A Chrysoporthe cubensis enzyme cocktail produced from a low-cost carbon source with high biomass hydrolysis efficiency

Low cost and high efficiency cellulolytic cocktails can consolidate lignocellulosic ethanol technologies. Sugarcane bagasse (SCB) is a low cost agro-industrial residue, and its use as a carbon source can reduce the costs of fungi cultivation for enzyme production. Chrysoporthe cubensis grown under solid state fermentation (SSF) with wheat bran has potential to produce efficient enzymatic extracts for SCB saccharification. This fungus was grown under submersed fermentation (SmF) and SSF with in natura SCB, pretreated with acid or alkali and with others carbon sources. In natura SCB induced the highest carboxymethylcellulase (CMCase), xylanase, β-xylosidase, α-galactosidase and mannanase activities by C. cubensis under SSF. In natura and washed SCB, inducers of enzyme production under SSF, did not induce high cellulases and hemicellulases production by C. cubensis in SmF. The C. cubensis enzymatic extract produced under SSF with in natura SCB as a carbon source was more efficient for lignocelulolic biomass hydrolysis than extracts produced under SSF with wheat bran and commercial cellulolytic extract. Chrysoporthe cubensis showed high potential for cellulases and hemicellulases production, especially when grown under SSF with in natura SCB as carbon source.

Enzymatic production by Chrysoporthe cubensis under submerged fermentation (SmF). Cellulase and hemicellulase enzymes production by C. cubensis under submerged fermentation (SmF) allowed to evaluate the effect of cell wall components (e.g., xylan, pectin) and simpler sugars (e.g. glucose, cellobiose) on regulating production of cellulolytic enzymes by this fungus.
Wheat bran, acid pretreated SCB and carboxymethylcellulose (CMC) induced the highest CMCase productions by C. cubensis, with 3.4, 2.7 and 2.6 U mL −1 , respectively, whilst CMC, wheat bran and pectin induced the highest β-glucosidase productions by this fungus, with 0.5, 0.4 and 0.3 U mL −1 , respectively (Table 2). Alkali pretreated SCB, wheat bran, acid pretreated SCB, CMC and xylan induced production of the highest xylanase activities under SmF, 37.2, 14.2, 11.0, 10.2 and 10.1 U mL −1 , respectively. The fact of CMC induced CMCase, β-glucosidase and xylanase enzyme activities of 2.6, 0.47 and 10.2 U mL −1 by C. cubensis, respectively, differed from results for Aspergillus genus fungi that produce xylan-degrading enzymes in the presence of substrates containing xylose and xylan, but not with cellulose 15 . CMCase and xylanase production by C. cubensis cultured in the medium with CMC indicates that, as occurs in T. reesei, this carbon source is involved in the activation of cellulase and hemicellulase production. Trichoderma reesei has the transcription factor ACEII that actives cellulase and hemicellulase production pathways in the presence of celullose 16 . This suggests that C. cubensis may have transcription factors with similar role to the ACEII of T. reesei.
Xylan induced higher xylanase production (10.1 U mL −1 ) by C. cubensis compared to CMCase production (0.3 U mL −1 ), agreeing with results for Fusarium graminearum. This fungus regulates xylanase production with transcriptional activator Xyr1 involved in activation of gene transcription to xylanases, but not cellulase 17 . Xyr1 orthologous genes are highly conserved in ascomycetes, suggesting that similar signaling pathways may occur in C. cubensis, an ascomycete 18,19 .
Alkali pretreated SCB induced production of the highest xylanase activity (37.2 U mL −1 ) by C. cubensis under SmF among the carbon sources evaluated. This can be explained by the fact that the alkaline pretreatment mainly solubilizes lignin from biomass, promoting a relative increase in the concentration of cellulose and hemicellulose 20 , inducers of xylanase production by this fungus (Table 2).
