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International Biodeterioration & Biodegradation 72 (2012) 88e93

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International Biodeterioration & Biodegradation

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

Effects of exogenous calcium or oxalic acid on Pinus taeda treatment with thewhite-rot fungus Ceriporiopsis subvermispora

André Aguiar a,b,*, André Ferraz a

aDepartamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo, CP 116, 12602-810 Lorena-SP, BrazilbDepartamento de Química, Biotecnologia e Engenharia de Bioprocessos, Campus Alto Paraopeba, Universidade Federal de São João Del-Rei, CP 131, 36420-000 OuroBranco-MG, Brazil

a r t i c l e i n f o

Article history:Received 25 April 2012Received in revised form12 May 2012Accepted 12 May 2012Available online 26 June 2012

Keywords:Ceriporiopsis subvermisporaBiopulpingManganese peroxidaseOxalic acidCalcium

* Corresponding author. Departamento de Químicade Bioprocessos, Campus Alto Paraopeba, UniversidadCP 131, 36420-000 Ouro Branco-MG, Brazil. Tel./fax:

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

0964-8305/$ e see front matter Crown Copyright �doi:10.1016/j.ibiod.2012.05.009

a b s t r a c t

Pinus taeda wood chips were treated with the biopulping fungus Ceriporiopsis subvermispora in calcium-or oxalic acid-amended cultures. The secretion of hydrolytic and oxidative enzymes was inhibited only inthe cultures having the highest concentration of calcium (1400 mg kg�1 wood). Calcium decreased theavailability of free oxalic acid, inhibited fungal growth, and reduced lignin mineralization and trans-formations. Oxalic acid amendment in the cultures was found not to affect the lignin mineralization andtransformations; however, it did inhibit the depolymerization reactions detectable in the residual ligninthat was retained in the biotreated wood. C. subvermispora presented catabolic activity for oxalic acid inthe cultures amended with 1660 mg acid kg�1 wood, whereas oxalic acid was synthesized when it wasamended at low amounts or initially absent in the cultures. These data suggest one ideal ratio of oxalicacid in C. subvermispora cultures and indicate that its exogenous addition does not necessarily accom-pany the further degradation of lignin.

Crown Copyright � 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Biopulping consists of treating wood chips with white-rot fungiprior to mechanical pulping to decrease the energy demands ofrefining and to improve the properties of the pulp and to decreasethe dependence on the chemicals used in chemical pulping. Cer-iporiopsis subvermispora, a selective basidiomycete used in lignindegradation, has been considered for utilization in industrial bio-pulping (Akhtar et al., 1998; Ferraz et al., 2008). Furthermore, thebiotreatment with this fungus has been reported to increase theenzymatic saccharification of lignocellulosic materials (Wan and Li,2011). This fungus is unique in its ability to cleave b-O-4 aryl-etherlinkages between lignin units during the early stages of wood decaywithout the extensive loss of cellulose mass (Guerra et al., 2002;Aguiar et al., 2010). Because the dominant oxidative enzyme thatthis fungus produces on wood chips is manganese peroxidase(MnP), it is believed that MnP plays a key role in the biodegradationof lignin by C. subvermispora (Vicuna et al., 1996; Aguiar et al., 2006,2010; Chi et al., 2007; Aguiar and Ferraz, 2008).

, Biotecnologia e Engenhariae Federal de São João Del-Rei,þ55 31 3741 3280.uiar@ufsj.edu.br (A. Aguiar).

2012 Published by Elsevier Ltd. All

It has been proposed that MnP oxidizes Mn2þ to Mn3þ, whichdissociate from the enzyme as an organic acid-chelated metal.Although Mn3þ is a strong oxidant that can dissociate from theenzyme and oxidize phenolic compounds, it cannot attack the non-phenolic units of lignin (Hofrichter, 2002; Aguiar and Ferraz, 2011).MnP generates phenoxy radicals, which then undergo a variety ofreactions, such as demethoxylation, quinone formation, and CaeCbcleavage, resulting in lignin depolymerization (Kapich et al., 1999;Hofrichter, 2002). In addition, MnP oxidizes non-phenolic ligninmodel compounds in the presence of Mn2þ via the peroxidation ofunsaturated fatty acids (Kapich et al., 1999; Cunha et al., 2010;Aguiar and Ferraz, 2011). The unsaturated fatty acids may be denovo synthesized by the fungus (Enoki et al., 1999) or produced byenzymes from the triglycerides present in the wood extractives(Gutiérrez et al., 2002). In the presence of chelating organic acidsand unsaturated fatty acids, fungal MnP is able to degrade non-phenolic lignin model compounds in vitro (Kapich et al., 1999)and to cause the fragmentation of lignin in milled hardwood(Cunha et al., 2010).

The production of MnP by the fungi on wood is typicallyaccompanied by the secretion of oxalic acid (Mäkelä et al., 2002;Aguiar et al., 2006; Aguiar and Ferraz, 2008), which may havea significant role in biopulping (Hunt et al., 2004). Oxalic acid isproduced by brown- and white-rot fungi (Espejo and Agosin, 1991;Hofrichter, 2002; Schilling and Jellison, 2005; Aguiar and Ferraz,

rights reserved.

