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International Biodeterioration & Biodegradation 64 (2010) 588e593

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Behavior of Ceriporiopsis subvermispora during Pinus taedabiotreatment in soybean-oil-amended cultures

André Aguiar a,b,*, Régis Mendonça c, Jaime Rodriguez c, André Ferraz a

aDepartamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo, Lorena, SP, BrazilbUniversidade Federal de São João Del-Rei, Campus Alto Paraopeba, Ouro Branco, MG, BrazilcBiotechnology Center, Universidad de Concepción, Concepción, Chile

a r t i c l e i n f o

Article history:Received 20 April 2010Received in revised form14 June 2010Accepted 15 June 2010Available online 15 August 2010

Keywords:Ceriporiopsis subvermisporaLipid peroxidationSoybean oilManganese peroxidaseWood biodegradation

* Corresponding author at: Universidade Federal dAlto Paraopeba, 36420-000 Ouro Branco, MG, Brazil.

E-mail addresses: andrepiranga@yahoo.com.br, ag

0964-8305/$ e see front matter Crown Copyright �doi:10.1016/j.ibiod.2010.06.011

a b s t r a c t

Pinus taedawood chipswere treatedwith the biopulping fungus Ceriporiopsis subvermispora in soybean-oil-amended cultures. The secretion of oxalic acid and the accumulation of thiobarbituric acid reactivesubstances were significantly increased in soybean-oil-amended cultures. By contrast, the secretion ofhydrolytic and oxidative enzymes was not altered in the cultures. Biotreated wood samples were charac-terized for weight and component losses as well as by in-situ thioacidolysis. Residual lignins were alsoextracted from biotreated wood using a mild-non-razing extraction procedure. The lignins were charac-terized by 31P nuclear magnetic resonance (31P-NMR) spectroscopy. Soybean oil amendment in the cultureswas found to affect lignin degradation routes; however, it inhibited depolymerization reactions detectablein the residual lignin that was retained in the biotreated wood. As a consequence, chemithermomechanicalpulping of the biotreated samples was not improved by soybean oil amendment in the cultures.

Crown Copyright � 2010 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Ceriporiopsis subvermispora is a white-rot fungus that causesselective delignification during the early stages of wood coloniza-tion and maintains cellulose in an almost undamaged state (Akhtaret al. 1998; Mendonça et al. 2002; Chi et al. 2007; Ferraz et al. 2008;Schmutzer et al. 2008). Manganese peroxidase (MnP) is the mainoxidative enzyme produced by this species during wood biodeg-radation (Lobos et al. 1994; Souza-Cruz et al. 2004; Aguiar et al.2006; Chi et al. 2007; Vicentim and Ferraz 2007). This enzymehas been considered as relevant for the biopulping process, becausethe cultures in which overproduction of MnP has been detectedalso provide improved benefits in biopulping (Vicentim and Ferraz2008). Commercial MnP (JenaBiosGmbH, Germany) has also beenused for direct wood chip pretreatment (Maijala et al. 2008). In thiscase, an energy savings of 11% was obtained during refining whenthe enzyme-treated wood chips were used.

Manganese peroxidase preferentially oxidizes Mn2þ, releasingchelatedMn3þ that in turn degrades phenolic lignin structures. Thisenzyme is also able to degrade non-phenolic lignin in the presenceof co-oxidants, such as unsaturated fatty acids (Hofrichter 2002).

e São João Del-Rei, CampusTel./fax: þ55 31 3741 3280.uiar@ufsj.edu.br (A. Aguiar).

2010 Published by Elsevier Ltd. All

To date, the most accepted mechanism for non-phenolic lignindegradation by C. subvermispora is based on the action of freeradicals that are formed during unsaturated fatty acid peroxidationinitiated by MnP (Jensen et al. 1996; Srebotnik et al. 1997;Watanabe et al. 2000; Daina et al. 2002; Cunha et al. 2010).

