7% of the total initially to a maximum of 37% on day 6 and then
decreased to 12% by day 14 (Fig. 8A). An unusually large portion
(44%) of the radioactivity in pulp SWKP*2 remained in the insoluble
residue after cellulase digestion and extraction of the cellulase
lignin. Although this material was apparently inaccessible to
cellulase, it was readily degraded by T. versicolor and
almost disappeared by day 10. The cellulase lignin, which was soluble
in alkali after cellulase digestion of the pulp, initially contained
42% of the total radioactivity, but this fraction decreased during the
fungal incubation and was 2% at day 14. Some radioactivity (range, 2 to 13%) was solubilized during the cellulase digestion and was not
precipitated by acidification. The amount of this material reached its
maximum value after 4 days of incubation and then decreased as the
radioactivity in the extracted pulp entering the cellulase digestion
step decreased. Overall, as the fungal treatment progressed, label
moved from the cellulase lignin and cellulase residue fractions into
the alkali-extractable fraction and then into the soluble and
CO2 fractions (Fig. 8A).
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FIG. 8.
Effect of T. versicolor 52J on the
distribution of 14C from lignin-labelled SWKP*2 and on the
kappa number of the pulp. (A) The vertical distance between pairs of
lines indicates the portion of the total radioactivity in each
fraction. (B) Comparison between the fraction of 14C
remaining in the pulp and kappa number. Data are the means of values
from three replicates ± standard errors.
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Laccase and MnP activities were measured in the filtrates obtained from
the pulp samples (Fig.
9). Both of these
enzymes appeared
early in the incubation period, and their maximum
activities were
reached by day 2. Laccase activity declined thereafter
and was
negligible by day 6. MnP persisted longer but almost
disappeared
by day 10.
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FIG. 9.
Laccase and MnP activities in cultures of T. versicolor 52J delignifying SWKP*2. Data are means of values from
three replicates; the bars indicate standard errors.
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Molecular size distribution of soluble, extractable, and cellulase
lignin fractions (i) Cellulase lignin.
The cellulase lignin
obtained from the untreated pulp SWKP*2 contained some
large-molecular-size material excluded from the gel filtration column
(Fig. 10, peak at an elution volume of
7 ml); most of the 280-nm-absorbing material eluted in a broad peak at
8 to 19 ml. The radioactivity tended to elute earlier than the
UV-absorbing material, suggesting that the molecular sizes of the
labelled materials were larger than the molecular sizes of the bulk of
the lignin.
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FIG. 10.
Gel filtration profile of cellulase lignin extracted
from SWKP*2 before incubation with the fungus. The smooth solid curve
shows the A280 profile, and the stepped curve
shows the 14C profile. All of the data are means of values
from three replicates ± standard errors; the dashed lines
indicate the standard errors for A280.
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After 2 days of incubation with the fungus, the amount of UV-absorbing
material eluting between 10 and 19 ml had decreased,
but the height of
the excluded peak (6.5 to 8 ml) had increased
slightly (Fig.
11). The
14C-labelled
materials also showed a small increase in the height
of the excluded
peak and a decrease in the amount eluting between
8 and 20 ml from day
0 to day 2. Between day 2 and day 12, the
heights of the
A280 curves decreased steadily; the absorbance
in the larger-molecular-size portion of the chromatogram (6.5
to 13 ml)
tended to decrease faster than the absorbance in the
smaller-molecular-size region (13 to 19 ml). At day 14, the peak
shifted toward earlier elution volumes (Fig.
11). The radioactivities
in all fractions of the chromatogram decreased at about the same
rate
from day 2 to day 6. After that the amount of
14C in the
larger-molecular-size fractions (eluting at 6 to 13 ml)
decreased
faster than the amount in the smaller-molecular-size
fractions.
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FIG. 11.
Gel filtration profiles of cellulase lignin prepared
from SWKP*2 after 0 to 14 days of incubation with T. versicolor. Data are the means of values from three replicates;
the bars indicate standard errors. Note that the panels on the right
have expanded scales.
|
|
(ii) Alkali extracts.