A. Aguiar, A. Ferraz / International Biodeterioration & Biodegradation 72 (2012) 88e93 89

2008, 2011) and is the only chelator that can removeMn3þ from theMnP-reaction site at physiological concentrations, thereby stabi-lizing the ion in solution (Hofrichter, 2002). In addition, oxalic acidcan also acidify the culture pH, providing a more appropriateenvironment for the action of the hydrolytic and oxidative enzymes(Hofrichter, 2002; Aguiar and Ferraz, 2011). A third role for oxalicacid that has been described for C. subvermispora is to serve asa source of H2O2, namely, via the oxidation of oxalic acid to producea CO2 radical by Mn3þ ions (Urzúa et al., 1998).

Oxalic acid is found in biodegradedwood in the free form (activefor MnP) and also as insoluble crystals of calcium oxalate (Akhtaret al., 1998). The presence of supposedly esterified oxalic acid inwood polysaccharides has been reported by Hunt et al. (2004) afterthe alkaline extraction of wood chips biodegraded byC. subvermispora and other white-rot fungi.

Understanding the mechanism of wood decay byC. subvermispora could be useful for improving the biopulpingprocess. In the present work, Pinus taeda wood chips were bio-degraded by this fungus in cultures amendedwith calcium or oxalicacid. The addition of Ca2þ aimed to limit the availability of freeoxalic acid for the evaluation of the role of this metabolite in thebiodegradation process, whereas the addition of exogenous oxalicacid aimed to alter the substrate to maximize the activity of MnPdue to the high content of the free chelating agent. The extracellularenzymes, wood component loss, and oxalic acid level (its produc-tion or degradation) were monitored in the cultures. The thio-acidolysis of the wood and 31P nuclear magnetic resonance(31P NMR) analyses of the isolated lignins were used for providinginformation on the structural characteristics of the biodegradedlignins under these culture conditions.

2. Materials and methods

2.1. Wood biodegradation

C. subvermispora (Pilat) Gilbn. & Ryv. (L14807 strain SS-3)cultures were maintained at 4 �C on 20 g l�1 malt extract (Oxoid,England), 2 g l�1yeast extract (Vetec, Brazil) and 20 g l�1 agar(Oxoid, England). P. taeda wood chips (2.5 cm � 1.8 cm � 0.2 cm)were inoculated with a mycelial suspension using procedures fromthe literature (Aguiar and Ferraz, 2008). Each Erlenmeyer flask wasfilled with 45 g of wood chips and 4.5 mg of blended mycelia(100 mg kg�1, both on a dry basis). The treated cultures alsoreceived an aliquot (5 ml) of calcium chloride or oxalic acid solu-tions. For the calcium-amended cultures, the concentrations cor-responded to 350, 700 or 1400 mg calcium kg�1 wood; theconcentrations corresponded to 415, 830 or 1660 mg oxalic acidkg�1 wood for the oxalic acid-amended cultures. Non-amendedcultures were also performed. After inoculation, the flasks wereshaken by hand and stored at 27 �C up to 2 weeks. A total of four 2-lErlenmeyer flasks were inoculated for each biodegradation condi-tion: three cultures were used for the enzyme extraction anddetermination, and another was employed for the oxalic aciddetermination. One set of wood chips was sterilized and not inoc-ulated to serve as a control for all of the treatments.

2.2. Enzyme extraction and assays

For the recovery of the extracellular enzymes, the culturedwoodchips were extracted with 200 ml of 50 mmol l�1 sodium acetatebuffer (pH 5.5) containing in 0.1 g l�1 Tween 60, according to Souza-Cruz et al. (2004). The extracts were assayed for MnP, LiP (ligninperoxidase) and laccase using phenol-red (Kuwahara et al., 1984),Azure B (Souza-Cruz et al., 2004) and ABTS (Elissetche et al., 2006)as the substrates, respectively. For hydrolytic enzymes,

carboxymethylcellulose and birch xylan were used to assay theendoglucanase (Wood and Bhat, 1988) and xylanase activities(Bailey et al., 1992), respectively. The enzymatic activities wereexpressed as IU kg�1 of dry wood initially contained in the cultures.Standard deviations were calculated from triplicate cultures thatwere analyzed independently.

2.3. Characterization of biotreated wood

After enzyme extraction, the wood chips were washed withwater, air dried and weighed. The untreated and biotreated woodchips were milled to pass through a 0.5-mm screen and extractedwith 95% ethanol for 6 h in a Soxhlet apparatus. The contents of thechemical components of the wood were determined by acidhydrolysis. The Klason-insoluble and -soluble lignins were deter-mined by gravimetry and spectrophotometry (at 205 nm), respec-tively. The soluble sugars were quantified by high-performanceliquid chromatography using an HPX-87H column heated at 45 �Cand eluted with 5 mmol l�1 sulfuric acid at 0.6 ml min�1. The totalpolyose and glucan values were derived from calculations consid-ering the acid-released monomers, xylose and glucose, respectively(Ferraz et al., 2000).