Glucose, xylose, lactose, galactose, arabinose and mannose did not induce cellulase and hemicellulase production by C. cubensis. These results are in agreement with reports that in the presence of preferred carbon sources the cellulase and hemicellulase production in fungi was inhibited by carbon catabolite repression 15 . 2.4 and 7.1 U g −1 , respectively. These activities were the largest ones produced by this fungus on all tested carbon sources. The β-glucosidase and α-arabinofuranosidase activities for cultivation on this substrate were 1.5 and 8.2 U/g respectively (Table 3). Chrysoporthe cubensis cultured on washed SCB produced lower CMCase, xylanase, β-xylosidase, α-galactosidase, α-arabinofuranosidase and mannanase activities (17.9, 500, 0.6, 0.4, 1.8 and 3.2 U g −1 , respectively) than those obtained by the cultivation on in natura SCB (Table 2). This differs from results for Trichoderma reesei, which showed higher enzyme production in cultivation with washed SCB compared to non-washed SCB 21 . Trichoderma reesei grown on in natura SCB with high glucose content showed lower enzyme production compared to that cultivated on washed, glucose-free SCB 21 . The greater induction of cellulase and hemicellulase production by C. cubensis when cultivated on in natura SCB compared to washed SCB can be explained by the absence of detectable glucose levels in the former SCB (Table 1). Glucose is a repressor molecule of cellulase and hemicellulase production by fungi, and its absence in the in natura SCB used for C. cubensis cultivation contributed to higher production in the washed material 22,23 . Moreover, the presence of xylose in the in natura SCB presented an activating effect on enzyme production compared to cultivation with washed SCB. This confirms the role of this molecule as an activator of cellulase and hemicellulase production in fungi 24 . In natura SCB contained sucrose (30.1 mg g −1 of dry SCB mass), a repressor of cellulase production in filamentous fungi, even in the presence of lignocellulosic material 25 . However, the sucrose concentrations found in the in natura SCB did not suppress cellulolytic production by C. cubensis. The xylanase, α-arabinofuranosidase, β-glucosidase and CMCase activities produced by C. cubensis under SSF with in natura SCB were higher than those obtained by  Table 2. Cellulase and hemicelulase activities produced by Chrysoporthe cubensis under submerged fermentation with different carbon sources (Carb.). SCB = sugarcane bagasse; IN-SCB = in natura sugarcane bagasse; W-SCB = washed SCB; Alk-SCB = alkali pretreated SCB; Ac-SCB = acid pretreated SCB; WBran = wheat bran, LBGum = locust bean gum. CMCase = carboxymethylcellulase; β-Glu = β-glicosidase; Xyl = xylanase; β-Xy l = β-xylosidase; α-Ara = α-arabinofuranosidase; α-Gal = α-galactosidase; MN = Mannanase; ND = not detected. Averages followed by the same letter do not differ by the Tukey test at 5% of significance. *Standard deviations lower than 0.001.
fungi of the Trichoderma genus used to produce commercial cellulolytic preparations, enhancing the potential for hydrolytic enzymes production by C. cubensis on in natura SCB 26 . In natura SCB was a better cellulase and hemicellulase production inducer by C. cubensis than wheat bran, the standard carbon source for enzyme production by this fungus 3,5,13 . This is an interesting result considering that in natura SCB is an abundant and inexpensive residue, does not require pretreatment or washing, and can be used in on site enzyme production in sugar and alcohol industries 9,27 . Acid pretreated SCB induced xylanase production (220 U g −1 ), but this material was a weak inducer of CMCase activity (2.5 U g −1 ), and did not induce β-glucosidase production by this C. cubensis, although this carbon source presents a high cellulose content. This finding is in disagreement with the report that cellulose is a strong cellulase production inducer in filamentous fungi 28 . Acid pretreated SCB had higher cellulose content than non-pretreated SCB, and therefore it was expected that this biomass would induce cellulase production by C. cubensis. This result further demonstrates the potential of this enzyme cocktail for pulp biobleaching processes. In these processes cellulase-poor xylanase cocktails act in the depolymerization of hemicelluloses precipitated on the pulp fiber surface and remove carbohydrate-lignin complexes generated during the Kraft process, without harming the cellulose fiber [29][30][31] .