The alkali extract obtained from pulp
before incubation with the fungus produced a low broad
A280 peak during gel filtration; the
radioactivity in the extract was too low to measure its distribution after chromatography (Fig. 12). After 2 days of incubation with the fungus, the gel chromatogram had an
asymmetrical A280 peak, with its maximum at an
elution volume of 17 ml. This peak was higher and more symmetrical
after 4 days of incubation and reached its maximum size on day 6. The
14C in the extracts eluted from the column in a peak having
the same shape and location as the A280 peak.
The relative height of the radioactivity peak at day 2 was less than
the relative height of the A280 peak, and the
radioactivity showed a bigger increase than the UV absorbance between
day 2 and day 4. The heights of both the A280
and 14C peaks decreased slightly from day 6 to day 10 and
more substantially between day 10 and day 12. The height of the
radioactivity peak continued to decrease between days 12 and 14, but
the A280 peak did not.
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FIG. 12.
Gel filtration profiles of alkali extracts from SWKP*2
after 0 to 14 days of incubation with T. versicolor. Data
shown are the means of values from three replicates; the bars indicate
standard errors.
|
|
(iii) Water-soluble materials.
Gel filtration of the dissolved
materials from the pulp suspension after 2 days of incubation with the
fungus partially resolved two peaks of UV absorbance, one centered at
17 ml and a higher one centered at 20 ml (Fig.
13). The soluble radioactivity eluted in a discrete peak centered at 17.5 ml. After 4 days of incubation with
the fungus, the height of the A280 peak at 17 ml
had tripled, but the height of the later peak had only increased by
20% and had shifted toward slightly earlier elution; a shoulder at 21 ml had disappeared. During further incubation with the fungus, the
heights of the A280 and 14C peaks
centered at 17 ml continued to increase with no shift in elution
volume.
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FIG. 13.
Gel filtration profiles of soluble materials in SWKP*2
suspensions after 0 to 14 days of incubation with T. versicolor. Data shown are the means of values from three
replicates; the bars indicate standard errors. Note that the panels on
the left have expanded scales.
|
|
| |
DISCUSSION |
The experiments reported here were carried out over several years
with labelled pulps prepared at different times and in slightly different ways. The delignifying activities of the fungal cultures also
varied from experiment to experiment. The results obtained for
mineralization of 14C show this variability clearly.
Incubation of HWKP*1 with T. versicolor for 5 days yielded
only 1% 14CO2, whereas a similar experiment
performed with an earlier preparation of labelled HWKP yielded 10%
14CO2 (27). Treatment of SWKP*1 for
14 days released less than 0.5% of the label as
14CO2, but incubation of SWKP*2 with the fungus
for the same period released 22% 14CO2. The
experiment performed with SWKP*2 also revealed more solubilization of
the label than the experiment performed with SWKP*1 did. It appears
that the delignifying activity of the T. versicolor culture used in the experiment with HWKP*1 and SWKP*1 was less vigorous than
normal; visual observation of the pulp brightness at the end of the
incubation period supported this conclusion. In contrast, the culture
used to treat SWKP*2 was more vigorous than usual; the pulp brightness
reached 53, and the final kappa number, 7, was at the low end of the
range observed in previous work (29). This variability in
the activity of the fungus, as well as the variability in the
properties of the labelled pulps, must be taken into account when the
significance of the results is assessed. Only effects that were seen
consistently can be relied upon.
One consistent result of all of the experiments was solubilization of
the labelled lignin and accumulation of soluble labelled products in
the culture medium. Soluble materials that absorbed light at 280 nm
also accumulated in the cultures (Fig. 13). Gel filtration indicated
that these soluble products had larger molecular sizes than single-ring
aromatic compounds like veratryl alcohol. The peak elution volumes of
the soluble products obtained from HWKP*1 and SWKP*2 both indicated
that oligomers containing about seven phenylpropane units were present.
Because the gel filtration column was calibrated with peptides and not
lignin molecules and because the structures of the soluble products are
unknown, molecular size estimates are very uncertain. It is clear,
nonetheless, that single-ring lignin degradation products did not
accumulate in the delignifying cultures.
Incubation with T. versicolor also consistently increased
the amounts of 14C and UV-absorbing material that could be
extracted from the pulps with alkali. Fungal treatment has been shown
to increase the effectiveness of alkali extraction to lower pulp kappa
numbers (30). The alkali-extractable lignin seemed to be an
intermediate between the initial insoluble form and the ultimate
soluble products, since the amount of extractable label increased early
in the fungal treatment and later decreased as the soluble label
accumulated. The apparent reconversion of alkali-extractable lignin to
the insoluble form between days 8 and 12 of treatment of SWKP*1 (Fig.