The content of b-O-4 linkages in the lignins was determined bythe quantification of thioacidolysis monomers from ethanol-extracted milled wood (Rolando et al., 1992). The monomers weredetermined using gas chromatography and were quantifiedaccording to Aguiar et al. (2010).

The solubility in 1% NaOHwas determined by treating 300mg ofmilled wood (not extracted with ethanol) with 15 ml 1% (w/v)NaOH solution for 1 h at 100 �C. After treatment, the mixture washot-filtered through a glass filter number 3. The solids weresuccessively washed with 50 ml of 1% NaOH cold solution and100 ml of cold water. The washed solids were dried to a constantmass at 105 �C. The solubility in 1% NaOH was calculated as thedifference between the initial and extracted dry masses divided bythe initial dry mass (Elissetche et al., 2001).

2.4. Lignin isolation and characterization by 31P NMR spectroscopy

The lignins were isolated by mild acidolysis from the ethanol-extracted milled wood under a nitrogen atmosphere using thedioxane method, which was adapted by Aguiar et al. (2010) fromEvtuguim et al. (2001). The lignin yields were calculated based onthe Klason lignin content. The recovery yields for these lignins were14.5% and 15.0% (g 100 g�1 Klason lignin) for the samples ofuntreated (control) and biotreated wood without amendment,respectively. The recoveries were 15.3%, 15.4% and 14.3% for thebiotreated wood amended with 350, 700 and 1400 mg calciumkg�1 wood, respectively, and 16.4%, 14.7% and 15.7% for the bio-treated wood amended with 415, 830 and 1660 mg oxalic acid kg�1

wood, respectively.The isolated lignins were characterized by 31P NMR spectros-

copy according to Granata and Argyropoulos (1995) using a Bruker250 MHz NMR spectrometer.

2.5. Extraction and determination of oxalic acid

The oxalic acid (free, acid- and alkali-extracted) content wasdetermined according to Aguiar and Ferraz (2008).

3. Results and discussion

The effect of oxalic acid on the action of MnP fromC. subvermispora was initially evaluated in the basis of the in vitroassays performed at various concentrations of oxalic acid (Fig. 1). A

0

10

20

30

40

0 1 2 3

Mn

P (

IU l

-1)

oxalic acid (mmol l-1

)

Fig. 1. Effect of oxalic acid on the MnP activity.

A. Aguiar, A. Ferraz / International Biodeterioration & Biodegradation 72 (2012) 88e9390

crude culture extract recovered from 2-week biotreated P. taedapresented a MnP activity of 32 IU l�1. This activity was detectedthrough the conventional phenol-red oxidation procedure inwhichthe final lactate and succinate concentrations were 15 mmol l�1

(Kuwahara et al., 1984; Souza-Cruz et al., 2004). The same assayperformed in the absence of lactate and succinate resulted in nooxidation of phenol-red, which is in agreement with a previousstudy inwhich theMnP activity had been evaluated in the presenceof several chelating agents (Kishi et al., 1994). In contrast, the sameassay performed in the presence of increasing oxalate concentra-tions resulted in the activities shown in Fig. 1. The maximal activitywas observed at an oxalate concentration of 1.5 mmol l�l, corre-sponding to approximately the same value detected in the presenceof 15 mmol l�l lactate and succinate. Previous data for the organicacid production by C. subvermispora indicated concentrationsvarying from 0.4 to 2.5 mmol l�l in liquid medium and0.5e3.2 mmol l�l in the water contained in solid-state cultures onwood; in contrast, other dicarboxylic acids were produced at lowerconcentrations (Urzúa et al., 1998; Aguiar et al., 2006). Takentogether, these data suggest that, oxalate is the major chelatingcompound supporting MnP cycling in C. subvermispora culturesunder physiological conditions.

In a series of subsequent experiments, the oxalate availability inthe cultures of C. subvermispora was disturbed by the addition ofCa2þ ions or external oxalic acid to evaluate the role of thischelating agent on the overall wood biodegradation process. In thecultures amended with Ca2þ ions, a significant portion of the oxalicacid produced by the fungus should precipitate as calcium oxalatedue to its low Kps value (2.3 � 10�9).

Table 1Enzymes, and mass and chemical component losses detected during the P. taeda treatm

Non-amended Ca2þ (mg kg�1 wood)a

0 350 700

Enzymatic activitiesb (UI kg�1 wood)Xylanase 565 � 188 (a) 834 � 99 (a) 840 � 287 (a)Endoglucanase 58 � 31 (a) 73 � 14 (a) 46 � 4 (a)MnP 142 � 51 (a) 218 � 32 (b) 164 � 14 (a)Laccase 0.3 � 0.4 (a) 0.3 � 0.2 (a) 1.3 � 0.3 (b)

Wood mass and component losses (% m/m)Mass 3.3 � 0.9 (a) 3.8 � 0.7 (a) 2.5 � 0.6 (a)Lignin 8.3 � 0.9 (a) 8.8 � 0.7 (a) 6.9 � 0.5 (a)Polyoses 5.1 � 0.9 (a) 5.8 � 0.7 (a) 4.8 � 0.4 (a)Glucan 4.7 � 0.9 (a) 6.7 � 0.7 (b) 6.1 � 0.5 (a)

a In each row, same letters indicate that the values do not differ, with 95% confidence, ab LiP activities were not detected in all of the cultures.