Locust bean gum (galactomannan) activated α-arabinofuranosidase production by C. cubensis under SSF, inducing higher values of this activity (16.5 U g −1 ) than the other carbon sources. The induction of α-arabinofuranosidase production by galactomannans in filamentous fungi under SSF is not well known. High enzyme production by C. cubensis under SSF with locust bean gum is in agreement with results obtained with T. reesei which reported CMCase, xylanase and mannanase production activated with this carbon source 32 . α-Arabinofuranosidases assist hemicellulose hydrolysis in the biomass saccharification process, since they catalyze the hydrolysis of α-arabinose bonds linking residues in the backbone of hemicellulose. These enzymes can be used to improve wine flavor, the main feature of this product 33, 34 . Specific activities of the extracts produced by C. cubensis under SSF and SmF with SCB. The specific activities of cellulases and hemicellulases from C. cubensis extracts cultured with SCB under SSF and SmF demonstrated that this fungus is a better cellulolytic enzyme producer under SSF than under SmF with in nature and washed SCB as carbon sources ( Table 4).
The production of enzyme cocktails with specific CMCase, β-glucosidase and xylanase activities generated by C. cubensis grown in SSF conditions with in natura SCB were 14.5, 4.3 and 90 times higher than those of the cocktails produced under SmF with the same material, respectively. The better cellulolytic production by C. cubensis on in natura SCB under SSF compared to SmF indicated advantages of its cultivation, because this process requires less water, no agitation and less energy demand 35 .
Chrysoporthe cubensis extract produced with alkali and acid pretreated SCB under SSF had higher specific xylanase activities (221 and 489 U mg −1 , respectively) than those of the extracts produced under SmF with the same carbon sources (185 and 68.9 U/mg, respectively) ( Table 4). Moreover, the extracts produced by this fungus with alkali and acid pretreated SCB under SmF showed higher specific activities of CMCase (11.5 and 16.9 U mg −1 , respectively) than those produced under SSF (2.2 and 5.7 U mg −1 respectively). These results indicate that the best culture condition (SSF or SmF) for both cellulase and hemicellulase production by C. cubensis varies with the carbon source as reported for Lentinusedodes and Pleurotus species 36 .

Chrysoporthe cubensis enzymatic profile and biomass enzymatic saccharification. The
Chrysoporthe cubensis extract produced under SSF with in natura SCB was applied to saccharification processes and compared to that produced by this fungus under SSF with wheat bran, the standard substrate used for enzyme production by this fungus 3, 13 , as well as a commercial extract. To compare the extracts applied in saccharification assays, the enzymatic activities were normalized relative to FPase activity, which is the total cellulose activity of  Table 4. Specific activities of cellulases and hemicellulases produced by Chrysoporthe cubensis under solid state fermentation (SSF) and submerged fermentation (SmF) with sugarcane bagasse (SCB). SCB = sugarcane bagasse; In-SCB = in natura SCB; W-SCB = washed SCB; Alk-SCB = alkali pretreated SCB, Ac-SCB = acid pretreated SCB. CMCase = carboxymethylcellulase; β-Glu = β-glicosidase; Xyl = xylanase; β-Xyl = β-xylosidase; α-Ara = α-arabinofuranosidase; α-Gal = α-galactosidase; MN = Mannanase; ND = not detected. The averages followed by the same letter do not differ significantly by the Tukey test at 5% of significance. *Standard deviations lower than 0.01.
the enzymatic complexes ( Table 5). The β-glycosidase/FPase ratios of the C. cubensis extracts were similar to those of the commercial extract Multifect CL 3 , but the Multifect CL extract had a CMCase/FPase ratio 3.1 and 4.3 times higher than that of the extracts produced by C. cubensis under SSF with in nature SCB and wheat bran, respectively ( Table 5). The Chrysoporthe cubensis extracts showed greater complexity of hemicelulolitic activities than the commercial product Multifect CL. This fungus secreted β-xilosidade, α-arabinofuranosidase and α-galactosidase, enzymes absent in the commercial extract. Furthermore, C. cubensis extracts showed xylanase/FPase ratios 9.8 and 7.5 times higher when grown with in natura SCB and wheat bran under SSF than the commercial enzyme extract, respectively. This result is interesting because the hemicellulases, xylanases, α-arabinofuranosidase and β-xylosidase are important in the saccharification of lignocellulosic materials such as SCB, which are rich in hemicelluloses 32,37 . The absence or lack of these enzymes limits the cellulases action in cellulose fiber hydrolysis, because the hemicellulases acts synergistically with cellulases in the hydrolysis of lignocellulosic materials 38 .
