Evaluation of Eucalyptus grandis Hill ex Maiden biopulping with Ceriporiopsis subvermispora under non-aseptic conditions

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1 Holzforschung, Vol. 62, pp. 1 7, 2008 Copyright by Walter de Gruyter Berlin New York. DOI /HF Evaluation of Eucalyptus grandis Hill ex Maiden biopulping with Ceriporiopsis subvermispora under non-aseptic conditions Fernando Masarin and André Ferraz* Escola de Engenharia de Lorena, Universidade de São Paulo, Lorena, SP, Brazil *Corresponding author. Escola de Engenharia de Lorena, Universidade de São Paulo, CP 116, Lorena, SP, Brazil Abstract In biopulping, efficient wood colonization by a selected white-rot fungus depends on previous wood chip decontamination to avoid the growth of primary molds. Although simple to perform in the laboratory, in largescale biopulping trials, complete wood decontamination is difficult to achieve. Furthermore, the use of fungal growth promoters such as corn steep liquor enhances the risk of culture contamination. This paper evaluates the ability of the biopulping fungus Ceriporiopsis subvermispora to compete with indigenous fungi in cultures of fresh or poorly decontaminated Eucalyptus grandis wood chips. While cultures containing autoclaved wood chips were completely free of contaminants, primary molds grew rapidly when non-autoclaved wood chips were used, resulting in heavily contaminated cultures, regardless of the C. subvermispora inoculum/wood ratio evaluated (5, 50 and 3000 mg mycelium kg -1 wood). Studies on benomyl-amended medium suggested that the fungi involved competed by consumption of the easily available nutrient sources, with C. subvermispora less successful than the contaminant fungi. The use of acidwashed wood chips decreased the level of such contaminant fungi, but production of manganese peroxidase and xylanases was also decreased under these conditions. Nevertheless, chemithermomechanical pulping of acidwashed samples biotreated under non-aseptic conditions gave similar fibrillation improvements compared to samples subjected to the standard biodegradation process using autoclaved wood chips. Keywords: biopulping; Ceriporiopsis subvermispora; mechanical pulping; non-aseptic cultures; primary molds. Introduction Biopulping is the fungal pretreatment of wood chips for the production of mechanical or chemical pulps (Akhtar et al. 1998). The concept of biopulping is based on the ability of a selected white-rot fungus to colonize and selectively degrade the lignin in wood, thereby leaving the cellulose relatively intact. While such selective wood biodegradation allows for pulp strengthening and energy savings in mechanical pulping (Akhtar et al. 1998; Scott et al. 2002; Guerra et al. 2005, 2006), reduced lignin content, chemical loads and pulping severity are benefits provided by the fungal pretreatment in chemical pulping. (Bajpai et al. 2001; Mendonca et al. 2002; Kang et al. 2003). To obtain the aforementioned benefits, effective wood colonization by a selected white-rot fungus is essential. During this step, some changes in wood structure and ultrastructure, which include opened pit membranes and increased secondary walls porosity (Akhtar et al. 1998), lignin depolymerization (Guerra et al. 2002, 2003, 2004; Choi et al. 2006) and oxalic acid esterification of the polysaccharides in secondary walls (Hunt et al. 2004), are produced within short biotreatment periods (15 30 days). Ceriporiopsis subvermispora has been selected as a suitable white-rot fungus for biopulping because it colonizes wood rapidly and causes preferential lignin degradation. Wood biodegradation with this species results in significant energy savings in subsequent mechanical pulping processes, even after short biotreatment periods (Akhtar et al. 1998). When growing on wood chips, this fungus secretes a wide range of metabolites, including major components such as organic acids (Hunt et al. 2004; Aguiar et al. 2006), manganese peroxidase (MnP) (Rüttimann et al. 1992; Souza-Cruz et al. 2004) and xylanase (Sethuraman et al. 1998; Heidorne et al. 2006). Detection of these extracellular metabolites has been