The amendment with Ca2þ ions at concentrations ranging from350 to 1400 mg Ca2þ kg�1 of wood resulted in an apparent inhi-bition of the fungal growth, as the mycelia covering the wood chipsurfaces diminished as a function of increasing Ca2þ concentrationsin the cultures. In contrast, the cultures amended with oxalic acid(415e1660 mg kg�1 of wood) presented no apparent effect on thefungal growth.

The levels of hydrolytic enzymes detected in the extractsrecovered from the above-mentioned cultures were similar to eachother, independent of the culture supplementation with Ca2þ oroxalic acid (Table 1). With regard to the oxidative enzymes, laccasewas detected only at minor amounts in most of the treatments,whereas the MnP activity was slightly increased in the culturescontaining the lowest Ca2þ concentration and in the two culturesamended with oxalic acid at 415 and 830 mg kg�1 of wood. At thehighest oxalic acid concentration, the level of MnP in the cultureswas twice the level observed in the non-amended cultures(Table 1). This high level of MnP may reveal some detoxificationmechanism of the fungus because the degradation of oxalic acid byMnP has been previously reported in cultures of C. subvermispora(Urzúa et al., 1998).

The mass and chemical component losses observed after thewood biotreatment were also similar among the examined cultureconditions, with the exception that the values observed for thecultures at the highest Ca2þ concentration resulted in a lowdegradation of the wood components, indicating a clear inhibitioneffect on fungal metabolism (Table 1). Another exception was thedecreased glucan loss in the cultures at the highest oxalic acidamendment (Table 1).

In contrast to the enzymes, the production of oxalic acid wasstrongly inhibited by the addition of Ca2þ to the cultures (Fig. 2).Oxalic acid can be found in biodegraded wood as water-extractableoxalate or as insoluble crystals of calcium oxalate that can beextracted by HCl solutions (Akhtar et al., 1998; Aguiar et al., 2006).The fraction extractable only with alkaline solutions has beenassigned to the oxalate esterified to polysaccharides in the plantcell wall (Hunt et al., 2004). However, at least part of the alkali-extractable oxalate could be assigned to the free oxalate that isreleased only after a certain degree of swelling of the fibers due tothe alkaline medium because untreated wood contains significantamounts of alkali-extractable oxalate (Fig. 2). This finding is rele-vant because the water-extractable oxalate should be considered tobe active during the biodegradation of wood and at least part of thealkali-extractable oxalate is also available for fungal metabolism.

The strong inhibition of oxalic acid production by the fungus athigh Ca2þ concentrations associated with the maintenance ofsignificant enzymatic activities in the cultures provided ametabolicsituation that was relevant to understand the mode of action of this

ent with C. subvermispora for 2 weeks in calcium- or oxalic acid-amended cultures.

Oxalic acid (mg kg�1 wood)a

1400 415 830 1660

493 � 92 (a) 664 � 158 (a) 826 � 218 (a) 794 � 109 (a)44 � 21 (a) 97 � 11 (a) 100 � 15 (a) 103 � 4 (a)91 � 6 (a) 221 � 20 (b) 232 � 42 (b) 413 � 83 (b)2.0 � 0.5 (b) 0 � 0 (a) 0 � 0 (a) 0 � 0 (a)

1.5 � 0.7 (b) 3.2 � 0.9 (a) 4.0 � 0.7 (a) 3.5 � 0.7 (a)5.2 � 0.7 (b) 9.6 � 0.8 (a) 9.5 � 0.7 (a) 9.4 � 0.6 (a)3.0 � 0.7 (b) 6.2 � 0.8 (a) 7.0 � 0.7 (a) 4.9 � 0.7 (a)4.3 � 0.7 (a) 4.2 � 0.9 (a) 4.3 � 0.7 (a) 2.1 � 0.7 (b)

nd different letters indicate that the values are significantly different (Dunnet’s test).

A. Aguiar, A. Ferraz / International Biodeterioration & Biodegradation 72 (2012) 88e93 91

fungus on the wood components. For example, the culture with thehighest concentration of Ca2þ imposed a condition on the fungus inwhich the total oxalic acid in the culture was approximately one-half of the values observed in the non-amended cultures. Thewater-extractable oxalate was even lower, attaining a concentra-tion of 0.6 mg kg�1 of wood (Fig. 2). Under this culture condition,the level of MnP was still significant (91 � 6 IU kg�1), but thelimited availability of oxalate to support the MnP cycling seems tobe responsible for the reduced ability of the fungus to degradelignin (Table 1). Moreover, the minimal lignin loss apparentlyaffected the overall degradation process, as the mass and polyoselosses were also low for this culture.