Unsaturated fatty acids, whether free or esterified, are found inwood extractives and can serve as a primary carbon source for thefungi during wood colonization (Wang et al. 1995; Gutiérrez et al.2002; Aguiar and Ferraz 2008). In some cases, free fatty acids canalso present inhibitory effects on the fungal growth (Venalainen et al.2004). In the particular case of C. subvermispora, Enoki et al. (1999)have demonstrated that cultures of this fungus produce some fattyacids from extractive-freemilledwood.With culturing time, the fattyacids were found to be converted into organoperoxides and thio-barbituric acid reactive substances (TBARS), suggesting the occur-rence of lipid peroxidation reactions.

The degradation of recalcitrant substances and lignin-derivedstructures by MnP and linoleic acid or Tween 80 indicates that theenzyme is able to oxidize high redox substrates once appropriatefatty acids are present (Bogan et al. 1996; Jensen et al. 1996; Kapichet al. 1999; Daina et al. 2002; Harazono and Nakamura 2005).In-vitro kraft-pulp delignification has been induced using a systemcomposed of MnP and unsaturated fatty acids (Bermek et al. 2002).In this case, the authors demonstrated that delignification wasimproved as a function of the number of double bonds in the fatty

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A. Aguiar et al. / International Biodeterioration & Biodegradation 64 (2010) 588e593 589

acids. In contrast, Kapich et al. (2010) found that a system composedof oleic acid/MnP acted efficiently to degrade a non-phenolic ligninmodel, whereas polyunsaturated fatty acids promoted lignin modeldegradation to a lesser extent. Manganese peroxidase is able torelease lignin fragments directly from lignocellulose substratesonce Mn2þ, H2O2, and unsaturated fatty acids are provided in thereaction medium (Hofrichter et al. 2001; Cunha et al. 2010). In-vivoamendment of soil with soybean oil or Tween 80 has also beenfound to enhance the degradation of polycyclic aromatic hydrocar-bons (PAH) by somewhite-rot fungi (Leonardi et al. 2008). The mainmechanism proposed in this case was based on improved fungalgrowth and increased bioavailability of the PAH.

Based on previous information, it was hypothesized thatlignin degradation and biopulping efficiency could be improved inC. subvermispora cultures that were amended with some source ofunsaturated fatty acids. Therefore, soybean oil, which is rich inunsaturated triglycerides (Zeitoun et al. 1991), was used as a sourceof unsaturated fatty acids in cultures of C. subvermispora growingon Pinus taeda wood chips. Extracellular enzymes, delignification,oxalic acid, and TBARS contents were monitored in the cultures.Thioacidolysis of the wood and 31P-NMR of the isolated ligninswere used to provide details of the structural characteristics of thebiodegraded lignins. Finally, selected culture conditions were usedto prepare biotreated wood samples that were pulped in a chem-ithermomechanical process to correlate the varied culture condi-tions with the biopulping efficiency.

2. Materials and methods

2.1. Wood biodegradation

C. subvermispora (Pilat) Gilbn. and Ryv. (L14807 strain SS-3)cultures were maintained at 4 �C on 20 g l�1 malt extract (Oxoid,England) and 7 g l�1 yeast extract (Vetec, Brazil). P. taeda woodchips (2.5 cm� 1.8 cm� 0.2 cm) were inoculated with a myceliumsuspension using procedures from the literature (Aguiar and Ferraz2008). Each Erlenmeyer flask was loaded with 45 g of wood chipsand 4.5 mg blended mycelium (100 mg kg�1, both on a dry basis).Amended cultures also received an aliquot of soybean oil thatwas previously dispersed inwater (90 g l�1; 20 min in an ultrasonicbath), corresponding to 0 (without oil), 2.6, 5.2, or 10.4 g soybeanoil kg�1 wood. After inoculation, the flasks were shaken by handand stored at 27 �C for up to 4 weeks. Five 2-l Erlenmeyer flaskswere inoculated for each biodegradation condition. Three cultureswere used for enzyme extraction and determination, one wasemployed for TBARS determination, and another was employed foroxalic acid determination. One set of wood chips was sterilizedbut non-inoculated and served as a control for all experiments.Scaled-up experiments were also performed using 2 kg of woodinside 20-l bioreactors. Wood chips biotreated in these bioreactorswere used in the pulping experiments. For this case, only twocultures conditionswere used, including non-amended and soybean-oil-amended cultures with the highest load, corresponding to10.4 g oil kg�1 wood.