successfully correlated with fungal growth (Vicentim and Ferraz 2007) and ultimately with the biopulping benefits (Ferraz et al. 2002). When biopulping experiments are performed on a laboratory scale, autoclaved or steam-treated wood chips may be inoculated with blended mycelium or spores, depending on the fungus species. In the case of C. subvermispora, fragmented mycelium has been used because this fungus does not produce spores in the culture conditions often used for inoculum production. In this case, inoculation of 5 mg of blended mycelium per kg of dry wood in 0.5% corn steep liquor (CSL)-supplemented cultures resulted in energy savings during mechanical pulping equivalent to non-supplemented cultures inoculated with 3000 mg kg -1 (Akhtar et al. 1998). In Eucalyptus grandis biopulping, inoculum levels of 5 mg kg -1 in 0.5% CSL-supplemented cultures and 500 mg kg -1 in non-supplemented cultures resulted in similar amounts of fungal biomass growth after 15 days (Souza- Cruz 2005). Despite such benefits, the use of CSL-supplemented cultures has failed to promote efficient wood colonization by C. subvermispora in large-scale wood biodegradation tests performed on a 50-t biopulping pilot plant installed in Brazil owing to culture contamination with naturally occurring microorganisms (unpublished data). This is a consequence of non-optimal decontamination of wood chips in a large-scale continuous process. In this context, the aim of the present study was to determine whether C. subvermispora would be able to

2 2 F. Masarin and A. Ferraz Article in press - uncorrected proof compete with indigenous fungi in cultures under nonaseptic conditions. Increasing inoculum/wood ratios in CSL-supplemented and non-supplemented cultures were evaluated and C. subvermispora growth was monitored by detection of typical titers for its extracellular metabolism. Acid washing of the eucalyptus wood chips was evaluated as a simple alternative decontamination procedure. Materials and methods Sampling and storage of wood chips E. grandis wood chips were sampled in a chemithermomechanical pulp (CTMP) mill located at Caieiras, SP, Brazil. Wood logs cut from 6 8-year-old trees were stored in the mill wood yard for periods not longer than 7 days. The logs were debarked, washed and chipped using the industrial mill facilities. The freshly cut wood chips were collected from the first section of the moving belt leaving the chipping machine and stored at -188C in clean polyethylene bags. Before use in biodegradation experiments, small amounts of these wood chips were thawed in clean polyethylene trays inside an aseptic chamber. Fungus, inoculum preparation and wood biodegradation C. subvermispora (Pilat) Gilbn. & Ryv. (L14807 SS-3 strain) cultures were maintained at 48C on 2.4% (wt/vol) potato dextrose broth (DIFCO, Maryland), 0.7% (wt/vol) yeast extract (Vetec, Brazil) agar slants containing a slice of wood chip. An aliquot of 200 ml of liquid medium containing potato extract broth (2.4% wt/vol) and yeast extract (0.7% wt/vol) was inoculated with 20 discs (8 mm in diameter) of C. subvermispora-precultured solid medium. This liquid culture was maintained at 278C without shaking for 12 days. The mycelium mat was then filtered and washed with 300 ml of autoclaved water. Mycelium obtained from several cultures was blended with 100 ml of autoclaved water in three cycles of 15 s. The mycelium suspension was used to inoculate wood chips in 2-l Erlenmeyer flasks. Prior to the biodegradation of autoclaved wood chips, 50 g (dry basis) of wood chips was immersed in water for 16 h. The surplus water was drained and the wood chips were autoclaved in 2-l Erlenmeyer flasks covered with cotton stoppers and aluminum foil at 1218C for 15 min. The moisture of autoclaved wood chips was approximately 55% (wt/wt). In the case of nonautoclaved fresh wood chips, thawed material was weighed inside an aseptic chamber (50 g, dry basis) and transferred to clean 2-l Erlenmeyer flasks. The moisture content of fresh wood chips was 48% (wt/wt) and autoclaved water was added to the flasks to increase this value to 55% (wt/wt). In experiments using acid-washed