In the oxalic acid-amended cultures, the quantification ofthe residual oxalate permitted the evaluation of whetherC. subvermispora regulates the extracellular concentration of oxalate,as previously reported for brown-rot fungi (Schilling and Jellison,2005). The total content of oxalic acid recovered from the non-amended cultures was 760 mg kg�1 of wood (Fig. 2). This valueincreased to 1200 mg kg�1 in the cultures amended with 415 mgoxalic acid kg�1 of wood, indicating that the fungus still secretedsignificant amounts of thismetabolite. For the culture amendedwith830mg oxalic acid kg�1 wood, an inhibition of the secretion or evena catabolic action on the oxalic acid should have occurred becausethe total content of oxalic acid recovered after two weeks of culti-vation did not exceed the values observed at the lowest amendmentlevel. At the highest concentration of oxalic acid added to thecultures, there was a clear degradation of the acid, as the totalamount recovered (1400 mg kg�1) was lower than the initial valueadded to the culture (1660 mg kg�1) (Fig. 2). Although this culturecondition induced the highest levels of MnP, the lignin loss was notenhanced (Table 1). This result corroborates previous studies indi-cating that the MnP levels in the cultures are not the single factorresponsible for the degradation of lignin in natural substrates, suchas wood or other lignocellulosic materials (Addleman et al., 1995;Vicentim and Ferraz, 2007).

The de novo synthesis or degradation of oxalic acid in theC. subvermispora cultures depended on the initial concentration ofexogenous oxalic acid and prompted the speculation that thefungus effectively balanced the concentration of oxalic acid in thecultures either via synthesis when this metabolite was not presentin sufficient amounts or through degradation when it was presentin excess. Thus, the fungus may have activated enzymes involved in

Fig. 2. Oxalic acid detected after P. taeda treatment with C. subverm

the synthesis (glyoxylate oxidase and oxaloacetase) or degradation(oxalate oxidase and oxalate decarboxylase) of oxalic acid becauseboth groups of enzymes were detected in C. subvermispora(Watanabe et al., 2005). As mentioned above, MnP can also playa significant role in the degradation of oxalic acid in C. sub-vermispora cultures (Urzúa et al.,1998). However, because the levelsof alkali-extractable oxalate were similar in all of the evaluatedcultures, the esterification of oxalate to polysaccharides (Hunt et al.,2004) seems not to be the strategy used by the fungus to avoid anexcess of oxalic acid in the cultures (Fig. 2).

The evaluation of the data for fungal growth, the secretion ofenzymes and oxalic acid and the losses of wood mass and chemicalcomponents indicate that some degree of inhibition of fungalmetabolismwas achieved in the cultures amendedwith the highestCa2þ concentration and that this included decreased oxalic acidproduction and lignin degradation. In the oxalic acid-amendedcultures, the highest level of exogenous acid induced a high levelof MnP secretion, which did not induce a significant increase inlignin degradation. Previous results also show that the productionof oxalic acid by C. subvermispora attained high concentrations(1200 mg kg�1 wood) in cultures amended with soybean oil(10.4 g kg�1); however, the delignification was not stimulated,revealing similar levels to those observed in the non-amendedcultures (Aguiar et al., 2010).

The mass and chemical component losses reported in Table 1reflect only the degradation of the wood components to carbondioxide and water or into water-soluble compounds that werereleased when the biotreated wood was washed at the end ofthe culture period. A better assessment of the transformation of theresidual wood components can be obtained by searching thestructural characteristics of the residual lignin or even by simpleassays, such as the solubility of the wood in 1% NaOH (rot-index).The solubility in 1% NaOH is a well-known titer that is indicative ofwood decay because the residual lignin and polysaccharides thatsuffered some depolymerization during the biotreatment becomemore soluble in alkaline solutions, increasing the 1% NaOH solu-bility value in biotreated wood (Elissetche et al., 2001).

The solubility in 1% NaOH of all of the wood samples evaluatedin this work is shown in Fig. 3. The solubility in 1% NaOH increasedfrom 7.8 � 0.3% in the undecayed wood to 10.7 � 0.2% in the woodbiotreated with C. subvermispora in the non-amended cultures. Inthe cultures amended with Ca2þ, the 1% NaOH solubility increased

ispora for 2 weeks in calcium- or oxalic acid-amended cultures.

0 4 8 12

Wood solubility in NaOH 1%

untreated

biotreated without additive

350 mg Ca2+ kg-1 wood

700 mg Ca2+ kg-1 wood

1400 mg Ca2+ kg-1 wood

415 mg OA kg-1 wood

830 mg OA kg-1 wood

1660 mg OA kg-1 wood

Fig. 3. Solubility in 1% NaOH of wood samples treated with C. subvermispora incalcium- or oxalic acid-amended cultures.

