2.1 Microorganism

This study was carried out with a new fungal strain Penicillium sp. FSDE15 obtained from the collection of cultures of the Laboratory of Applied Microbiology (UFPB, Brazil). The strain was isolated by screening soil samples from sugarcane plantation located at Santa Rita, Paraiba, Brazil (6°99′20.67′’S 35°02′55.61′’) and stored at 4 °C on potato dextrose agar plates (Sigma Aldrich, St. Louis, USA). The microorganism was maintained in Petri dishes containing PDA medium under sterile conditions at 37 °C for seven days. Spore suspension was counted at 10⁶ spores/mL according to Santos et al. (2021).

2.2 Cellulases production by submerged state fermentation (SFm)

The basal medium was adapted from Mandels and Weber medium (MW) (1969). Three different lignocellulosic residues, wheat bran (WB), sugarcane bagasse (SB) and sugarcane straw (SS), were used as carbon source. Before their use, each residue underwent milling processes in a knife mill (Wiley Mill, Solab, SL-31) and sieving (mesh 32). Subsequently, residues were submitted to thermal pretreatment in autoclave (121 °C, 1 atm for 15 min). A volume of 50 mL of MW medium were added to 125 mL Erlenmeyer flasks, as well as 5% (w/v) of pretreated WB, SB and SS, separately, final pH was 5.7. The medium was autoclaved for 15 min at 121 °C and, after cooling, received an inoculum of 10⁶ spore/mL. Enzymatic production under SmF occurred in duplicates in rotary shaker (Solab, SL-223) at 150 rpm, 37 °C for 8 days. Every 24 h, two flasks were removed and the sample were filtrated using Whatman n° 1 filter paper and centrifuged at 5000 rpm, 10 °C for 5 min (Quimis, Q222RT). Afterwards, the pH of all centrifuged samples was measured. The crude enzymatic extract was used for determination of enzymatic activities.

2.3 Enzymes activities measurement

CMCase activity was assayed in triplicate according to Santos et al. (2021). Calibration curve for CMCase was performed using different glucose concentrations, each level in three replicates (Miller, 1959). One unit (U) of CMCase activity corresponded to 1 µmol of glucose released per minute.

β-glucosidase activity was determined using 2 mM p-nitrophenyl-β-D-glucopyranoside (PNPG, Sigma-Aldrich) in 50 mM sodium acetate buffer (pH 5.0) and 0.125 mL of the crude extract. The trials were in triplicate according to Gao et al. (2008). Calibration curve for β-glucosidase was made similarly as CMCase, although using p-nitrophenol as substrate (Shi et al., 2018). One unit of β-glucosidase was defined as the releasing of 1 µmol of p-nitrophenol per minute.

2.4 Experimental design and optimization of cellulase production by response surface methodology (RSM)

In order to optimize the cultivation conditions and enhance CMCase and β-glucosidase production by Penicillium sp. FSDE15, experimental design was performed using RSM by the software Statistica (Statsoft 7.0, EUA). Then a 2³ experimental design was used with 3 repetitions in the central point and 8 unique combinations, totaling 11 assays. The factors were inoculum concentration (X₁) (10⁵ –10⁷ spores/mL), temperature (X₂) (30–40 °C) and wheat bran concentration (X₃) (1–5%). Each factor in the design matrix was studied at three different levels (-1, 0, +1) and their level values are shown in Table 1. The variables or responses were activity of CMCase (U/mL) and β-glucosidase (U/mL) are depicted in Table 2.

The fungal strain was cultivated in 125 mL Erlenmeyer flasks containing 50 mL of modified MW medium with 5% (w/v) wheat bran for 4 days, under constant agitation in orbital shaker at 150 rpm. The enzymatic extract was centrifuged at 12.000 rpm at 10 °C for 5 min and enzymatic activity was assayed.

2.5 Enzymatic profile analysis with SDS-PAGE electrophoresis

For CMCase profile analysis, the cell-free supernatant was assayed using polyacrylamide denaturing gel (12% w/v; 0.75 mm) (Padilha et al., 2015). The β-glucosidase gel was washed with distilled water and incubated at 55 °C for 15 min in 0.5 mM 4-methyl-lumbelliferyl β-D-glucopyranoside (MUG) diluted in citrate buffer 50 mM (pH 4.8). The molecular mass markers used were those for low molecular weight ranges (GE, USA) including phosphorylaseb (97 kDa), serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and α-Lactalbumin (14.4 kDa). Finally, for enzymatic activity visualization and photography, the CMCase gel was submerged in Congo Red (0.1 %) for 15 min and decolored in NaCl 1 M, while β-glucosidase gel was visualized under UV 365 nm (UltraBright transilluminator) (Ng et al., 2010).