wood chips, fresh wood chips were weighed and transferred to clean 0.5-l Erlenmeyer flasks containing 300 ml of 3.2 mm sulfuric acid solution at ph 2.5. Acid treatment was performed at 808C for 1 h, followed by simple draining of the resulting liquor (Lundqvist et al. 2006). The weight loss of the wood chips after acid washing was determined as 1.0% (wt/wt). Acidwashed wood chips were transferred to 2-l Erlenmeyer flasks under aseptic conditions. In a group of control experiments, acid-washed wood chips were autoclaved at 1218C for 15 min. In all cases, wood chips were inoculated with blended mycelium corresponding to an inoculum/wood ratio of 5, 50 or 3000 mg dry mycelium kg -1 dry wood. In CSL-supplemented cultures, 0.5% (wt/wt on a dry wood basis) CSL (Corn Products, Brazil) was added to 2-l Erlenmeyer flasks. The flasks were manually shaken and then stored stationary at 278C for periods varying from 3 to 28 days. For each culture condition, four flasks were inoculated for each biodegradation period. Three flasks were used for enzyme extraction and determination and one flask for ph determinations. Some biodegradation experiments were repeated under selected culture conditions, as described in Table 1. In this case, biotreated wood chips were directly used in CTMP pulping experiments without any washing or air-drying. Extracellular enzymes produced during biodegradation were extracted with 50 mm sodium acetate buffer (ph 5.5) supplemented with Tween 60 (0.1 g l -1 ). The entire contents of each flask was extracted with 200 ml of extraction solution for 5 h at 10"18C (Souza-Cruz et al. 2004). The crude extracts were recovered after centrifugation at 10"18C and 3500=g for 15 min. Extracted wood chips were washed with water and airdried. MnP activity was measured by phenol red oxidation. The reactions were performed in 30-ml assay tubes containing 1.5 ml of 50 mm sodium succinate buffer at ph 4.5, 1.5 ml of 50 mm sodium lactate, 0.5 ml of 1.0 mm MnSO 4,0.5mlof1gl -1 phenol red, 0.25 ml of 10 g l -1 bovine serum albumin, 0.5 ml of enzymatic extract and 0.25 ml of 2.0 mm H 2 O 2. At 1-min intervals, 1 ml of this reaction mixture was added to 30 ml of 6.5 M NaOH solution and the absorbance was measured at 610 nm (absorptivity of 22 mm -1 cm -1 for oxidized phenol red; Kuwahara et al. 1984). Laccase activity was determined with 2,2-azinobis-(3-ethylbenzenthiazoline-6-sulfonic acid) (ABTS) as substrate. Reactions were carried out in 1-ml cuvettes containing 0.3 ml of 200 mm sodium phosphate/100 mm citric acid buffer at ph 5.0, 0.1 ml of water, 0.5 ml of enzyme extract and 0.1 ml of 1.0 mm ABTS. Reactions were monitored at 420 nm for up to 5 min at 10-s intervals (absorptivity of 36 mm -1 cm -1 for oxidized ABTS; Bourbonnais et al. 1998). Endoglucanases and xylanases were assayed with carboxymethyl cellulose (Wood and Bhat 1988) and birchwood xylan (Bailey et al. 1992), respectively. For ph determinations, the entire contents of one culture (50 g of biotreated wood chips) was soaked with 300 ml of distilled water (ph previously adjusted to 7.0) for 48 h. The ph was measured in the resulting aqueous extract. Determination of metal content of wood chips Fresh and acid-washed wood chips were milled to pass through a 0.5-mm screen. Milled samples (500 mg) were hydrolyzed with 6 ml of concentrated sulfuric acid in a Digesdahl Hach digester heated for 5 min at 4408C. Then 3 ml of 30% (w/w) hydrogen peroxide was added and the mixture was heated at 4408C for an additional 3 min. After cooling, the solution was diluted to 50 ml with deionized water. This solution was analyzed in an ICP-GBC Integra XM instrument to determine the manganese, iron, calcium and copper contents. Table 1 Biotreatment conditions and sample denomination in CTMP biopulping experiments. Sample Pre-treatment Inoc/wood Culture denomination ratio a supplement b CTRL Autoclaved No CTRL AW Acid-washed No Bio A-CSL Autoclaved 5 Yes Bio A-NS Autoclaved 500 No Bio AW-CSL Acid-washed, 5 Yes non-autoclaved Bio AW-NS Acid-washed, 500 No non-autoclaved a Oven-dry basis, mg mycelium kg -1 wood. b 0.5% corn steep liquor, wt/wt on an oven-dry basis.