0 1500 3000 4500

control

without additive

350 mg Ca/kg

700 mg Ca/kg

1400 mg Ca/kg

415 mg OA/kg

830 mg OA/kg

1660 mg OA/kg

aliphatic-OH

non-condensed aromatic-OH

guaiacyl-OH

p-hydroxy-aromatic-OH (x 10)

carboxyl (x 10)

total aromatic-OH

total condensed aromatic-OH

µmol g-1 Klason lignin

Fig. 4. Contents of the major functional groups detected in the lignins isolated fromP. taeda treated with C. subvermispora for 2 weeks in calcium- or oxalic acid-amendedcultures, as revealed by 31P NMR of the phosphitylated derivatives. The contents of thecarboxyl and p-hydroxy-aromatic OH groups were multiplied tenfold for bettervisualization.

A. Aguiar, A. Ferraz / International Biodeterioration & Biodegradation 72 (2012) 88e9392

to a lower extent, reaching a maximum of 9.5% at the lowest Ca2þ

concentration; in the oxalic acid-amended cultures, these valuesincreased up to 11.8� 0.8% as a function of the increased oxalic acidconcentrations added to the cultures. These data suggest that theresidual polymers in the Ca2þ-amended cultures were less depo-lymerized than those in the non-amended cultures and in thecultures amended with oxalic acid.

Regarding the structural characteristics of the residual lignin,the untreated and biotreated wood samples were characterized byin situ thioacidolysis. The thioacidolysis monomer yield is a well-known titer that is indicative of lignin depolymerization (Choiet al., 2006; Aguiar et al., 2010), and decreases in the monomeryield of thioacidolysis can be a consequence of lignin trans-formation via the cleavage of aryl-ether linkages. However, fungal-induced CaeCb cleavage and simple Ca-oxidation would alsodecrease the monomer yield in this analytical technique (Rolandoet al., 1992). This fact is relevant because, in addition to aryl-ethercleavage, Ca-Cb cleavage and simple Ca-oxidation have beenobserved during the biodegradation of lignin model compounds byC. subvermispora (Srebotnik et al., 1997) and during the in vitrodegradation of a non-phenolic ligninmodel compound by theMnP/linoleic acid system (Kapich et al., 1999). The data in Table 2 showthat, in the non-amended cultures, the residual lignin produced29% fewer monomers after thioacidolysis than the untreated wood,indicating that the residual lignin of the biotreated wood waspartially depolymerized. In a previous work, the decrease of thio-acidolysis monomer yields was progressive over time, attaining50% fewer monomers after four weeks of biotreatment (Aguiaret al., 2010). The extent of depolymerization of the residual lignin

Table 2Yield of thioacidolysis monomers detected after 2 weeks of P. taeda treatment withC. subvermispora in calcium- or oxalic acid-amended cultures.

Wood sample Yield of thioacidolysismonomers (mmol g�1 ofKlason lignin)

Untreated (control) 1024 � 24Biotreated in non-amended cultures 723 � 63 (�29%)

Biotreated in cultures amended with:350 mg of Ca2þ kg�1 wood 860 � 7 (�16%)700 mg of Ca2þ kg�1 wood 842 � 3 (�18%)1400 mg of Ca2þ kg�1 wood 906 � 16 (�11%)415 mg of oxalic acid kg�1 wood 935 � 62 (�9%)830 mg of oxalic acid kg�1 wood 998 � 16 (�2%)1660 mg of oxalic acid kg�1 wood 1086 � 16 (þ6%)

was progressively diminished in the Ca2þ- and oxalic acid-amended cultures, as shown in Table 2.

Residual lignins were also extracted from biotreated wood usinga mild-non-razing extraction procedure (Evtuguim et al., 2001) forthe subsequent characterization using 31P NMR. Additional ligninstructural characteristics were revealed by a 31P NMR spectroscopicevaluation of the phosphitylated lignin derivatives for the detectionof aliphatic and aromatic OH groups and carboxyl functional groups(Granata and Argyropoulos, 1995).

In our previous work, the pattern of transformation of theresidual lignin in the C. subvermispora-biotreated samples for fourweeks involved a decrease of the aliphatic and phenolic OHcontents and an increase of the carboxyl groups (Aguiar et al.,2010). These lignin transformations were inhibited in thecalcium-amended cultures. The decrease of the OH contents andincrease of the carboxyl groups were less pronounced in thecultures amended with 700 and 1400 mg kg�1 wood. In contrast,the addition of Ca2þ did not inhibit the formation of carboxylgroups, evenwith the inhibition of the lignin depolymerization andmineralization (Fig. 4).

For the oxalic acid-amended cultures, the trend of a decrease inthe aliphatic and phenolic OH contents and increase in the carboxylgroups were maintained in the extracted lignins. However, thelevels of functional groups were unaffected because of the variedconcentrations of exogenous oxalic acid (Fig. 4).

4. Conclusion

The cultures of C. subvermispora on P. taeda were disturbed bythe addition of Ca2þ ions or oxalic acid. Both of the cultureamendments affected fungal metabolism, and the most significantchange was an increased production of MnP in the culturesamended with the highest oxalic acid concentration. However,lignin degradation was not stimulated, even though the MnP levelswere increased. In contrast, when the oxalic acid became unavail-able to the fungus through the addition of Ca2þ ions, somerepression of lignin degradation was detected. In general, theresults suggest that the fungus minimized the effects of the addi-tion of external sources of Ca2þ or oxalic acid to the cultures. In thecase of high levels of oxalic acid in the cultures, the fungus clearly

A. Aguiar, A. Ferraz / International Biodeterioration & Biodegradation 72 (2012) 88e93 93

degraded a portion of the exogenous oxalic acid. Therefore, the datasuggest that oxalic acid addition to the cultures can be useful tostimulate MnP secretion but not to increase lignin degradation.