2.2. Enzyme extraction and assays

For enzyme recovery, cultured wood chips were extractedaccording to Souza-Cruz et al. (2004). Extracts were assayed forMnP, LiP, and laccase using phenol-red (Aguiar et al. 2006), Azure B(Souza-Cruz et al. 2004) and ABTS (Bourbonnais et al. 1998) assubstrates, respectively. For hydrolytic enzymes, carboxymethyl-cellulose and birch xylan were used to assay endoglucanase (Woodand Bhat 1988) and xylanase activities (Bailey et al. 1992), respec-tively. Enzymatic activities are expressed as IU kg�1 of dry wood

initially contained in the cultures. Standard deviations were calcu-lated from triplicate cultures that were analyzed independently.

2.3. Determination of weight and chemical componentlosses of decayed wood

After enzyme extraction, the wood chips were washed withwater, air-dried, and weighed. Untreated and biotreated wood chipsweremilled to pass through a 0.5-mmscreen and extractedwith 95%ethanol for 6 h in a Soxhlet apparatus. Klason insoluble and solublelignins were determined by acid hydrolysis as described by Ferrazet al. (2000). After acid hydrolysis, soluble sugars were quantifiedby HPLC using an HPX-87H column, heated at 45 �C and eluted with5 mM sulfuric acid at 0.6 mlmin�1. Total polyoses and glucan valuesderived from calculations considering the acid-released monomers,glucose and xylose (Ferraz et al. 2000).

2.4. Extraction and determination of oxalic acid and TBARS

Oxalic acid (free, acid- and alkali-extracted) and TBARS weredetermined according to Aguiar and Ferraz (2008).

2.5. Thioacidolysis

The content of b-O-4 linkages in the lignins was determined bythe quantification of thioacidolysismonomers, according to Rolandoet al. (1992). The thioacidolysis residue obtained was dissolvedin 1 ml dichloromethane, and 10 ml of this solution was mixedwith 5 ml pyridine and silylated with 50 ml N,O-bis-(trimethylsilyl)-trifluoroacetamide at 60 �C for 1 h. The silylated products wereanalyzed in an HP 5890 gas chromatograph coupled to a flameionization detector (GC-FID) using a DB-5 column (5%-phenyl-95%-methylsiloxane, 30 m� 0.53 mm i.d.� 0.5-mm film thickness).Nitrogen was used as the carrier gas at 33 cm s�1 and the injectortemperaturewas 240 �C. The ovenwasmaintained initially at 140 �Cfor 1 min, and then the temperature was increased at 3 �Cmin�1

to 240 �C. After 1 min, the oven temperature was increased at30 �Cmin�1 to 290 �C. The final temperature was maintained for7 min. The injection volume was 1 ml in split mode (1:30).

The concentrations of main thioacidolysis monomers werecalculated according to Onnerud and Gellerstedt (2003), consid-ering a yield of 76% obtained upon the thioacidolysis of a b-O-4lignin model compound (guaiacylglycerol b-guaiacyl ether).The response factor (based on the internal standard) and the molarmass of the phenylpropane unit utilized were 1.5 and 187 gmol�1,respectively (Rolando et al. 1992).