3 Biopulping under non-aseptic conditions 3 Solubility in 1% NaOH After enzyme extraction, 5 g of air-dried wood chips was milled in a knife mill to pass through a 0.5-mm screen. The milled sample (300 mg) was treated with 15 ml of 1% (wt/vol) NaOH solution at 1008C for 1 h. The resulting solid was filtered though a porous glass filter (no. 3), then washed with 50 ml of 1% NaOH and subsequently with water until the ph of the filtrate was neutral. The washed residue was oven dried to constant weight at 1058C. The dry weight values before and after 1% NaOH extraction were used to calculate the solubility in 1% NaOH (Tappi standard T212 om-02). Incubation of primary mold-contaminated wood chips on benomyl-amended medium Wood chips selected from heavily contaminated cultures in which primary molds were easily distinguished were placed on Petri dishes containing benomyl-amended medium. This medium consisted of 2.4% (wt/vol) potato extract broth, 0.7% (wt/vol) yeast extract, 2% (wt/vol) agar and 4 mg l -1 benomyl wmethyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate; Aldrich, St. Louisx previously dissolved in 10 mm HCl. Petri dishes were incubated at 278C for 10 days. The growth of contaminant molds or C. subvermispora mycelium was visually inspected every day. Chemimechanical pulping of the wood chips Chemimechanical pulping of untreated and biotreated E. grandis wood chips was performed in two steps. Wood chips biotreated under selected culture conditions (Table 1) were first cooked in alkaline sulfite liquor (50 g of wood chips and 300 ml of cooking liquor corresponding to 10% Na 2 SO 3 and 5% NaOH, both wt/ wt on a dry wood basis). Cooking was performed in an autoclave heated to 1218C for 2 h. Cooked wood chips were blended in a 3.5-l aluminum blender for 1 h at 1.66% (w/v) consistency. Fibrous material was centrifuged, giving a final pulp consistency of approximately 30%. The second step consisted of fiber refining in a Jokro mill (Regmed, Brazil). Each pot of the Jokro mill was filled with 20 g of fibrous material (dry basis) and 180 ml of fresh cooking liquor. The fibrous material was refined for periods varying from 60 to 220 min to obtain pulps with fibrillation degrees in the range of SR. After refining, the pulps were washed with water and screened in a 0.15-mm slot screen. Pulp passing the 0.15-mm slot was collected on a 200-mesh screen and centrifuged to give a pulp consistency of approximately 30%. The SR degree of 2 g l -1 pulp suspensions was determined on a SRIP-Regmed Shopper-Riegler instrument. Handsheets with a basic weight of g m -2 were prepared with CTMP pulps obtained from untreated or biotreated wood chips. Handsheets were assayed for their strength properties in terms of tensile index (Tappi standard T494 om-01) and tear index (Tappi standard T414 om-04). C. subvermispora was observed from 14 days of biodegradation. An inoculum/wood ratio as low as 5 mg kg -1 provided efficient colonization of autoclaved wood chips, which is in agreement with previous work indicating that CSL is an efficient fungal growth promoter in biopulping (Akhtar et al. 1998). The visual observations indicating efficient wood colonization were further confirmed by detection of typical titers for C. subvermispora extracellular metabolism (Figure 1a). Overall, xylanases and MnP were found to predominate as extracellular enzymes, while laccases and endoglucanases were produced in minor amounts, corroborating previous studies (Sethuraman et al. 1998; Souza-Cruz et al. 2004; Vicentim and Ferraz 2007). ph values decreased as a function of cultivation time from ph 5.9 in the undecayed control to ph 3.3 in the wood chips degraded for 28 days (Figure 1a). Increasing inoculum/wood ratios had a significant effect on MnP production, which was three-fold higher in cultures inoculated with 3000 mg kg -1 compared to cultures inoculated with 5 mg kg -1. Laccases, xylanases and endoglucanases were less affected, since maximal activities were increased by factors of 1.2, 1.8 and 1.7, respectively, in the same cultures. The kinetic behavior of MnP production, however, was always similar to that illustrated in Figure 1a, regardless the inoculum/wood ratio evaluated. The solubility of wood samples in 1% NaOH solution was evaluated as an indication of the structural changes Results and discussion Wood colonization and extracellular metabolism of C. subvermispora Autoclaved wood chips supplemented with 0.5% CSL were quickly colonized by C. subvermispora at all inoculum/wood ratios evaluated (5, 50 and 3000 mg mycelium kg -1 wood). A discrete mycelial mass was visible from 14 days of culture, while abundant mycelium on the wood chip surfaces was observed after 28 days. The typical eucalyptus wood-chip brightening induced by Figure 1 Extracellular titers produced by C. subvermispora during the biodegradation of autoclaved E. grandis wood chips. (a) Wood chips supplemented with 0.5% corn steep liquor, inoculated with 5 mg mycelium kg -1 wood; (b) non-supplemented wood chips, inoculated with 3000 mg mycelium kg -1 wood. SD values were calculated from data obtained for three independent cultures.