Acknowledgments

We wish to recognize Anderson Guerra (in memoriam) forassistance in the thioacidolysis and 31P NMR analyses. The technicalassistance of J.S. Canilha, J.M. Silva and J.C. Tavares is alsoacknowledged. This research was supported by FAPESP, CAPES,CNPq and SCTDE/SP. A. Aguiar is thankful to FAPESP for a studentfellowship under contract 03/04465-6.

References

Addleman, K., Dumonceaux, T., Paice, M.G., Bourbonnais, R., Archibald, F.S., 1995.Production and characterization of Trametes versicolor mutants unable to bleachhardwood kraft pulp. Applied and Environmental Microbiology 61, 3687e3694.

Aguiar, A., Ferraz, A., 2008. Relevance of extractives and wood transformationsproducts on the biodegradation of Pinus taeda by Ceriporiopsis subvermispora.International Biodeterioration & Biodegradation 61, 182e188.

Aguiar, A., Ferraz, A., 2011. Mechanisms involved in the biodegradation of ligno-cellulosic materials and related technological applications. Química Nova 34,1729e1738.

Aguiar, A., Souza-Cruz, P.B., Ferraz, A., 2006. Oxalic acid, Fe3þ-reduction activity andoxidative enzymes detected in culture extracts recovered from Pinus taedawood chips biotreated by Ceriporiopsis subvermispora. Enzyme and MicrobialTechnology 38, 873e878.

Aguiar, A., Mendonça, R., Rodríguez, J., Ferraz, A., 2010. Behavior of Ceriporiopsissubvermispora during Pinus taeda biotreatment in soybean-oil-amendedcultures. International Biodeterioration & Biodegradation 64, 588e593.

Akhtar, M., Blanchette, R.A., Myers, G., Kirk, T.K., 1998. An overview of biome-chanical pulping research. In: Young, R., Akhtar, M. (Eds.), Environmentallyfriendly technologies for the pulp and paper industry. John Wiley and Sons,New York, pp. 309e383.

Bailey, M.J., Biely, P., Poutanen, K., 1992. Inter-laboratory testing of methods forassay of xylanase activity. Journal of Biotechnology 23, 257e270.

Chi, Y., Hatakka, A., Maijala, P., 2007. Can co-culturing of two white-rot fungiincrease lignin degradation and the production of lignin-degrading enzymes?International Biodeterioration & Biodegradation 59, 32e39.

Choi, J.W., Choi, D.H., Ahn, S.H., Lee, S.S., Kim, M.K., Meier, D., Faix, O., Scott, G.M.,2006. Characterization of trembling aspen wood (Populus tremuloides L.)degraded with the white rot fungus Ceriporiopsis subvermispora and MWLsisolated thereof. Holz als Roh-und Werkstoff 64, 415e422.

Cunha, G.G.S., Masarin, F., Norambuena, M., Freer, J., Ferraz, A., 2010. Linoleic acidperoxidation and lignin degradation by enzymes produced by Ceriporiopsissubvermispora grown on wood or in submerged liquid cultures. Enzyme andMicrobial Technology 46, 262e267.

Elissetche, J.P., Ferraz, A., Parra, C., Freer, J., Baeza, J., Rodriguez, J., 2001. Biodegra-dation of Chilean nativewood species,Drimyswinteri andNothofagus dombeyi, byGanoderma australe.World Journal ofMicrobiology&Biotechnology 17, 577e581.

Elissetche, J., Ferraz, A., Freer, J., Mendonça, R., Rodríguez, J., 2006. Thiobarbituricacid reactive substances, Fe3þ reduction and enzymatic activities in cultures ofGanoderma australe growing on Drimys winteri wood. FEMS MicrobiologyLetters 260, 112e118.

Enoki, M., Watanabe, T., Nakagame, S., Koller, K., Messner, K., Honda, Y.,Kuwahara, M., 1999. Extracellular lipid peroxidation of selective white-rotfungus, Ceriporiopsis subvermispora. FEMS Microbiology Letters 180, 205e211.

Espejo, E., Agosin, E., 1991. Production and degradation of oxalic acid by brown rotfungi. Applied and Environmental Microbiology 57, 1980e1986.

Evtuguim, D.V., Pascoal Neto, C., Silva, A.M.S., Domingues, P.M., Amado, F.M.L.,Robert, D., Faix, O., 2001. Comprehensive study on the chemical structure of

dioxane lignin from plantation Eucalyptus globuluswood. Journal of Agriculturaland Food Chemistry 49, 4252e4261.

Ferraz, A., Rodríguez, J., Freer, J., Baeza, J., 2000. Estimating chemical composition ofbiodegraded pine and eucalyptus by DRIFT spectroscopy and multivariateanalysis. Bioresource Technology 74, 201e212.

