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Next Article 
Applied and Environmental Microbiology, April 1999, p. 1367-1371, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Fungal Degradation of Lipophilic Extractives in
Eucalyptus globulus Wood
Ana
Gutiérrez,1,*
José C.
del Río,1
María Jesús
Martínez,2 and
Angel T.
Martínez2
Instituto de Recursos Naturales y
Agrobiología de Sevilla, Consejo Superior de
Investigaciones Científicas, E-41080
Seville,1 and Centro de Investigaciones
Biológicas, Consejo Superior de Investigaciones
Científicas, E-28006 Madrid,2 Spain
Received 2 October 1998/Accepted 5 January 1999
 |
ABSTRACT |
Solid-state fermentation of eucalypt wood with several fungal
strains was investigated as a possible biological pretreatment for
decreasing the content of compounds responsible for pitch deposition
during Cl2-free manufacture of paper pulp. First,
different pitch deposits were characterized by gas chromatography (GC)
and GC-mass spectrometry (MS). The chemical species identified arose from lipophilic wood extractives that survived the pulping and bleaching processes. Second, a detailed GC-MS analysis of the lipophilic fraction after fungal treatment of wood was carried out, and
different degradation patterns were observed. The results showed that
some basidiomycetes that decreased the lipophilic fraction also
released significant amounts of polar extractives, which were
identified by thermochemolysis as originating from lignin
depolymerization. Therefore, the abilities of fungi to control pitch
should be evaluated after analysis of compounds involved in deposit
formation and not simply by estimating the decrease in the total
extractive content. In this way, Phlebia radiata,
Funalia trogii, Bjerkandera adusta, and
Poria subvermispora strains were identified as the most
promising organisms for pitch biocontrol, since they degraded 75 to
100% of both free and esterified sterols, as well as other lipophilic
components of the eucalypt wood extractives. Ophiostoma
piliferum, a fungus used commercially for pitch control,
hydrolyzed the sterol esters and triglycerides, but it did not appear
to be suitable for eucalypt wood treatment because it increased
the content of free sitosterol, a major compound in pitch deposits.
| |
INTRODUCTION |
Biotechnology has been
introduced into pulp and paper manufacturing, the first nonfood
industrial use of plant biomass (7). Xylanase-aided
elementary chlorine-free (ECF) bleaching of paper pulp is the
best example of the applications developed in recent years
(22). However, other aspects of paper pulp manufacturing also offer promising avenues for using microorganisms and
enzymes; one of these is biological control of the so-called pitch
deposits. Accumulation of wood extractives in pulp and paper mills
(pitch deposits) results in low-quality pulp and blockages that cause shutdowns of operations and important economic losses (15). The increasing need for recirculating water in pulp mills and the need
to reduce effluents in order to meet environmental protection requirements are leading to increases in the concentrations of pitch
compounds in the production process. This situation and the use of
totally chlorine-free (TCF) and ECF bleaching processes based on
the replacement of Cl2 by milder chemical oxidants or enzymes result in greater pitch deposition problems in mills. Traditionally, pitch deposits during pulping processes have been reduced by debarking and seasoning logs and wood chips and by adding
pitch control agents (1, 5). However, often the results are
far from satisfactory. Alternatively, biological control of pitch
deposits by treatment of pulp with enzymes (9-11) and
treatment of wood with different microorganisms (2, 6, 8,
12), have been suggested in recent years. These biotechnological
approaches have involved mainly treatment of pine wood with
Ophiostoma piliferum and related species, as well as some
basidiomycetes. The studies of pitch removal performed with
basidiomycetes, such as Phanerochaete chrysosporium,
Poria subvermispora (synonym, Ceriporiopsis
subvermispora), and Phlebiopsis gigantea, are in the
preliminary stages and are often associated with studies of the use of
these fungi for so-called wood biopulping (i.e., biological removal of
lignin for paper pulp manufacturing). A white strain of O. piliferum (Cartapip from Clariant) has been used
commercially to depitch some types of wood prior to pulping.
However, successful use of Cartapip to control pitch deposition
in Kraft pulp obtained from eucalypt wood (which is extensively used as
a raw material for paper pulp manufacturing in Spain, Portugal,
Brazil, and other countries) has not been reported. Moreover, no
information about biological depitching of this type of wood with other
fungal species is available.
Designing effective biotechnological solutions for wood
extractive removal requires thorough characterization of the
compounds responsible for pitch deposition. In this context, the
first aim of this work was to identify the specific constituents of
pitch deposits during Kraft pulping of Eucalyptus globulus
wood compared with wood extractives obtained from this eucalypt
species. Then, the main aim was to evaluate the viability of
biotechnological solutions for eliminating these problematic pitch
compounds by analyzing in detail the patterns of removal of the main
lipophilic compounds present in eucalypt wood by a selection of fungal strains.
| |
MATERIALS AND METHODS |
Samples.
