Effect of wood flour as carbon source on cellulases and xylanases production by white-rot-fungi native from Misiones

There is a need to explore lignocellulosic materials to select an adequate substrate for lignocellulolytic enzyme production. Utilization of some residues provides an opportunity to produce high yields of lignocellulolytic enzymes in a simple medium. The aim of the present work was to study the effect of wood flour as a carbon source on the cellulolytic and xylanolytic secretion of white-rot fungi native from Misiones. Fungi were incubated with 5 g Pinus sp. wood flour/L and 5 g Eucalyptus sp. wood flour/L as carbon sources in a reciprocal shaker at 80 rpm and 29oC for 15 days. Total cellulase, endo-1,4-β-glucanase, β-glucosidase, cellobiohydrolase and endo-1,4-β-xylanase activities were determined in culture supernatants. Tested fungi showed high endo-1,4-β-xylanase activity between 6 and 12 days. Total cellulase showed the highest activity between 12-15 culture days. The test did not show differences among Pycnoporus sanguineus LBM 014, P. sanguineus BAFC 2126, Irpex lacteus BAFC 1171, Irpex sp. LBM 032, Irpex sp. LBM 034 and Lenzites elegans BAFC 2127, showing the highest activity for this group regard the others strains. I. lacteus BAFC 1171 was the strain with major endo-1,4-β-glucanase activity at day 9 (847 U/L). In the case of β-glucosidase and cellobiohydrolase, P. sanguineus BAFC 2126 was the strain with the highest activity between 12-15 culture days (18 U/L), and between 9-15 culture days (39 U/L), respectively. Wood flour proved to be a suitable carbon source to produce hydrolytic enzymes. I. lacteus BAFC 1171 and P. sanguineus BAFC 2126 have potential for cellulase production whereas P. sanguineus LBM 008 is a good endo-1,4-β-xylanase producer. Indexing terms/


INTRODUCTION
The availability of fossil fuel resources and the increasing energy demand are the main driving forces in the search for alternative energy sources. The large-scale replacement of petroleum fuels by biofuels, such as ethanol from lignocellulosic materials appears to be a powerful approach to meet the growing energy demands [1]. The lignocellulosic material from softwood source contains about 44% cellulose, 21% hemicellulose and 28% lignin [2], while hardwood has 40% cellulose, 17% hemicellulose and 21% lignin [3]. To breakdown polymeric sugars in an environmental friendly process, it is necessary to decrease the cost of cellulases and hemicellulases production, to increase volumetric productivity, to use cheaper substrates and to produce enzymes with high stability [4].
There are many microorganisms able to hydrolyze cellulose and hemicellulose. The description and analysis of new enzymes with distinctive biochemical properties from the rich and vast biodiversity of our region can reveal peculiar and worthy capacities. White-rot fungi have the ability to degrade most of wood components due to their capacity to synthesize hydrolytic extracellular enzymes. Potential applications of lignocellulolytic enzymes in industrial and environmental biotechnology require huge amounts of these enzymes at the lowest cost possible [5]. The enzyme cost is one of the factors determining the economics of a biocatalytic process and it can be reduced with optimum conditions for their production [6][7]. Hence, finding new microbial strains able to produce cellulases and xylanases and increasing the hydrolytic activity of fungal culture extracts is a way to lower the cost of enzymes used in hydrolysis of polymeric sugars to fermentable sugars.
Submerged liquid cultures (SLC) are widely used for enzyme production [8][9][10]. Synthetic media, such as carboxymethyl cellulose, xylan, cellulose powder and glucose, are usually used for cellulolytic and xylanolytic enzyme production [11][12][13]. However the cost of raw materials constitutes a large part of the total production cost [14][15]. Hence, there is a need to explore lignocellulosic materials to select an adequate substrate for enzyme synthesis to fully express and correctly evaluate the lignocellulolytic potential of fungi. Utilization of some residues provides an opportunity to produce high yields of lignocellulolytic enzymes in a simple medium [5].
The aim of the present work was to study the effect of wood flour as a carbon source on the cellulolytic and xylanolytic secretion of white-rot fungi native from Misiones.

Culture conditions
Fungi were first cultured on malt extract agar for 5 days at 29ºC. To prepare the pre-inoculum, two 78 mm 2 agar-plugs from each fungus were cut and transferred to 100 mL Erlenmeyer flasks containing 20 mL of modified Mandels media [12] and 5 g glucose/L, incubated at 29ºC in static conditions for 13 days. The pre-inoculum was washed with sterile distilled water and transferred to 250 mL Erlenmeyer flasks containing 60 mL of modified Mandels medium at pH 5 with 5 g PWF/L and 5 g EWF/L as carbon sources. The flasks were placed in a reciprocal shaker at 80 rpm and 29ºC for 15 days. At regular intervals of 72 h, samples of culture supernatants were collected and stored at -20°C. Each experiment was performed in duplicate.

Statistical analysis
Two-way ANOVA with multiple range test was performed using the Statgraphic Centurion program (StatPoint, Inc.).

Compositional analysis
The chemical composition of both PWF and EWF was determined and the results are tabulated in Table 1.