4 4 F. Masarin and A. Ferraz Article in press - uncorrected proof induced by the fungus in these short biodegradation periods. Solubility in 1% NaOH is widely known as a rot index and involves partial solubilization of extractives and low-molecular-mass hemicelluloses and lignin (Tappi standard T212 om-02). The solubility in 1% NaOH of eucalyptus wood chips biotreated in CSL-supplemented cultures increased from 10.9"0.5% (wt/wt) in the undecayed control to 14"1% and 14.7"0.1% in samples biotreated for 28 days in cultures inoculated with 5 and 3000 mg mycelium kg -1 wood, respectively. While cultures containing autoclaved wood chips were completely free of contaminants, primary molds were found to grow rapidly when non-autoclaved wood chips were used, resulting in heavily contaminated cultures, independent of the C. subvermispora inoculum/wood ratio used (5, 50 and 3000 mg mycelium kg -1 wood). Contaminant fungi grew rapidly during the first 10 days of culture, with abundant sporulation observed thereafter. Such contamination inhibited C. subvermispora growth, as indicated by the absence of characteristic titers for C. subvermispora extracellular metabolism in these cultures. No MnP and laccase activities or decrease in ph were detected up to 28 days of biodegradation, while xylanases and endoglucanases were produced at very low levels (maximum of 64 and 43 IU kg -1 dry wood, respectively). Furthermore, wood chips did not brighten as a function of culture time and the solubility in 1% NaOH remained constant over the entire biodegradation period (10.9"0.5% and 11.2"0.5% in undecayed controls and 28-day biotreated samples, respectively). Spore production by the contaminant fungi after the first 10 days of culture suggests that they grew at the expense of small amounts of easily available carbon sources present in the wood tissues or CSL nutrients. As previously reported by Levy (1987), this behavior is typical for primary molds, which are unable to degrade polymeric wood cell-wall components. Based on these observations, another group of experiments was carried out on non-supplemented wood chips. In this case, only the highest C. subvermispora inoculum/wood ratio was used (3000 mg kg -1 ). Contaminant fungi grew on nonautoclaved fresh wood chips, even in the absence of CSL and, typical titers for extracellular C. subvermispora metabolism were detected only in control cultures containing autoclaved wood chips (Figure 1b). The solubility of biotreated samples in 1% NaOH increased from 10.9"0.5% in the undecayed control to 15"2% in the autoclaved sample biotreated for 28 days, while it remained constant when fresh (non-autoclaved) wood chips were used. Taken together, these results indicate that both CSL and easily available carbon sources present in the wood tissues are suitable substrates for the contaminant fungi to establish their short growth phase. Further efforts to evaluate the nature of competition between the primary molds and C. subvermispora included incubation of heavily contaminated C. subvermisporainoculated wood chips on nutritive Petri dishes amended with 4 mg l -1 benomyl. Benomyl has long been used to inhibit the growth of primary molds, while allowing the development of basidiomycetes (Score et al. 1998). In these Petri dishes, an abundant C. subvermispora mycelium mass grew from the periphery of the wood chips after 7 days of culture (data not shown). This result demonstrates that C. subvermispora mycelium or clamydospores (Saxena et al. 2001) remained viable on wood chips, even after intense contamination by primary molds. Based on such a finding, we may conclude that C. subvermispora was not killed by the contaminant fungi. Instead, the fungi involved seem to compete by consumption of the easily available nutrient sources, with C. subvermispora being clearly less successful than the contaminant fungi. Acid washing of wood chips as a simple decontamination procedure Considering that C. subvermispora was not able to colonize non-autoclaved fresh E. grandis wood chips during the culture periods required for biopulping, hot-acid washing of the wood chips was evaluated as a possible decontamination procedure. Acid washing of wood chips has become an important option for pulp mills to recycle water in their systems, since it removes part of the