2.6. Lignin isolation and characterization by 31P NMR spectroscopy

Lignin was isolated by mild acidolysis from milled wood ina nitrogen atmosphere using the dioxane method, which wasadapted from Evtuguim et al. (2001) with some modifications.Extractives-freemilledwood (10 g, dried under silica) was placed ina 500-ml three-necked flask that was fittedwith a reflux condenser,a nitrogen bubbler, and a thermometer. The solvent, 100 ml ofdioxane/water in a 9:1 mixture containing 0.2 M hydrochloric acid,was added to the flask. The reaction mixture, under nitrogen, washeated in a silicone bath and refluxed at 85e90 �C for 30 min.Subsequently, the mixture was allowed to cool in a nitrogenatmosphere to approximately 50 �C. The liquid phase was removedand the solid residue was subjected to the next extraction with100 ml of the acidic dioxane/water solution for 30 min, as describedabove. Two more extractions were carried out in the same manner.The final (fourth) extractionwas performed without the addition ofhydrochloric acid in the dioxane/water mixture. The four extracts

Table 1Enzymes, weight and component losses and thiobarbituric acid reactive substances(TBARS) detected during P. taeda treatment by C. subvermispora for 2 weeks in thesoybean oil-amended cultures.

Soybean oil (g kg�1 wood)a

0 2.6 5.2 10.4

Enzymatic activitiesb (UI kg�1 wood)Xylanase 565� 188 (a) 760� 40 (a) 699� 153 (a) 600� 245 (a)Endoglucanase 58� 31 (a) 59� 11 (a) 44� 13 (a) 30� 13 (a)MnP 142� 51 (a) 254� 49 (b) 230� 24 (a) 154� 35 (a)

Wood weight and component losses (% w/w) and TBARS accumulation (mg kg�1

of wood)Weight 3.3� 0.9 (a) 2.5� 0.7 (a) 2.9� 0.8 (a) 2.1� 0.7 (a)Lignin 8.3� 0.9 (a) 8.3� 0.7 (a) 9.2� 0.8 (a) 8.0� 0.7 (a)Polyoses 5.1� 0.9 (a) 5.1� 0.7 (a) 5.9� 0.8 (a) 5.1� 0.7 (a)Glucan 4.7� 0.9 (a) 2.8� 0.7 (b) 4.0� 0.8 (a) 1.9� 0.7 (c)TBARS 54� 6 63� 14 79� 11 106� 3

a In each row, same letters indicate that values do not differ with 95% confidence,different letters indicate values significantly different (Dunnet test).

b LiP and laccase activities were not detected in all cultures.

Fig. 1. Oxalic acid detected during P. taeda biodegradation with C. subvermispora for 2weeks in the soybean-oil-amended cultures.

A. Aguiar et al. / International Biodeterioration & Biodegradation 64 (2010) 588e593590

were combined and concentrated to 20 ml at reduced pressure.Next, the ligninwas precipitated by addition of the dioxane solutionto cold water (about 600 ml). This mixture was maintained over-night at 10 �C. Isolated lignins were filtered andmaintained at 55 �Covernight. Their yields were calculated based on the Klason lignincontent. The recovery yields for these lignins were 14.5%, 15.0%,15.1%, 16.1, and 16.0% (g 100 g�1 Klason lignin) for samples fromuntreated (control) and biotreated wood with 0, 2.6, 5.2, and 10.4 goil kg�1 wood, respectively.

Characterization of isolated lignins using 31P NMR spectroscopywas carried out according to Granata and Argyropoulos (1995)using a Bruker 250 MHz NMR spectrometer.