The commercial extract Multifect more efficiently released reducing sugars (18.6 μmol ml −1 ) than the extracts produced by C. cubensis under SSF with wheat bran and in natura SCB (7.95 and 7.98 μmol/mL, respectively) in saccharification assays of the microcrystalline cellulose Avicel (Fig. 1). This can be justified by the higher CMCase/FPase ratio presented by the commercial cocktail compared to C. cubensis extracts, since endoglucanases start the saccharification process hydrolyzing the inner parts of the cellulose fiber, releasing smaller chains which are hydrolyzed by cellobiohydrolases 39 .
The cocktail produced by C. cubensis under SSF with in natura SCB released more glucose (2.8 g L −1 ) in saccharification of alkali pretreated SCB than the commercial extract and that the extract produced by this fungus under SSF with wheat bran, 2.3 and 1.98 g L −1 , respectively (Fig. 2a). The extract produced by C. cubensis under SSF with in natura SCB released more xylose from the alkali pretreated SCB (3.91 g L −1 xylose) than the commercial extract and the extract from this fungus cultivated under SSF with wheat bran (0.72 and 2.97 g L −1 xylose, respectively) after 72 hours of saccharification (Fig. 2b).
Greater effectiveness of the C. cubensis extract produced under SSF with in natura SCB than that of the commercial extract regarding glucan and xylan hydrolysis was mainly due to greater richness of hemicellulase  Table 5. Comparative analysis of cellulase and hemicellulase activities of the extracts produced by Chrysoporthe cubensis under SSF with in natura sugarcane bagasse (SCB) or wheat bran in comparison to Multifect CL 3 . The values displayed were obtained dividing each total enzymatic activity by total FPase activity found in each cellulolytic extract. activities in the C. cubensis extract compared to the commercial cocktail. The hydrolysis of hemicellulose by xylanase, α-arabinofuranosidase, β-xylosidase, mannanase and α-galactosidase enzymes from C. cubensis facilitates the access of cellulase enzymes to cellulose. Hemicellulose acts as a barrier, preventing access of cellulases to cellulose, and the hydrolysis of this polymer is essential for efficient saccharification of plant biomass 40 . The superior ability for saccharification of alkali pretreated SCB by the C. cubensis extract produced under SSF with in natura SCB in relation to that produced on wheat bran agrees with the results reported for Aspergillus niger and T. reesei, which produced more efficient enzyme extracts for SCB saccharification when grown with this substrate than with wheat bran 19 . This is due to the fact that fungi can produce enzyme complexes adjusted to the presents fractions in the biomass used to cultivate this fungus 25 .
Chrysoporthe cubensis cultured under SSF with in natura SCB as the sole carbon source produced a more effective enzymatic extract for saccharification of alkali pretreated SCB than that produced by the same fungus cultivated on a standard carbon source (wheat bran) as well as the commercial cellulolytic extract. This extract showed better saccharification performance of alkali pretreated SCB due to a more complete hemicellulolytic arsenal, demonstrating the importance of these enzymes in saccharification of lignocellulosic materials.

Conclusions
Enzyme production by Chrysoporthe cubensis cultured with sugarcane bagasse (SCB) was studied for the first time, and this carbon source can be used to efficiently produce cellulases and hemicellulases by this fungus. The enzyme production by C. cubensis with in natura SCB is important because this is a low-cost material, does not require washing, can be used for enzyme production by the sugar and alcohol industry and it induced production of an enzyme cocktail production which was effective for SCB saccharification.