2.6 Statistical analysis

The statistical analysis was performed by variance analysis (ANOVA) aiming to obtain the regressions’ significance at a 95 % level of confidence. Softwares such as Graph Prism 5.0 (GraphPad software, CA, EUA) and Statistica (Statsoft 7.0, EUA) were used to analyze the data obtained.

3.1 CMCase and β-glucosidase production by SmF

All three carbon sources (WB, SB and SS) used in our study induced growth and production of CMCase and β-glucosidase by the Penicillium sp. FSDE15. The initial activity of CMCase and β-glucosidase before 24 h, in all culture media, was zero. When the medium was composed of WB, maximum CMCase and β-glucosidase yields were 1.03 (96 h) and 1.68 (168 h), respectively (Fig. 1A). With the SB medium, maximum values of enzymatic activity observed were after 120 and 168 h culture to CMCase (1.38 U/mL) and β-glucosidase (0.55 U/mL), respectively (Fig. 1B). Although, when the SS medium was used, the highest enzymatic activity was 1.02 U/mL of CMCase and 0.39 U/mL of β-glucosidase after 120 h growth (Fig. 1C).

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Fig. 1

Enzymatic activity (EA) of Penicillium sp. FSDE15 in basal media containing wheat bran (a), sugarcane bagasse (b) and sugarcane straw (c).

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Regarding the pH variation, alkalinization of the WB medium was directly related to the reduction in CMCase activity, where the pH increased from 5.7 (96 h) to 7.4 (120 h) and reduced about 35% of the enzyme activity (Fig. 1). On the order hand, highest β-glucosidase activity were observed at pH 8.2, 5.7 and 6.6, when the medium WB, SB and SS were used, respectively.

3.2 Cellulase production by RSM

The results presented in Table 2 showed a wide variation of CMCase activity (0 to 1.3 U/mL) and β-glucosidase activity (0.52 to 6.5 U/mL), which reflects the importance to optimize the process parameters. Both CMCase and β-glucosidase showed highest activity in the presence of lower levels of WB (1%) (w/w), inoculum concentration of 10⁵ spores/mL and temperatures of 41 and 33 °C, respectively (Table 2).

In this study, R² for CMCase and β-glucosidase were 0.96 and 0.95, respectively. Analysis of variance (ANOVA) was used to assess the fit of the polynomial model to CMCase and β-glucosidase activity. The results tabulated in Table 3 showed that calculated F value was 1.28 for CMCase and 0.92 for β-glucosidase (P < 0.05 at 95% confidence level), indicating that the model was significant only to CMCase.

Statistical analysis showed that the two independent variables (WB concentration and temperature) were significant for CMCase. For β-glucosidase, independent variable temperature and interaction between temperature-WB concentration was significant at 5% significance level, as observed in X₃ and X₂ bars (Fig. 2A) and X₂ and X₂*X₃ bars (Fig. 2B).

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Fig. 2

Pareto chart showing the significance of variables X1 (inoculum concentration), X2 (temperatura) and X3 (wheat bran concentration) on the CMCase (a) and β-glucosidase (b) activity.

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The final response function to predict CMCase activity was: $$y=0.684-0.131X_1+0.164X_2-0.356X_3-0.024X_1{X}_2+0.046X_1{X}_3+0.061X_2{X}_3-0.001X_1{X}_2{X}_3$$

For β-glucosidase, the final response function for activity prediction was: $$y=2.995+0.213X_1-{1.488X}_2-0.813X_3-0.443X_1{X}_2+0.148X_1{X}_3+1.183X_2{X}_3-0.76{3X}_1{X}_2{X}_3$$

Fig. 3 shows the optimized response surface, built based on the model for the CMCase activity. It is possible to verify that with the increase in temperature and decrease in the concentration of WB, CMCase activity increases, for any spore concentration used, obtaining a maximum value of 1.3 U/mL under the conditions studied in this work.

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Fig. 3

Effect of temperature and wheat bran concentration on CMCase activity for the fixed spore concentration of 10⁵.

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3.3 Enzymatic profiling by SDS-PAGE electrophoresis

The enzymatic profile of Penicilium sp. FSDE15 in the crude enzymatic extract by zymogram presented two isoforms of CMCase and one isoform of β-glucosidase. The CMCase isoforms were highly different in molecular weight with 150 kDa and between 20 and 30 kDa. β-glucosidase isoform weighed closely to 70 kDa (Fig. 4).

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Fig. 4

Enzymatic profile of cellulases from Penicilium sp. FDSE15. CMCase and β-glucosidase isoforms are shown in lanes 1 and 2, respectively. M: molecular marker (bp).