2.7. Chemithermomechanical pulping (CTMP) of wood samples

Wood chips were cooked at 121 �C for 2 h in alkaline sulfiteliquor containing 12% Na2SO3 and 6% NaOH, as w/w of dry wood.Four flasks containing 50 g wood chips (dry basis) and 300 ml ofliquor were cooked simultaneously. The content of one of the flaskswas washed several times with tap water until the remaining waterreached a neutral pH. Washed wood chips were air-dried andweighed, and their moisture content was determined using anOhaus infrared balance. Initial and final dry weights of these woodchips were used to calculate the pulping yield of the cooking stage.Cooked wood chips from the other three flasks were washed andfiberized for 1 h in a 3.5-l commercial blender (Sire, Brazil) at lowconsistency (1.7% w/v). Fiberized material was washed with waterand screened in a 0.15-mm slot screen. The screened pulp wascentrifuged, yielding a pulp consistency of approximately 30%(w/w). The rejected materials retained in the screen (fiber bundles)were weighed and fiberized for an additional hour in a Jökro mill(Regmed, Brazil) at a consistency of 11.1% (20-g fiber bundles and180 ml of alkaline-sulfite liquor in each Jökro pot). The resultingfibers were washed and screened in a 0.15-mm slot screen. Thescreened pulp was centrifuged and mixed with the pulp that wasprepared in the blender-fiberizing step. The mixed fiberized pulpwas then refined in alkaline-sulfite liquor for periods varying from90 to 180 min in a Jökro mill under the same conditions describedearlier. Refined pulps were suspended in water and used forSchoppereRiegler determinations (SRIP analyzer, Regmed, Brazil)as well as for hand-sheet preparation. Handsheets (150 gm�2) wereassayed for their strength properties following the TAPPI standardsfor tensile index (T 494 om-01) and tear index (T 414 om-04).

3. Results and discussion

Fungal growth was improved in wood chips that were amendedwith soybean oil, as the apparent mycelial mass on the wood chipsurfaces was proportional to the soybean oil concentrations in thecultures. This first observation suggests that the soybean oil couldhave served as an extra carbon source in the cultures, corroboratingprevious studies that have indicated a rapid consumption of lipo-philic extractives inwood by C. subvermispora (Gutierrez et al. 2002;Mendonça et al. 2002). By contrast, the level of hydrolytic andoxidative enzymes detected in the extracts recovered frombiotreatedwoodwas similar for all soybean oil concentrations testedand for non-supplemented cultures (Table 1). Manganese peroxi-dase was the main phenoloxidase detected in the cultures and wasthe only extracellular activity found to be slightly increased whenthe soybean oil concentration was 2.6 g oil kg�1 of wood. Degrada-tion of the wood components, evaluated based on their weight andchemical component losses, was also similar among the examinedculture conditions (Table 1). An exception was the decreased glucanloss that was observed in the culture with the highest soybean oilconcentration.

Accumulation of TBARS in biotreated wood increased signifi-cantly in soybean-oil-amended cultures (Table 1). TBARS are wellknown titers indicative of the occurrence of lipid peroxidationreactions in vivo (Masaphy et al. 1996; Enoki et al. 1999; Cunha et al.2010). Progressive levels of TBARS as a function of the increasedconcentrations of soybean oil were clear evidence of the intenseextracellular oxidative activity in these cultures. However, thisincreased oxidative activity did not increase lignin loss valuesobserved for the same cultures (Table 1).

In contrast to the enzyme production, the secretion of oxalicacid was significantly increased in the cultures that were supple-mented with soybean oil (Fig. 1). Direct production of oxalicacid from lipids by Aspergillus niger has been reported (Rymowiczand Lenart 2003). Likewise, it is probable that part of the soybeanoil added to the cultures served as a carbon source for oxalic acidproduction in the C. subvermispora cultures.

Sequential extractions of the wood chips with water, acid, andalkaline solutions served to discriminate soluble, insoluble, andesterified oxalate present in biodegraded wood, respectively (Huntet al. 2004; Aguiar and Ferraz 2008). Oxalic acid has several rolesin the cultures of wood-decay fungi. It acts as an efficient chelatingagent to release unstable Mn3þ ions from the MnP enzyme. It canalso acidify the culture pH, providing a more appropriate environ-ment for the action of the hydrolytic and oxidative enzymes. A thirdrole for oxalic acid has been described for C. subvermisporadto serveas a source of H2O2, namely, via oxidation to a CO2 radical by Mn3þ

ions (Urzúa et al. 1998). The highest amounts of oxalic acid wereextracted with 0.1 M HCl. Thus, the oxalic acid was found