nonprocess metals from the wood chips entering the pulping processes (Lundqvist et al. 2006). As shown in Table 2, this procedure removed significant amounts of Ca (19%), Fe (24%) and Mn (36%) from the wood chips. The same procedure presumably removed part of the easily available carbon sources from the wood tissues, since free sugars and soluble polysaccharides are released and washed out (Lundqvist et al. 2006). Acid-washed wood chips were biotreated in non-supplemented cultures inoculated with 3000 mg mycelium kg -1 wood, as well as in CSL-supplemented cultures inoculated with 5 mg kg -1. The acid washing affected C. subvermispora metabolism, since control cultures on standard autoclaved wood chips (Figure 1b) differed from cultures on acid-washed/autoclaved wood chips (Figure 2a). Production of MnP and xylanase decreased by factors of three and two, respectively, when acid-washed wood chips were used. This change seems to be related to the lower initial wood ph (4.9), as well as to the removal of metals (Table 2) and free sugars during acid washing, and the resulting slower fungal growth (Manubens et al. 2003; Vicentim and Ferraz 2007). On the other hand, acid washing resulted in some decontamination of the wood chips, and both non-supplemented and CSL-supplemented cultures on these eucalyptus chips were less contaminated than previous cultures on non-autoclaved fresh wood. Typical titers for extracellular C. subvermispora metabolism were observed in these cultures (Figure 2b). In non-supplemented cultures, xylanases and MnP were produced at similar levels compared to the acid-washed/autoclaved control (Figure 2a,b). However, CSL-supplemented cultures presented more contami- Table 2 Selected metal content of fresh and acid-washed wood chips from E. grandis. Wood chips Metal content (mg kg -1 wood) Ca Fe Mn Cu Fresh 589"8 54.2" " "0.08 Acid-washed 477" " " "0.06 Values were calculated on an oven-dry basis. SD values were calculated from three independent determinations per sample.

5 Biopulping under non-aseptic conditions 5 nant fungi and proportionally less C. subvermispora growth. In the latter cultures, the highest MnP and xylanase activities detected after 28 days of culture (80 and 430 IU kg -1, respectively) were lower than values observed in the non-supplemented culture (Figure 2b). Wood chip brightening was observed only after 28 days of culture in acid-washed wood chips and solubility in 1% NaOH was not increased, even in control cultures containing the acid-washed/autoclaved wood chips. This group of experiments demonstrates that acid washing resulted in some decontamination of the wood chips, but fungal metabolism was significantly affected. Furthermore, a reduced number of spores of contaminant fungi resisted the acid treatment and CSL supplementation of the cultures improved the growth of these contaminants. CTMP pulping of biotreated wood samples Figure 2 Extracellular titers produced by C. subvermispora during the biodegradation of acid-washed E. grandis wood chips on non-supplemented cultures. (a) Acid-washed/autoclaved wood chips; (b) acid-washed/non-autoclaved wood chips. Inoculum/wood ratio of 3000 mg mycelium kg -1 wood in both cultures. SD values were calculated from data obtained for three independent cultures. Some culture conditions previously evaluated were selected to prepare biotreated wood samples for pulping experiments (Table 1). This pulping study was driven by the hypothesis that a certain level of contamination may be acceptable for biopulping purposes. In these experiments, acid-washed wood chips were quickly colonized by C. subvermispora and low contamination levels were observed. Even the CSL-supplemented cultures were significantly less contaminated than those described for prior experiments. CTMP pulping of these eucalyptus wood chips was performed using a two-stage pulping procedure as described elsewhere (Guerra et al. 2005). The data in Figure 3 show that SR values for biopulps prepared from autoclaved wood chips biotreated for 28 days in CSLsupplemented and non-supplemented cultures were Figure 3 Refining level and strength properties of CTMP pulps prepared from E. grandis wood chips. SD values for tensile and tear indexes were calculated from data obtained for three independently prepared handsheets per sample.