Pitch deposits after TCF and ECF bleaching were
obtained from eucalypt Kraft pulp mills in Huelva and Pontevedra,
Spain. The TCF sequence used included two oxygen delignification
stages, a chelation stage, an H2O2 stage under
pressure (with oxygen), and a final H2O2 stage.
The ECF sequence used included an oxygen delignification stage,
followed by two ClO2 stages with an intermediate alkaline
extraction stage. E. globulus wood chips were ground to
sawdust. The different pitch deposits and sawdust were Soxhlet extracted with acetone for 6 h (21). The acetone
extracts were evaporated to dryness and redissolved in chloroform
before they were analyzed by gas chromatography (GC) and GC-mass
spectrometry (MS).
Fungal strains and wood treatment conditions.
The first
screening of fungi for removal of eucalypt extractives under
solid-state fermentation conditions was carried out with 73 strains
(18). Many of these strains were isolated from fruiting bodies growing
on eucalypt wood in forests or in log piles and identified. Additional
strains were obtained from the Centraalbureau voor Schimmelcultures
(Baarn, The Netherlands), Wageningen Agricultural University
(Wageningen, The Netherlands), and the fungal culture collection of the
Centro de Investigaciones Biológicas (Madrid, Spain).
Treatment of wood with all of the fungi (in duplicate) was carried out
in flasks containing 2 g (dry weight) of small wood chips (1 to 2 by 10 to 20 mm) and 5 ml of water (sterilized at 120°C) that were
inoculated with two portions of mycelium from cultures on 2% malt
extract agar. After 40 days of incubation at 28°C and a constant
humidity, the wood was dried with air at 60°C, milled, and extracted
with acetone as described above. Then, the Klason lignin content was
estimated after hot-water extraction (21). Wood treatment
with the 14 most promising strains was repeated in quadruplicate to
confirm the results obtained in the first screening.
GC and GC-MS.
The GC analysis were performed with a
Hewlett-Packard model HP 5890 GC equipped with a flame ionization
detector by using a high- temperature polyimide-coated fused-silica
capillary column (5 m by 0.25 mm; type DB5-HT; film thickness, 0.1 µm; J & W Scientific). The injector and detector temperatures were
300 and 350°C, respectively. The oven temperature was programmed to
increase from 100°C (1 min) to 350°C (3 min) at a rate of
15°C/min. Samples were injected in the splitless mode. Helium was
used as the carrier gas. A mixture of standard compounds (palmitic
acid, sitosterol, cholesteryl oleate, and triheptadecanoin) was used to
construct a calibration curve for quantitation of wood extractives at
concentrations ranging from 0.1 to 1 mg/ml. The correlation
coefficient was greater than 0.99 in all cases. Peaks were quantified
by determining areas.
GC-MS analyses were performed with a Varian model Star 3400 GC equipped
with an ion trap detector (Varian model Saturn 2000) by using a type
DB-5HT capillary column (15 m by 0.25 mm; film thickness, 0.1 µm; J & W Scientific). Helium was used as the carrier gas. Samples were
injected directly into the column with an autoinjector (Varian model
8200) by using a septum-equipped programmable injector system. The
temperature of the injector during injection was 120°C, and 0.1 min
after injection the temperature was programmed to increase to 380°C
(10 min) at a rate of 200°C/min. The oven temperature was programmed
to increase from 120°C (1 min) to 380°C (5 min) at a rate of
10°C/min. The temperatures of the ion trap detector and the transfer
line were set at 200 and 300°C, respectively. Compounds were
identified by comparing their mass spectra with mass spectra in the
Wiley and Nist libraries, by performing mass fragmentography, and, when
possible, by using standards.
Thermochemolysis.
A thermochemolysis analysis was performed
with a Varian model Saturn 2000 GC-MS coupled to a Curie point
pyrolyser (Horizon Instruments Ltd.) by using a type DB-5 column (30 m
by 0.25 mm; film thickness, 0.25 µm). A finely divided sample was
deposited onto ferromagnetic wire, mixed with approximately 0.5 µl of
tetramethylammonium hydroxide (25% [wt/wt] aqueous solution)
(14), inserted into the glass liner, and then immediately
located in the pyrolyser, and pyrolysis was carried out at 610°C. The
temperature of the chromatograph was programmed to increase from 40°C
(1 min) to 300°C (20 min) at a rate of 6°C/min. The temperature of
the injector, which was equipped with a liquid carbon dioxide cryogenic
unit, was programmed to increase from 30°C (1 min) to 300°C at
a rate of 200°C/min, while the GC-MS interface was kept at 300°C.
| |
RESULTS AND DISCUSSION |
Lipophilic compounds in pitch deposits and E. globulus wood.