3500

3750

4000

4250

4500

0 7 14 21 28Biotreatment time (days)

slyxordyh citahpilA

(ug lo

m1-

)ningil nosalk fo

100

200

300

400

slyxobraC

(ug lo

m1-

)ningil nosalk fo

aliphatic OHcarboxyl

A

0

400

800

1200

1600

2000

0 7 14 21 28Biotreatment time (days)

slyxordyh citamor

A(u

g lom

1-)ningil nosalk fo

totalin guaiacyl unitsin condensed unitsin p-hydroxy units

B

Fig. 2. Contents of the major functional groups detected in lignins isolated fromP. taeda that was treated with C. subvermispora, as revealed by 31P-NMR of the phos-phytilated derivatives. (A) Carboxyl and aliphatic hydroxyl groups, and (B) aromatichydroxyl groups.

A. Aguiar et al. / International Biodeterioration & Biodegradation 64 (2010) 588e593 591

predominantly as insoluble oxalate crystals. The high concentrationof calcium in P. taeda wood (Souza-Cruz et al. 2004) would explainthe insolubilization of most of the oxalic acid produced.

To evaluate the structural transformations in residual ligninduring these short biotreatment periods, biotreated wood sampleswere initially characterized using in-situ thioacidolysis. Residuallignins were also extracted from biotreated wood by a mild-non-razing extraction procedure (Evtuguim et al. 2001) and character-ized by 31P-NMR.

The time course of lignin transformation during P. taeda bio-treatment was initially studied in non-amended cultures. Data forlignin loss and yield of thioacidolysismonomers are shown inTable 2.The thioacidolysis monomer yield was determined as an indicatorof lignin depolymerization (Choi et al. 2006). This yield decreasedsignificantly in the two first weeks of biotreatment, whereas ligninloss was only 0.6% and 3% after 1 and 2 weeks of biotreatment,respectively. These data suggest that lignin depolymerization(demonstrated by decreases in the yields of thioacidolysis mono-mers) was not followed by immediate mineralization. After 4 weeksof biotreatment, the lignin loss reached a high value (12.3%)when theyield of thioacidolysis monomers was half of the initial values.In previous reports, residual lignin depolymerization induced byC. subvermispora has also been demonstrated based on derivatizationfollowed by reductive cleavage (Guerra et al. 2002, 2004; Vicentimand Ferraz 2007). Decreases in monomer yield in thioacidolysis canbe a consequence of lignin transformation via the cleavage of aryl-ether linkages. However, fungal-induced Ca-Cb cleavage and simpleCa-oxidation would also decrease monomer yield in this analyticaltechnique (Rolando et al. 1992). This fact is relevant because,in addition to aryl-ether cleavage, Ca-Cb cleavage and simpleCa-oxidation have been observed during the biodegradation of ligninmodel compounds by C. subvermispora (Srebotnik et al. 1997)and during in-vitro degradation of a non-phenolic lignin modelcompound by the MnP/linoleic acid system (Kapich et al. 1999).Additional lignin structural characteristics were revealed bya 31P-NMR spectroscopic evaluation of phosphytilated lignin deriv-atives. This technique enables the sensitive detection of carboxylfunctional groups as well as aliphatic and aromatic hydroxyl groups(Granata and Argyropoulos 1995).

31P-NMR analysis showed that the contents of aliphatic hydroxylgroups and all types of aromatic hydroxyl groups diminished withincreasing biotreatment time (Fig. 2). In contrast, the presence ofcarboxyl functional groups increased during the 4-week biotreat-ment period (Fig. 2a). These data suggest that significant changes inthe structure of residual lignins occurred simultaneously with thelignin depolymerization reactions. It is noteworthy that the increaseof carboxylic acid contents was not proportional to the decrease inaliphatic hydroxyls. For example, the carboxyl content increasedby 10 mmol g�1 of lignin after one week of biotreatment, whilealiphatic hydroxyls decreased by 110 mmol g�1 of lignin during thesame period. When the biotreatment was extended to 4 weeks, theincrease in carboxyl content corresponded to 280 mmol g�1, whereasthe aliphatic hydroxyl decrease corresponded to 420 mmol g�1 oflignin (Fig. 2a). These data indicate that the oxidation of side chainsin lignin, via Ca-Cb cleavage, to form new carboxylic acids was only

Table 2Lignin losses and yield of thioacidolysis monomers detected during P. taeda treat-ment by C. subvermispora in non-amended cultures.