Cellulolytic and xylanolytic activities
The FPA activities are shown in Table 2. These activities were used for ANOVA. The EXs activities are shown in Table 3. Most of the tested fungi showed high EXs activity between 6 and 12 days without significant differences. Twelve of the twenty four strains exhibited the highest EXs activity between 6 and 9 days of incubation. Trametes sp. LBM 004 produced 42 U/L on the 6 th culture day, P. sanguineous LBM 008 41 U/L on the day 9 and T. elegans LBM 017 46 U/L on the 12 th culture day. These activities were used for ANOVA. Results from ANOVA are given in Table 4. Main effects (Time and Strain) and interaction (Time x Strain) were statistically significant for total cellulases and EXs (p˂0.05). Multiple range tests were performed to determine statistical differences between each level of main effects. The strains exhibiting the highest FPA were tested for EGs, BGLs and CBHs ( Figure 1). ANOVA test was performed for EGs, BGLs and CBHs. The analysis showed significant differences at 95% for each factor (Strain and Time) and interaction (Strain x Time). The multiple range tests showed that I. lacteus BAFC 1171 was the strain with highest EGs activity at 9 th day (847 U/L), with statistical differences among the other strains. Regarding BGLs and CBHs, P. sanguineus BAFC 2126 was the strain with the highest significant activity between 12-15 culture days (18 U/L), and between 9-15 culture days (39 U/L), respectively.
For an industrial application it is important to obtain high enzyme activity in the shortest possible period of time. P. sanguineus LBM 008 showed the highest activity over a longer period of time (3 th to 15 th culture day). This result was lower compared with that reported by Falkoski et al. [27] for P. sanguineus grown on sugarcane bagasse as carbon source. These results agree with Diorio et al. [28] using different lignocellulosic substrates, where EGs and EXs showed lower activity using wood wastes.
Analyzing the bar graphs in Figure 1, it can be seen that BGLs activity curves for P. sanguineus LBM 014 and Irpex sp. LBM 034 displayed a negative skewed behavior both with the maximum at 12 th culture day (34 and 8 U/L respectively), whereas EGs showed a positive skewed behavior, with a maximum for P. sanguineus LBM 014 on the 3 rd culture day (562 U/L) and Irpex LBM 034 on the 9 th culture day (541 U/L). It must be pointed out that the enzyme activity for Irpex sp. LBM 034 growing on wood flour as substrate was higher than using wood chips as substrate (1 U/L) for 4 to 6 days [29]. CBHs from P. sanguineus LBM 014 reached the maximum value on the 15 th culture day (16 U/L). Regarding this strain, each enzyme displayed maximum activity at different times. CBHs from Irpex sp. LBM 034 exhibited two peaks, 33 and 26 U/L on the day 3 and 15, respectively.
Irpex sp. BAFC 1171 and Irpex sp. LBM 032 showed two peaks of EGs, for Irpex sp. BAFC 1171 on the 3 rd and 9 th days (667 and 847 U/L) and for Irpex sp. LBM 032 on the 6 th and 15 th days (821 and 762 U/L). CBHs had similar behavior as EGs for Irpex sp. LBM 032 whereas Irpex sp. BAFC 1171 showed a bell-like behavior with the maximum value at 9 th day (42 U/L). Irpex sp. BAFC 1171 exhibited a positive skewed behavior for BGLs with a maximum at 9 th day, however Irpex sp. LBM 032 had an incremental pattern, rising slowly until the last day (10 U/L), when the three enzymes had similar and maximum activity.
For P. sanguineus BAFC 2126, EGs secretion pattern had a negative skewed behavior with a maximum at the 12 th day (291 U/L) and BGLs showed two peaks, at days 6 and 15 (15 and 17 U/L, respectively). This strain had a normal shape behavior with a maximum for CBHs (129 U/L-day 9). Yoon et al. [30] found similar results for FPA (80 U/L on the 6 th culture day), and EGs activity on the 9 th culture day (203 U/L) for P. sanguineous grown in medium supplied with sugarcane bagasse, but lower BGLs activity (17 U/L) on the 15 th culture day. P. sanguineous BAFC 2126 showed higher CBHs activity (129 U/L) compared with L. elegans (76 U/L) and Irpex sp. LBM 032 presented in this and another work [27].
EGs from L. elegans had normal bell shape pattern throughout the time with a peak at 9 th culture day (528 U/L), whereas CBHs and BGLs increased gradually until rising the maximum at day 15 (10 U/L for BGLs).
Dynamic knowledge of enzyme is important for efficient biotechnological applications [6]. According to the present results, PWF and EWF proved to be suitable carbon source to produce hydrolytic enzymes. I. lacteus BAFC 1171 and P. sanguineus BAFC 2126 have potential for cellulase production when growing on wood flour as a carbon source, since both fungi not only present filter paper activity, but also produce EGs, BGLs and CBHs. P. sanguineus LBM 008 secreted high EXs activity over an extended period of time. This function is remarkable because some biotechnological application require mixtures of cellulases and hemicellulases for maximum benefits [31]. To accomplish an efficient saccharification process, high activities of EGs, BGLs and CBHs are necessary to degrade cellulose and high EXs activity to degrade hemicellulose, thus increasing yields to obtain bioetanol by fermenting 5 and 6 carbon sugars. Nevertheless, it is still necessary to explore other variables to improve these levels of cellulase and xylanase using wood flour as substrate.