Pitch deposits were obtained after TCF
and ECF bleaching of E. globulus wood. These industrial
processes include the use of H2O2 and
ClO2, respectively, as bleaching agents (20).
Two types of organic fractions were distinguished on the basis of
acetone solubility. The acetone-insoluble fractions contained
mainly salts of fatty acids and minor amounts of ellagic acid salts
(4). The compositions of the acetone-soluble fractions of
the different pitch deposits analyzed by GC and GC-MS are
summarized in Table 1, which also shows
the results for extractives obtained from E. globulus
wood. This analysis was possible because a method for analysis of this
type of compounds was optimized previously (13). The results
obtained showed that many of the chemical species found in eucalypt
wood extracts survive the pulping and bleaching processes, since they
were identified in pitch deposits. In all of the samples (wood
and pitch deposits), the different steroid compounds identified
(Fig. 1) accounted for more than 70% of
the total lipophilic compounds in the acetone extracts. Several steroid
ketones (compounds VII to X), which have been reported to be sterol
oxidation products previously (16), were present at
relatively high levels in pitch deposits, but they were also detected
in wood. Triglycerides were absent from both types of deposits since
they were hydrolyzed during Kraft cooking. The composition of the pitch
deposits produced after TCF bleaching was very similar to the
composition of E. globulus wood extractives. The
deposits produced after ECF bleaching had a very different composition.
No sitosterol, the main sterol present in E. globulus wood, was found in either the free form or the
esterified form, whereas only a saturated sterol (stigmastanol)
remained in the deposits because of its higher resistance to
oxidation. The same thing happened with unsaturated fatty acids, which
were absent after ClO2 treatment. Based on the results
described above, during the screening of fungi to determine whether
they remove extractives from E. globulus wood prior
to Kraft cooking special emphasis was placed on the biological removal
of free and esterified sterols.
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TABLE 1.
Hydrocarbons, fatty acids, waxes, sterols, ketones,
sterol esters, and triglycerides in extracts from E. globulus wood and pitch deposits after ECF and
TCF bleachinga
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FIG. 1.
Main free sterols (sitosterol [compound I],
stigmastanol [compound III], and fucosterol [compound V]),
esterified sterols (sitosterol esters [compound II], and stigmastanol
esters [compound IV]), steroid hydrocarbon (stigmasta-3,5-diene
[compound VI]), and ketones (stigmast-4-en-3-one [compound VII],
stigmasta-3,5-dien-7-one [compound VIII], stigmastan-3-one [compound
IX], and stigmastane-3,6-dione [compound X]) in acetone extracts
from E. globulus wood and pitch deposits during
manufacture of Cl2-free eucalypt Kraft pulp. See Table 1
for compound abundance values.
|
|
Fungal treatment of E. globulus wood.
The
first screening for extractive removal by treatment of
E. globulus wood with fungi under sterile
solid-state fermentation conditions was performed by examining
different species of basidiomycetes, ascomycetes, and conidial fungi
(18). The ascomycetes included strains belonging to eight
Ophiostoma species, five Ceratocystis species, and two Mollisia species (the latter were isolated
from eucalypt wood). The basidiomycetes included members of three
Pleurotus species and two Phlebia species, as
well as strains of Funalia trogii, Bjerkandera
adusta, P. chrysosporium, Crepidotus
variabilis, and Melanotus hepatochrous (the two latter
organisms were isolated from eucalypt wood). Finally, strains of
Paecilomyces sp., Penicillium megasporum
(isolated from resin of Eucalyptus tereticornis), and several lipase-producing Fusarium species were among the
conidial fungi investigated.