Methods
Biomass composition analysis. The chemical composition of the in natura, washed, acid pretreated and alkali pretreated SCB were determined using a modified Klason lignin method derived from the TAPPI Standard Method T222 om-98 41 . Extractive-free biomass (0.3 g) was incubated at 30 °C with 3 mL of 72% H2SO4 for 1 h with occasional mixing. The slurry was then transferred into a penicillin bottle containing 84 mL of deionized water and the flask sealed with a rubber stopper and aluminum seal. The bottle was placed in an autoclave calibrated at 118 °C for 1 h, then the slurry was filtered through a medium coarseness sintered glass filter for gravimetric determination of acid-insoluble lignin. Concentrations of biomass sugars (arabinose, galactose, glucose, xylose, and mannose) in the filtrate were quantified using high-performance liquid chromatography (HPLC), while acid-soluble lignin was determined by absorption measurements at 205 nm 42 . The HPLC system Dionex DX-300 (Dionex Co. -Sunnyvale, CA, USA) was equipped with a Carbopac PA1 column and a pulsed amperometric detector with a gold electrode. Prior to injection, samples were filtered through 0.45-mm HV filters and a volume of 20 μL loaded into the chromatograph system. The column was pre-equilibrated with a NaOH solution, 300 mM, and elution was carried out at a flow rate of 1.0 mL min −1 at room temperature.

Biomass washing and pretreatment. Sugarcane bagasse (SCB) was provided by the Jatiboca Sugar and
Alcohol Plant in Urucânia, Minas Gerais state, Brazil. This material was dried, ground (particle size less than 1 mm) and stored at −80 °C for further use. Four types of SCB were obtained from the sample described: washed SCB, acid pretreated SCB, alkali pretreated SCB and in natura SCB, which was not subjected to washing or pretreatments. The washed SCB was obtained by washing of the in natura SCB with distilled water until the contents of glucose, xylose and sucrose were not detectable by HPLC analysis. This material was dried at 70 °C until reaching constant weight. NaOH 1% (w/v) or H 2 SO 4 1% (w/v) were used to pretreat 25 g of in natura SCB with 10% (w/v) solids loading, generating the alkali pretreated SCB and the acid pretreated SCB, respectively. The pretreatments were performed in an autoclave at 120 °C for 60 min. These pretreated materials were separated into solid and liquid fractions using a Buchner funnel fitted with filter paper. The solid fraction was washed thoroughly with distilled water, sealed in a hermetic vessel to retain moisture and stored at −20 °C.
Microorganism. The fungus C. cubensis LPF-1 used in this study was obtained from the mycological collection of the Forest Pathology Laboratory of the Universidade Federal de Viçosa in Viçosa, Minas Gerais State, Brazil. The fungus was maintained on PDA (potato dextrose agar) plates at 28 °C and subcultured periodically.

Submerged fermentation (SmF).
Chrysoporthe cubensis was grown in liquid medium using 1% (w/v) glucose, mannose, xylose, arabinose, galactose, cellobiose, lactose, xylanbirch wood, pectin, locust bean gum, carboxymethylcellulose, wheat bran, acid pretreated SCB, alkali pretreated SCB, washed SCB and in natura SCB as carbon sources. Cultivation was conducted in 125 mL Erlenmeyer flasks with 50 mL of culture medium composed of (g L The flasks were autoclaved at 120 °C for 20 minutes, inoculated with 0.5 mL of a spore suspension (2.2 × 10 6 spores mL −1 ) and placed in a shaker for seven days at 180 rpm and 28 °C. The samples were centrifuged at 10,000 × g for 20 minutes and the supernatant used as enzyme extracts. Solid state fermentation (SSF). Chrysoporthe cubensis was grown under SSF to evaluate the effect of different carbon sources on enzyme production in this cultivation condition. Chrysoporthe cubensis was cultured under solid state fermentation (SSF) using washed and in natura sugarcane bagasse (SCB), acid and alkali pretreated SCB, wheat bran and locust bean gum as the main carbon source. The fermentations were carried out in 125 mL Erlenmeyer flasks containing 5 g (dry weight) of the substrate moistened with culture media presenting the following composition, in g L −1 : NH 4 NO 3 , 1.0; KH 2 PO 4 , 1.5; MgSO 4 , 0.5; CuSO 4 , 0.25 and yeast extract, 2. Furthermore, MnCl 2 (0.1 mg L −1 ), H 3 BO 3 (0.075 mg L −1 ), Na 2 MoO 4 (0.02 mg L −1 ), FeCl 3 (1.0 mg L −1 ) and ZnSO 4 (3.5 mg L −1 ) also were added to the medium as trace elements.

Inoculum preparation for solid state fermentation (SSF).