6 6 F. Masarin and A. Ferraz Article in press - uncorrected proof Figure 4 Refining level and strength properties of CTMP pulps prepared from acid-washed E. grandis wood chips. higher than those observed in control pulps over the refining periods examined. These results are in agreement with previous studies showing facilitated fibrillation of biopulps compared to their controls (Akhtar et al. 1998; Guerra et al. 2005). This faster fibrillation of biopulps might increase the process throughput or save energy during the refining step, because a reduced refining time was necessary to achieve a desired fibrillation level (Guerra et al. 2005). The use of a low inoculum/ wood ratio (5 mg kg -1 ) in CSL-supplemented cultures was found to compensate the efficient fungal growth observed in non-supplemented cultures inoculated with a higher mycelium ratio (500 mg kg -1 ), as shown by the similar improvements in fibrillation observed in both biotreated samples (Figure 3a). As expected, improved fibrillation of the biopulps resulted in paper sheets with a higher tensile index compared to the controls (Figure 3b). However, at similar SR values, analogous tensile index values were observed for control and biopulps. Similar behavior for control and biopulps was observed in terms of tear index (Figure 3c). In fact, graphs of tensile versus tear index dispersion for these pulps indicated that their mechanical properties did not differ significantly (Figure 3d). Previous results reported by Guerra et al. (2005) already demonstrated that the strength improvements in CTMP are less significant than in RMP and TMP processes (Akhtar et al. 1998; Scott et al. 2002). Accordingly, the major benefit obtained from wood biotreatment in CTMP pulping of E. grandis was the improved fibrillation. Biopulps prepared from acid-washed (non-autoclaved) wood chips also showed improved fibrillation (Figure 4a). Similar refining curves for the controls (CTRL in Figure 3a and CTRL AW in Figure 4a) suggests that acid washing of the wood chips was of minor relevance for pulp fibrillation. Graphs of tensile versus tear index dispersion for the pulps prepared from acid-washed wood chips suggest that the biopulps were slightly stronger than their control pulps (CTRL AW) and even the autoclaved control pulps (Figures 4b and 3d, respectively). Conclusion Fresh wood chips inoculated with C. subvermispora in CSL-supplemented or non-supplemented cultures suffered from contamination by primary molds that limited C. subvermispora growth. This limited growth was apparently due to the inability of C. subvermispora to compete with primary molds for the easily available nutrient sources present in the wood tissues or from CSL. Acid washing of the wood chips was efficient for reducing, but not abolishing, the presence of contaminant fungi in the biopulping experiments. Secretion of MnP and xylanases by C. subvermispora was decreased when acid-washed wood chips were used as substrate. However, CTMP biopulping benefits obtained from biotreated samples under these conditions were similar to those obtained from samples subjected to the standard biodegradation process when autoclaved wood chips were used. Acknowledgements This work was supported by FAPESP, CNPq, CAPES and SCTDE/SP. We wish to thank J.M. Silva and J.C. Tavares for technical assistance. Dr. J. Freer from Renewable Resource Laboratory, University of Concepción, Chile is acknowledged for help with the metal analysis of untreated E. grandis wood samples. Permission to use the laboratory facilities of Melhoramentos Papéis Ltda is acknowledged. References Aguiar, A., Souza-Cruz, P.B., Ferraz, A. (2006) Oxalic acid, Fe 3q - reduction activity and oxidative enzymes detected in culture extracts recovered from Pinus taeda wood chips biotreated by Ceriporiopsis subvermispora. Enzyme Microb. 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