2) is anomalous, and I can offer no explanation for it. On the gel
filtration column, the 14C and A280
peaks from the alkali extracts eluted slightly earlier than the peaks
from the water-soluble materials, although the elution patterns for the
two fractions overlapped greatly (Fig. 14). This suggests that the alkali
extracts included some larger molecules than the water-soluble
fractions did. The apparent molecular size distribution of the alkali
extracts did not change much from day 4 to day 14 of treatment of
SWKP*2, from day 4 to day 12 of treatment of SWKP*1, or from day 2 to
day 5 of treatment of HWKP*1.
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FIG. 14.
Comparison between gel filtration profiles of labelled
materials in the water-soluble fraction and the alkali extract from
SWKP*2 treated for 6 days with T. versicolor. Data are means
of values from three replicates; the bars indicate standard errors.
|
|
Cellulase digestion makes much of the residual lignin in kraft pulps
extractable and hence available for characterization. This technique
was used only with pulp SWKP*2. With this pulp, the 14C and
A280 gel filtration profiles for the cellulase
lignin extract differed; a larger portion of the 14C than
of the A280 eluted in the large-molecular-size
excluded peak. Almost all of the A280 came from
the unlabelled pilot plant pulp in the mixture, whereas the
14C came from the laboratory pulp. Because the labelled
wood was milled to a powder before pulping, the pulping reagents should have penetrated the wood particles faster than they penetrated the
chips used in the pilot plant digestion. To compensate for this effect,
the cooking time for the pulping of the radioactive wood powder was
shorter than the regular cooking time for chips. However, the amount of
compensation needed was not known exactly, and SWKP*2 could have been
under- or overcooked. The labelled pulp was too scarce and too valuable
to measure its kappa number. In this case the
A280 behavior is probably more representative of
normal residual lignin.
Residual lignins isolated by cellulase digestion of kraft pulps are
contaminated with proteins (15). The protein contamination can be decreased by reprecipitating the lignins from solutions in
dimethylacetamide and in alkali (15). Such lignin
purification steps were not used in this study because they might have
lowered the yield of cellulase lignin, especially the
smaller-molecular-size portion. Contaminating proteins may have made
minor contributions to the yields of cellulase lignin reported in Table
1 and to the A280 profiles in Fig. 10 and 11.
Because the specific absorptivity at 280 nm of proteins is only about
one-tenth the specific absorptivity at 280 nm of lignin, the effect of
protein contamination on the A280 results should
have been negligible.
The cellulase lignin apparently underwent net polymerization during the
first 2 days of fungal treatment. The amounts of both 14C
and A280 in the large-molecular-size excluded
peak increased, and the amounts eluting later decreased (Fig. 11).
Polymerization in the early stages of degradation of dehydrogenative
polymerizate by white rot fungi has been observed previously (9,
25) and attributed to oxidative coupling catalyzed by
phenol-oxidizing enzymes (25). Methanol is released from
demethoxylation of phenolic rings in the residual lignin within the
first few days of pulp treatment by T. versicolor, catalyzed
by MnP (22). The polymerization and demethoxylation of the
lignin probably both result from oxidation of the phenolic rings in the
lignin by MnP and laccase. After the second day of treatment, both the
large-molecular-size and the smaller-molecular-size parts of the
cellulase lignin gradually disappeared. The large-molecular-size
fraction of the cellulase lignin disappeared at least as fast as the
smaller molecules, and between days 6 and 12, the large-molecular-size
peak disappeared faster than the smaller-molecular-size material. Both
the 14C and the A280 measures of lignin
followed this pattern.