The assays consisted of cultivation with washed SCB, in natura SCB (non-washed SCB), acid pretreated SCB, alkali pretreated SCB, wheat bran and locust bean gum with final moisture contents of 80, 80, 90, 90, 75 and 60%, respectively. The flasks were autoclaved at 120 °C for 20 min and then inoculated with 3 mL (containing 1.5 × 10 7 spores mL −1 ) of the inoculum obtained as aforementioned. The flasks were maintained at 30 °C in a temperature controlled chamber and the enzymatic extraction performed after seven days of fermentation. Enzymes secreted during SSF were extracted with sodium acetate buffer, 50 mM, pH 5, at a ratio of 10:1 (buffer/dry substrate), under agitation of 150 rpm for 60 min at room temperature. Solids were separated by filtration through a nylon cloth followed by centrifugation at 15000 g for 10 minutes, and the clarified supernatants were frozen and stored for subsequent enzymatic analysis. Experiments were carried out with three replicates for each medium composition and each incubation time.
Enzymatic assays. All enzymatic assays were carried out in sodium acetate buffer, 100 mM, pH 5, at 50 °C in triplicate and the mean values calculated. Relative standard deviations of measurements were below 5%. FPase and endoglucanase activities were determined using Whatman No. 1 filter paper and carboxymethilcellulose as substrates respectively 43 . The total reducing sugar liberated during the enzymatic assays were quantified by the dinitrosalicylic acid (DNS) method 44 using glucose as a standard.
Xylanase and mannanase activities were determined in beechwood xylan (final concentration of 1% w/v) and locust bean gum (0.4% w/v), respectively. The enzymatic reactions were initiated with addition of 100 mL of enzyme extract diluted to 400 μL substrate solution with the polysaccharide prepared in buffer. The reaction mixtures were incubated for 30 min and the amount of reducing sugars released determined by the DNS method using xylose and galacturonic acid as standards. Activities β-glucosidase, β-xylosidase, α-galactosidase and arabinofuranosidase were measured using ρPNβ-Glc, ρNPβ-Xyl, ρNPα-Gal and ρNPα-Ara as substrates, respectively.
The reaction mixtures contained 30 μL enzyme solution was diluted 50 μL synthetic substrate solution (1 uM final concentration) and 20 buffer μL. The reaction mixture was incubated for 15 min and quenched with 100 μL of a sodium carbonate solution (0.5 M). Absorbance was measured at 410 nm and the amount of ρ-nitrophenol released assessed by a standard curve. One enzyme activity unit (U) was defined as the amount of enzyme which released a μmol of the product (equivalent glucose, xylose and ρ-nitrophenol) per minute under assay conditions used for all activities.
Protein determination. Protein concentration in the enzymatic extracts was determined by the Coomassie Blue binding method using bovine serum albumin as the standard 45 .
Biomass saccharification. The crude enzymatic extracts produced by C. cubensis and com-mercial cellulase (Multifect ® CL) were applied in a biomass saccharification experiment. The C. cubensis enzymatic extract were concentrated 5-fold before the experiment using an Amicon Ultra-filtration system (Millipore Co. -Billerica, MA, USA) and an YM-10 (Cut-off Mr 10,000 Da) membrane filter. Enzymatic saccharification of alkali-treated sugarcane bagasse was performed in 2 mL sample tubes at an initial solid concentration of 2% dry matter (w/v) in 1.5 mL of 50 mM sodium acetate buffer at pH 4.5. Enzyme loading was specified as 10 FPase units per gram of biomass with the addition of sodium azide (10 mM) and tetracycline (40 μg mL −1 ) to the reaction mixture to inhibit microbial contamination. The reaction was carried out in an orbital shaker at 250 rpm and 50 °C for different time intervals up to 72 h. These samples were immediately heated to 100 °C to denature the enzymes, cooled and then centrifuged for 5 min at 15,000 g. Products of the saccharification assays were analyzed by high performance liquid chromatography (HPLC) with a Shimadzu series 10 A chromatograph. The HPLC was equipped with an Aminex HPX-87P column (300 × 7.8 mm) and refractive index detectors. The column was eluted with water at a flow rate of 0.6 mL min −1 and 80 °C.