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4.1 Cellulase production using different agroindustrial wastes

In this study, the agroindustrial wastes (WB, SB and SS) induced the production of CMCase and β-glucosidase by Penicillium sp. FSDE15. While the SB medium was considered exceptional for CMCase activity, β-glucosidase yield was low for this carbon source. Thus, the WB medium was selected for the experimental design.

Before the experimental design by RSM, the highest CMCase and β-glucosidase activity were 1.03 U/mL and 1.68 U/mL in the WB medium, respectively. Zimbardi et al. (2013) investigated the action of agroindustrial wastes such as corn straw, wheat bran, corn cob, cassava meal and sugarcane bagasse in the production of CMCase and β-glucosidase. They observed that wheat bran was the best induction source for the production of these enzymes. Studies presented by Kumar et al. (2011) demonstrated that the highest production of CMCase occurred in 126 h, pH 4.6, and in a carbon source (a combination of wheat bran, corn bran and kinnow peel at the proportion of 2:1:2, respectively) concentration of 65 g/L in SmF. Prasanna et al. (2016) reported that the highest β-glucosidase activity presented by Penicillium sp. was 1.04 U/mL, when the culture medium presented a mixture of cellulose at 0.5%, lactose at 0.5% and sawdust at 0.5% and that at the morphological changes, enzyme secretion and product stability in the medium can be affected by pH change during the growth of microorganisms.

The present study showed that the highest CMCase production by the microorganism evaluated in WB, SB and SS medium was observed at pH 5.7, showing that these enzymes are more active at acidic pH, independent of the carbon source. Sohail et al. (2009) showed that the maximum CMCase yield by A. niger MS82 was obtained at pH 4.0, while at pH 5 enzyme production decreased drastically and remained constant up to 7. Unlike CMCase, the highest levels of β-glucosidase were obtained at pH 4.0, followed by a reduction in its activity as pH increases to 5, and a continuous activity decrease while pH reaches 7.

Sulyman et al. (2020) demonstrated that the optimum pH for maximum CMCase and β-glucosidase production by Aspergillus niger in the submerged fermentation was 4.0. Prasanna et al. (2016) observed that the highest CMCase and β-glucosidase production by Penicillium sp. was at pH 5.0. The highest β-glucosidase activity by Penicillium roqueforti ATCC 10,110 occurs in pH 5.5 (Neves et al., 2020).

The results obtained after experimental design by RSM demonstrate a significant effect on the optimization of CMCase and β-glucosidase activity. After carrying out the experimental design by RSM, using WB as carbon source, the largest CMCase activity was observed at 41 °C (1.3 U/mL), with an increase of 26% in relation to the initial values of 1.03 U/mL for this same carbon source.

Matkar et al. (2013) optimized SmF conditions for Aspergillus sydowii using solely lactose as carbon source and reported an optimal CMCase activity (0.39 U/mL) at 40 °C. Gomathi et al. (2012), on the other hand, studied CMCase production by Aspergillus flavus using wheat bran as carbon source and registered the highest production of cellulases (0.8 U/mL) at 30 °C.

For β-glucosidase, optimal activity was observed at 33 °C (6.5 U/mL), with an increase of 287% in relation to the initial values of 1.68 U/mL for WB medium. Saini et al. (2015) obtained similar results in optimization studies with Penicillium oxalicum, where temperature of cultivation was held at 28 °C. Our results are also comparable to those obtained by Singhvi et al. (2011) in SmF experiments with mutant strains (EMS-UV-8, 5.1 U/mL and EU-2D-21, 3.1 U/mL) and the parent strain (6.8 U/mL) of Penicillium janthinellum.

Few studies have been made to compare the β-glucosidase production between the widely used T. reesei and other fungal strains, like Penicillium and Aspergillus. Singhania et al. (2013) performed comparative studies of SmF between a reference strain of T. reesei and four strains of Penicillium. The maximum yield of 0.03 U/mL of β-glucosidase was obtained from the T. reesei strain, while the Penicillium strains produced between 0.97 and 2.45 U/mL. Results like these demonstrate the importance of screening studies for finding novel biological sources of industrially applicable enzymes.

The β-glucosidase is as a “rate-limiting” enzyme due to its key role in determining efficiency of biomass hydrolysis. If its amount during hydrolysis is low, cellobiose concentration rises and provokes feedback inhibition of the other components of the cellulolytic system, reducing the rate of glucose release (Monteiro et al., 2020).

4.2 Enzymatic profiling

Various fungi are known for producing multiple cellulases. The presence of different isoforms of CMCase can be explained by different genes that encode enzymes of the same type and this can happen when the fungus grow in different culture media and culture conditions (Bonfá et al., 2018). The existence of multiple isoforms may be important for the microorganism’s ability to respond well and rapidly to environmental changes (Oberoi et al., 2014).