Wide differences were observed in the extent of wood extractive removal
by the different fungi. While some of the fungi, such as
Pleurotus eryngii, Paecilomyces sp., F. trogii, Ophiostoma valdivianum, and Mollisia
melaleuca, reduced the total extractive content by 50 to 70%, a
significant increase in the extractive content was observed in wood
treated with Coniophora puteana, C. variabilis,
P. subvermispora, and other organisms. However, close
examination of the acetone extracts obtained from the biotreated woods
revealed that some of the fungi that decreased the total extractive
content reduced only the polar fraction, while the lipophilic
fraction (the main fraction responsible for pitch deposition, as shown
above) remained unchanged. The opposite occurred with other fungi which
increased the total acetone extract content. The results obtained with
two representative fungi are shown in Table
2. In this experiment the acetone
extracts obtained from the biotreated woods were
fractionated into lipophilic (chloroform-soluble) and polar
(chloroform-insoluble) fractions. O. valdivianum reduced the
total acetone extract content due to a drastic decrease in the polar
fraction content; however, the lipophilic fraction content was barely
modified. In contrast, C. variabilis reduced the lipophilic fraction content, although it increased the total acetone extract content due to an increase in the polar compound content (Table 2). The
origins of the polar compounds were determined by thermochemolysis. We
found that the acid/aldehyde ratio of vanillyl compounds, an indicator
of lignin oxidative degradation (14), was higher in the
polar fraction from wood treated with C. variabilis. This, together with the decrease in lignin content estimated by the Klason
method, suggested that the polar compounds described above probably
arose from fungal alteration of lignin. We deduced from this study that
the abilities of some fungi to control pitch should be evaluated by
specifically analyzing compounds involved in deposit generation and not
simply estimating the decrease in the total amount of extractives.
Next, the compositions of lipophilic extractives from E. globulus wood treated with the different fungal species were
analyzed by GC and GC-MS. Two representative chromatograms are shown in Fig. 2. A total of 73 species were
investigated initially, and the species that significantly degraded
total extractives and/or significantly decreased the content of
problematic lipophilic compounds, as well as caused a limited loss of
wood weight (18), were selected for more detailed
quantitative study (Table 3). Different
patterns of extractive degradation were observed with the fungal
strains used. Some of the fungi, including O. piliferum and
O. valdivianum, reduced the sterol ester content but
simultaneously increased the content of free sterols, mainly
sitosterol, which was probably related to the fatty acyl-sterol
esterase activity detected (unpublished results). A similar
pattern has been found previously during pine wood treatment with
Ophiostoma ainoae (19). O. piliferum (Cartapip strain from Clariant) has been
reported to be useful for reducing pitch problems in mechanical pulping of pine wood (6), as well as in spruce sulfite pulping
(8). However, this strain proved to have limited
utility in the case of eucalypt wood because it was not able to degrade
the free sterols released, which, as shown above, are among the
problematic compounds in this type of wood. This fungus and other
ascomycetous fungi have also been reported to remove 60 to 70% of
resin acids from pine wood (12, 23), whereas some
basidiomycetes can completely degrade these compounds (19).
Some of the basidiomycetes assayed, such as P. chrysosporium, degraded both sterols and sterol
esters but significantly increased the content of triglycerides, which were probably derived from fungal metabolism (24).
The ability of liquid cultures of P. chrysosporium to
degrade sterol esters from aspen (Populus tremuloides),
which also cause pitch problems during pulping of this type of wood
(3), has been described by Leone and Breuil (17).
During the present study we found for the first time that a
number of fungi, including Phlebia radiata, F. trogii, B. adusta, P. subvermispora,
and C. variabilis, efficiently degrade the lipophilic
compounds that have been identified as the compounds responsible for
pitch deposition during manufacture of Cl2-free pulp from
eucalypt wood. One of these fungi, P. subvermispora, has been reported to be an efficient degrader of resin acids in pine
wood (8). Further experiments are being carried out to establish the time course of extractive removal and to scale up wood
treatment with the most promising strains that remove problematic lipophilic compounds.
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FIG. 2.
Gas chromatograms for lipophilic extracts from
E. globulus wood after treatment with two fungi and the
corresponding control. The same sample volume for the lipophilic
fraction obtained from the same amount of wood was injected in each
case, and the relative chromatographic responses are shown. Figure 1
and Table 3 show the chemical structures and abundance values for the
different steroids (compounds I to X).
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TABLE 3.
Fungal degradation of E. globulus wood
extractives: percentage of total extract and main lipophilic
fraction contents in wood treated with different fungi and a
control, as determined by GCa
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| |
ACKNOWLEDGMENTS |
We thank J. M. Barrasa (University of Alcalá, Madrid,
Spain) for fungal strains, Javier Romero (Centro de
Investigación, ENCE, Pontevedra, Spain) for samples of eucalypt
wood and pitch deposits, and Clariant (Barcelona, Spain) for a
sample of Cartapip (O. piliferum white strain).
This research was carried out with the financial support of the
European project "Wood Extractives in Pulp and Paper Manufacture: Technical and Environmental Implications and Biological Removal" (FAIR contract CT95-560) and the Spanish Biotechnology Programme.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: IRNAS, CSIC,
P.O. Box 1052, E-41080, Seville, Spain. Phone: 34954624711. Fax:
34954624002. E-mail: anagu{at}irnase.csic.es.
| |
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Applied and Environmental Microbiology, April 1999, p. 1367-1371, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