The dynamics of the various pools measured in this study suggest that
there is a sequential transformation from the cellulase lignin and
insoluble residue form to the alkali-extractable form to the soluble
form and finally to CO2. Thus, the amounts of the cellulase
lignin and insoluble residue, initially the most abundant forms,
decreased throughout the treatment. The nearly complete disappearance
of these fractions indicates that almost all of the lignin in the pulp
was accessible to the fungus. The alkali-extractable fraction first
increased and later decreased, behavior typical of an intermediate
pool. The abundance of the soluble fraction increased throughout the
treatment, and this fraction contained most of the label at the end of
the experiment. Carbon dioxide is clearly the ultimate product of
oxidative biodegradation; it is not clear whether all of the
lignin-derived carbon would have been mineralized to CO2 if
the incubation with the fungus had been long enough. Possibly some
molecules in the soluble pool are recalcitrant to further metabolism.
The gel filtration results suggest that the transition from the
cellulase lignin form to the alkali-extractable form involves
considerable fragmentation of the lignin molecules. Smaller decreases
in average molecular size accompany the transition from the
alkali-extractable form to the soluble form.
Lignin degradation products in the size range of the single-ring
aromatic molecules from which lignin is polymerized were noticeably
absent from both the alkali-extractable and soluble fractions. Possibly
such fragments were rapidly taken up and metabolized by the fungus; if
that is true, the 22% yield of 14CO2 is an
upper limit for the production of small fragments from lignin.
The soluble lignin degradation products had apparent molecular sizes in
the size range of phenylpropane oligomers. The molecular size
distribution did not change with time of incubation; there was no
evidence of net polymerization or depolymerization. If these soluble
products contained phenolic hydroxyls, oxidative polymerization,
catalyzed by the laccase and MnP present in the cultures during the
first two-thirds of the incubation period, would be expected. The
resistance of the soluble products to polymerization suggests that they
did not contain free phenols. Whether the phenolic rings were removed
by reactions like alkyl-phenyl cleavage or destroyed by ring opening or
the phenolic groups were tied up by intramolecular ether formation, by
glycosylation (19), or by other derivatization reactions
could not be determined from the information available. A soluble
lignin derivative produced from bagasse by Lentinus edodes
was characterized previously; this material has few phenolic hydroxyls
but many carboxyls and is highly condensed (32).
Various explanations for the resistance of residual lignin to removal
from kraft pulps have been offered (14-16, 36). If the
polysaccharides are removed by enzymatic hydrolysis, the residual lignin becomes soluble in dilute alkali, showing that it is not inherently insoluble. Either chemical attachment of the lignin to
polysaccharides or physical entrapment of the lignin molecules in the
polysaccharide matrix is possible. Treatment of kraft pulps with
xylanase or leaching at a high pH makes removal of a part of the
residual lignin easier; removal of hemicelluloses which physically
obstruct the diffusion of lignin molecules out of the fiber walls is
implicated in both cases (20). In the case of pulp
delignification by T. versicolor, the observed
depolymerization of the lignin should facilitate its diffusion out of
the polysaccharide network of the fiber walls and could also liberate
lignin fragments from lignin-carbohydrate complexes. The enzymes
responsible for lignin depolymerization and solubilization are not
known with certainty. The combination of laccase with the synthetic
mediator 2,2'-azinobis(3-ethylbenzthiozoline-5-sulfonate) has been
shown to solubilize 14C from lignin-labelled HWKP
(5). Laccase and MnP were detected in the cultures during
the early stages of delignification, but lignin solubilization
continued after these enzyme activities had disappeared (Fig. 9). MnP,
or laccase with a mediator, can delignify kraft pulps, but
delignification to the extent achieved by living T. versicolor cultures requires repeated treatments interspersed by
alkaline extractions, which seem to reactivate the lignin for enzyme
attack (6, 18). These differences between the effects of
known enzymes and the effects of whole cultures of T. versicolor provide circumstantial evidence that additional, unknown enzymes participate in pulp delignification. Chemical characterization of the soluble and alkali-extractable products of pulp
delignification should allow more definitive identification of the
delignifying enzymes.
| |
ACKNOWLEDGMENTS |
I thank Michelle Ricard for technical assistance and Sylvie
Renaud and Pak Wong for help with pulping the labelled wood.
| |
FOOTNOTES |
*
Mailing address: Paprican, 570 boul. St-Jean,
Pointe-Claire, QC, Canada H9R 3J9. Phone: (514) 630-4101, ext. 2244. Fax: (514) 630-4134. E-mail: reid{at}paprican.ca.
| |
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Appl Environ Microbiol, June 1998, p. 2117-2125, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