Biotreatment time (weeks)

0 1 2 4

Lignin loss (%) 0 0.6� 0.1 3.0� 0.3 12.3� 0.7Yield of thioacidolysis monomers

(mmol g�1 of Klason lignin)1024� 24 767� 11 723� 63 530� 28

one of the reaction routes dictating the decrease in aliphatichydroxyl content. The formation of new keto-groups after aryl-ethercleavage or simple Ca oxidation would explain the excessivedecrease in aliphatic hydroxyl groups (Kapich et al. 1999).

Three reactionmechanisms could explain the decrease of aromatichydroxyl groups, as shown by 31P-NMR (Fig. 2b): (a) direct conversionof free phenolic minor substructures in lignin to quinones, as hasbeen postulated in earlier work (Guerra et al. 2002); (b) aromaticnuclei opening, resulting in muconic acid substructures, as has beenobserved forwood biotreatment by L. edodes (Crestini et al.1998); and(c) fungi could detoxify phenols by methylation (Chen et al. 1982;Lamar and Dietrich, 1990).

Thioacidolysis reactions and 31P-NMR characterization ofresidual lignins were also performed in the samples that werebiotreated for 2 weeks in the soybean-oil-amended cultures.As discussed previously (Table 2), the yield of thioacidolysismonomers decreased from 1024 mmol g�1 in the untreated controlto 723 mmol g�1 in the sample treated for 2 weeks in the non-amended cultures. In soybean-oil-amended cultures, this effectwas progressively diminished, because the yield of thioacidolysismonomers gradually increased from 723 mmol g�1 in the non-amended culture to reach 1073 mmol g�1 in the sample treated inthe culture with the highest soybean oil concentration (Table 3).Taken together with the lignin loss data (Table 1), these datasuggest that, in the non-amended cultures, part of the lignin wascompletely mineralized (detected as lignin loss) and the residualpolymer retained in the wood was transformed through depoly-merization and side chain oxidations. By contrast, in the culturesamended with soybean oil, part of the lignin was equally miner-alized, but the remaining lignin present in wood did not undergointense depolymerization.

Table 3Yield of thioacidolysis monomers detected after 2 weeks of P. taeda treatment byC. subvermispora in soybean oil-amended cultures.

Wood Sample Yield of thioacidolysis monomers(mmol g�1 of Klason lignin)

Untreated 1024� 24Biotreated in non-amended cultures 723� 63Biotreated in soybean oil-amended cultures:2.6 g of oil kg�1 of wood 941� 205.2 g of oil kg�1 of wood 913� 1510.4 g of oil kg�1 of wood 1073� 10

Table 4CTMP pulp properties of P. taeda that was biotreated with C. subvermispora.

Sample Time requiredto reach25�SR (min)

Tensile indexat 25 �SR(Nmg�1)

Tear indexat 25 �SR(mNm2 g�1)