Ferraz, A., Guerra, A., Mendonça, R., Masarin, F., Vicentim, M.P., Aguiar, A.,Pavan, P.C., 2008. Technological advances and mechanistic basis for fungalbiopulping. Enzyme and Microbial Technology 43, 178e185.

Granata, A., Argyropoulos, D.S., 1995. 2-Chloro-4,4,5,5-tetramethyl-1,3,2-dioxa-phospholane, a reagent for the accurate determination of the uncondensed andcondensed phenolic moieties in lignins. Journal of Agricultural and FoodChemistry 43, 1538e1544.

Guerra, A., Mendonça, R., Ferraz, A., 2002. Characterization of the residual lignins inPinus taeda biodegraded by Ceriporiopsis subvermispora by using in situ CuOoxidation and DFRC methods. Holzforschung 56, 157e160.

Gutiérrez, A., del Río, J.C., Martinez-Inigo, M.J., Martinez, M.J., Martinez, A.T., 2002.Production of new unsaturated lipids during wood decay by ligninolyticbasidiomycetes. Applied and Environmental Microbiology 68, 1344e1350.

Hofrichter, M., 2002. Review: lignin conversion by manganese peroxidase (MnP).Enzyme and Microbial Technology 30, 454e466.

Hunt, C., Kenealy, W., Horn, E., Houtman, C., 2004. A biopulping mechanism:creation of acid groups on fiber. Holzforschung 58, 434e439.

Kapich, A.N., Jensen, K.A., Hammel, K.E., 1999. Peroxyl radicals are potential agentsof lignin biodegradation. FEBS Letters 461, 115e119.

Kishi, K., Wariishi, H., Marquez, L., Dunford, H.B., Gold, M.H., 1994. Mechanism ofmanganese peroxidase compound II reduction. Effect of organic acid chelatorsand pH. Biochemistry 33, 8694e8701.

Kuwahara, M., Glenn, J.K., Morgan, M.A., Gold, M.H., 1984. Separation and charac-terization of two extracellular H2O2-dependent oxidases from ligninolyticcultures of Phanerochaete chrysosporium. FEBS Letters 169, 247e250.

Mäkelä, M., Galkin, S., Hatakka, A., Lundell, T., 2002. Production of organic acids andoxalate decarboxylase in lignin-degrading white rot fungi. Enzyme andMicrobial Technology 30, 542e549.

Rolando, C., Monties, B., Lapierre, C., 1992. Thioacidolysis. In: Lin, S., Dence, C.W.(Eds.), Methods in lignin chemistry. Springer-Verlag, Heidelberg,pp. 334e349.

Schilling, J.S., Jellison, J., 2005. Oxalate regulation by two brown rot fungi decayingoxalate-amended and non-amended wood. Holzforschung 59, 681e688.

Souza-Cruz, P.B., Freer, J., Siika-Aho, M., Ferraz, A., 2004. Extraction and determi-nation of enzymes produced by Ceriporiopsis subvermispora during biopulpingof Pinus taeda wood chips. Enzyme and Microbial Technology 34, 228e234.

Srebotnik, E., Jensen, K., Kawai, S., Hammel, K.E., 1997. Evidence that Ceriporiopsissubvermispora degrades nonphenolic lignin structure by a one-electron-oxidation mechanism. Applied and Environmental Microbiology 63,4435e4440.

Urzúa, U., Kersten, P.J., Vicuña, R., 1998. Manganese peroxidase-dependent oxida-tion of glyoxilic and oxalic acids synthesized by Ceriporiopsis subvermisporaproduces extracelular hydrogen peroxide. Applied and Environmental Micro-biology 64, 68e73.

Vicentim, M.P., Ferraz, A., 2007. Enzyme production and chemical alterations ofEucalyptus grandis wood during biodegradation by Ceriporiopsis subvermisporain cultures supplemented with Mn2þ, corn steep liquor and glucose. Enzymeand Microbial Technology 40, 645e652.

Vicuna, R., Larraın, J., Lobos, S., Salas, C., Salas, L., 1996. Culture conditions of Cer-iporiopsis subvermispora determine the pattern of MnP isoenzymes. In:Srebotnik, E., Messner, K. (Eds.), Biotechnology in the pulp and paper industry,recent advances in applied and fundamental research. Facultas-Unversitätsverlag, Vienna, pp. 345e350.

Wan, C., Li, Y., 2011. Effectiveness of microbial pretreatment by Ceriporiopsis sub-vermispora on different biomass feedstocks. Bioresource Technology 102,7507e7512.

Watanabe, T., Hattori, T., Tengku, S., Shimada, M., 2005. Purification and charac-terization of NAD-dependent formate dehydrogenase from the white-rotfungus Ceriporiopsis subvermispora and a possible role of the enzyme inoxalate metabolism. Enzyme and Microbial Technology 37, 68e75.

Wood, T.M., Bhat, K.M., 1988. Methods for measuring cellulase activities. Methods inEnzymology 160, 87e113.