The aim of this study was to verify the viability of lignocellulosic substrates to obtain renewable energy source, through characterization of the cellulolytic complex, which was obtained by solid state fermentation using
The growing demand for energy for transportation and industrial processes stimulates the search for new energetic renewable matrixes to replace fossil fuels that have limited reserves, turning feasible the use of agroindustrial residues that besides being abundant reduce the environmental impact [
The lignocellulosic biomass is the largest source of carbohydrate, since it is the main plant cellular wall. It consists of lignins chains, cellulose, and hemicellulose, which are intertwined and chemically linked by noncovalent forces and by covalent crossed connections, becoming a substrate of difficult hydrolysis [
The cellulolytic enzymes are synthesized by microorganisms like bacteria and fungi.
The greatest difficulty for the use of the lignocellulosic residues is represented by the physical barrier formed by the lignin, which prevents the use of the native cellulose; thus the enzymes cannot penetrate this barrier easily. The separation of the lignin may be achieved through physical, chemical, or biological treatments or their combination. The chemical treatment is usually used through acid or alkaline hydrolysis [
The most used techniques in the cellulose biomass hydrolysis are the chemical and enzymatic methods. The chemical hydrolysis presents advantages because of its high rate and unnecessary pretreatment, but the enzymatic hydrolysis is superior to it, in several aspects, as, for instance, before the possibility to be performed at low temperatures (45°C–50°C) and atmospheric pressure, there is no subproducts formation, increasing the yield of fermentable sugar production. The enzymatic reactions may occur under mild conditions of pH (4.8) not causing corrosion problems in equipment. Thus, to reach high conversion of cellulose it is necessary to have high concentrations of enzymes, increasing the cost of production. Therefore, the study of microorganisms, which produce high productivity cellulases, is very important, as well as the development of economic production techniques [
Fermentation in solid state consists in the process of microbial growing in solid substrate, with enough moisture to guarantee the cells growing and metabolism, and does not exceed the maximum retention capacity of water of the solid matrix, that is, exempt of free water [
The filamentous fungi present better capacity of growing under conditions of low levels of water.
According to Leu and Zhu [
In this context, the objective was to study the saccharification of lignocellulosic residues by the cellulases obtained by fermentation in solid state using
The microorganism used was
The inoculum was prepared in Erlenmeyer flasks of 1000 mL with 50 mL of medium agar filter paper, to which 1 mL of spores suspension was added, resulting from the growth in test tubes. The Erlenmeyer flasks were kept in an oven at 30°C for 7 d, for later suspension with the addition of Tween 80 0.1% sterilized, which were filtered in cotton, for later use as inoculum.
The corncob was the substrate used in the fermentation processes, which was delignified according to the method adapted from Sukumaran et al. [
The delignified substrate was submitted to drying in an oven at 35°C during 24 h for total removal of moisture, obtaining a 50% delignified substrate.
The corncob was used as the source of carbon, which was passed through sieve whose opening was 1.18 mm (14 mesh). To this substrate was added 30% (v/w) of macro- and micronutrients solution, adapted from the method described by Aguiar et al. [
Solid state fermentation for the production of cellulolytic enzymes (cellulolytic complex) was carried out in Erlenmeyer flasks of 250 mL with 10 g of medium culture, 0.5 g filter paper, and 1.0 mL of spores suspension (inoculum), containing 109 spores·mL−1. The concentration of spores in suspension was estimated by counting in microscope, using a Neubauer chamber. The experiments were incubated at 30°C for 192 h.
The lignocellulosic substrates corncob (C), eucalyptus sawdust (ES), or filter paper (FP) delignified were used in the saccharification assays. The enzymes of the cellulolytic complex (CC) were obtained from the bran of solid state fermentation with
The saccharification of the substrates was performed by using a 22 Full Factorial Design (FFD) with three central points. The studied variables were temperature and pH, according to the matrix of experiments showed in Table
Enzymatic activity (U/g) of the cellulolytic complex, obtained from the fermented bran, under pH and temperature conditions tested in the Full Factorial Design with three central points.
Experiment |
|
pH | Substrate | ||
---|---|---|---|---|---|
Filter paper | Eucalyptus sawdust | Corncob | |||
1 | 40 | 4.4 | 10.634d | 8.241c | 9.200b |
2 | 40 | 5.2 | 9.629cd | 10.146d | 10.785c |
3 | 60 | 4.4 | 8.421bc | 7.256b | 8.271a |
4 | 60 | 5.2 | 7.762ab | 6.631a | 8.992b |
5 | 50 | 4.8 | 6.649a | 9.391d | 9.543b |
6 | 50 | 4.8 | 6.984a | 10.118d | 9.359b |
7 | 50 | 4.8 | 6.880a | 10.044d | 9.086b |
In the same column, different letters mean statistical difference at 5% significance.