Untreated control 124 34.6 2.45Biotreated in non-amended culture 114 34.6 2.26Biotreated in the soybean

oil-amended culture112 36.0 2.43

A. Aguiar et al. / International Biodeterioration & Biodegradation 64 (2010) 588e593592

Fig. 3 shows the content of lignin functional groups found insamples that were biotreated for 2 weeks in the soybean-oil-amended cultures. Similar to what was observed for the non-amended cultures, the lignins isolated from the wood samples thatwere biotreated in the amended cultures also presented decreasedlevels of aromatic hydroxyls when compared with the untreatedsample. Interestingly, the decrease of aliphatic hydroxyl groupswasmore pronounced in the soybean-oil-amended cultures. Consid-ering that the thioacidolysis yield did not decrease significantly inthese biotreated samples, Ca oxidation and Ca-Cb cleavage wouldnot explain the decrease of aliphatic hydroxyls. This finding isalso corroborated by the decrease of carboxyl groups in soybean-oil-amended cultures, especially at high concentrations. In general,it appears that soybean oil amendment in the cultures affected thelignin degradation routes but minimized the depolymerizationreactions.

3.1. Chemithermomechanical pulping (CTMP) of untreatedand biotreated P. taeda

P. taeda biotreatment in non-amended cultures and under thehighest soybean oil concentration was scaled up to 2 kg and thebiotreated wood chips were cooked to prepare CTMP pulps(Table 4). The pulps were obtained using a mild cooking step fol-lowed by mechanical fiberizing and refining. Blender fiberizing ofpre-cooked wood chips has been proposed as a simple method toprovide pulps with strength properties similar to those observedin pulps prepared by industrial disk refiners (Ruzinsky and Kokta2000; Vicentim and Ferraz 2008). The cooking yield was almostthe same for the untreated control and the biotreated samples(89.3e89.8%). The fiber bundles obtained after blender fiberizingwere further refined in a Jokro mill for beating periods from 90 to

Fig. 3. Contents of the major functional groups detected in lignins isolated from P.taeda that was treated with C. subvermispora for 2 weeks in soybean-oil-amendedcultures, as revealed by 31P-NMR of the phosphytilated derivatives. Contents ofcarboxyl and p-hydroxy-aromatic OH groups were multiplied tenfold for bettervisualization.

180 min, yielding pulps with refining levels ranging from 9 to 60�SR(Schopper-Riegler). In general, the SR values in biopulps increasedmore rapidly than did those in the control pulp. To achieve 25 SR,the untreated control pulp required 124min of beating, while thebiopulps from non-amended and soybean-oil-amended culturesrequired 114 and 112 min, respectively. Alternatively, improvedrefining levels can be obtained from biopulps at a fixed beatingtime. However, the biotreatments in non-amended and soybean-oil-amended cultures presented very similar benefits. Tensile and tearresistance were also evaluated for the refined pulps. As expected,tensile resistance increased during beating and tear indexes pre-sented the opposite tendency. The biopulps prepared from thewood chips that were biotreated in soybean-oil-amended culturespresented a slight increase in the tensile index and similar values forthe tear index. The slight increase in the tensile index observed forthe biopulp from the soybean-oil-amended culture correlates withthe lower glucan loss values observed in these cultures (Table 1),suggestingminor damage to the cellulose fibrils in this biotreatmentcondition. The limited pulp-strength improvement in bioCTMPpulps has been previously reported (Guerra et al. 2005). The presentwork corroborates this previous finding, showing that the strengthproperties of biopulps from P. taeda prepared in a CTMP processwere only slightly improved when compared to the control pulps.

In summary, increased fungal growth, as well as intense accu-mulation of TBARS and oxalic acid, were observed in soybean-oil-amended cultures. In contrast, enzyme secretion and lignin losseswere almost unaffected, while lignin depolymerization reactionswere inhibited in the soybean-oil-amended cultures. One conse-quence of this altered degradation routes was that CTMP pulping ofthe biotreated samples was not improved by soybean oil addition tothe cultures.

Acknowledgements

We wish to recognize Anderson Guerra (in memoriam) for helpwith the thioacidolysis and 31P NMR analyses. The technical assis-tance of J. S. Canilha, J. M. Silva, and J. C. Tavares is also acknowl-edged. This research was supported by FAPESP, CAPES, CNPq, andSCTDE/SP. A. Aguiar is thankful to FAPESP for a student fellowshipunder contract 03/04465-6.

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