The experiments were made in Erlenmeyer flasks of 300 mL, containing 5 g of substrate and 75 mL of citrate buffer 0.05 mol·L−1, with pH variable according to the experimental planning. The mixture was submitted to a thermostatic bath (temperature variable according to the factorial design) for 10 min for medium adaptation, after 5 g of fermented bran containing the cellulolytic complex or 5 mL of commercial enzyme with dilution of 1 : 100 (v/v) was added. The saccharification was made during 24 h, without agitation. The experiments were performed in triplicate. The control experiments were performed by using citrate buffer to replace the source of enzymes.
The enzymatic activity of the cellulolytic complex and of the commercial enzyme was evaluated according to the filter paper assay (FPU) adapted from Ghose [
The efficiency of saccharification was evaluated through the reducing sugars content after filtration, using the 3.5-dinitrosalicylic acid (DNS) method by spectrophotometer at 546 nm, using glucose as standard [
The results were showed in units, where one enzymatic unit (U) is defined as a quantity of enzyme which is able to release 1
The results of the enzymatic activity obtained in the planning were analyzed through analysis of variance (ANOVA), with the estimated effects and regression coefficients being obtained.
Figures
Concentration of reducing sugars formed (a) and enzymatic activity (b) during the time of saccharification, under experimental conditions of the temperature 40°C and pH of 4.4 (Experiment 1), where FP is filter paper, C is corncob, ES is eucalyptus sawdust, CC is cellulolytic complex, and CE is commercial enzyme.
The cellulolytic complex behavior was similar in all substrates studied during the saccharification period. This behavior was not observed for the commercial cellulase, which presented low conversion values when eucalyptus sawdust was used. Therefore, it was verified that the commercial enzyme has different degrees of specificity among substrates, while the cellulolytic complex did not present difference of specificity among the substrates, since no considerable differences in the values of enzymatic activity among the available substrates were presented (Figures
Concentration of reducing sugars formed (a) and enzymatic activity (b) during the time of saccharification, under experimental conditions of the temperature 40°C and pH of 5.2 (Experiment 2), where FP is filter paper, C is corncob, ES is eucalyptus sawdust, CC is cellulolytic complex, and CE is commercial enzyme.
Concentration of reducing sugars formed (a) and enzymatic activity (b) during the time of saccharification, under experimental conditions of the temperature 60°C and pH of 4.4 (Experiment 3), where FP is filter paper, C is corncob, ES is eucalyptus sawdust, CC is cellulolytic complex, and CE is commercial enzyme.
Concentration of reducing sugars formed (a) and enzymatic activity (b) during the time of saccharification, under experimental conditions of the temperature 60°C and pH of 5.2 (Experiment 4), where FP is filter paper, C is corncob, ES is eucalyptus sawdust, CC is cellulolytic complex, and CE is commercial enzyme.
Concentration of reducing sugars formed (a) and enzymatic activity (b) during the time of saccharification, under experimental conditions of the temperature 50°C and pH of 4.8 (Experiment 5), where FP is filter paper, C is corncob, ES is eucalyptus sawdust, CC is cellulolytic complex, and CE is commercial enzyme.
It was verified that the greatest enzymatic activities were obtained in initial times of saccharification, regardless of the source of enzyme used (cellulolytic complex or commercial cellulase). A high decrease in enzymatic activity was observed until 12 h of reaction, with later stabilization (Figures
The maximum enzymatic activity of the cellulolytic complex on filter paper was shown in Experiment 1 (10.634 U/g), with temperature of 40°C and pH 4.4; thus it did not show significant difference (
The cellulolytic complex action on eucalyptus sawdust showed maximum enzymatic activity in the lower level of temperature (40°C) and higher level of pH (5.2), with value of 10.146 U/g. These conditions show no significant difference (
The saccharification of the corncob substrate by the cellulolytic complex was favored under temperature of 40°C and pH 5.2 and this condition showed significant difference (
Annamalai et al. [
Corncob and eucalyptus sawdust substrates show the lowest enzymatic activities in the experiments corresponding to temperature of 60°C. It is verified that the cellulolytic complex obtained a similar behavior in all substrates used, since this enzymatic form has greater performance in temperature between 40°C and 50°C and pH between 4.8 and 5.2. Gokhale et al. [
By comparing maximum enzymatic activities obtained in the experiments of the FFD with the three substrates studied and by using, as source of enzymes, the cellulolytic complex obtained via SSF, it was verified that there was no significant difference (
Table
Enzymatic activity (U/mL) of the commercial cellulase under the conditions of pH and temperature tested in the
Experiment |
|
pH | Substrate | ||
---|---|---|---|---|---|
Filter paper | Eucalyptus sawdust | Corncob | |||
1 | 40 | 4.4 | 12.807bc | 3.276d | 26.87b |
2 | 40 | 5.2 | 13.563cd | 3.272d | 25.308b |
3 | 60 | 4.4 | 8.805a | 1.369b | 10.423a |
4 | 60 | 5.2 | 11.315b | 1.181a | 16.099a |
5 | 50 | 4.8 | 15.771e | 3.692e | 25.456b |
6 | 50 | 4.8 | 15.419de | 2.914c | 26.281b |
7 | 50 | 4.8 | 15.528de | 3.121d | 24.310b |
In the same column, different letters mean statistical difference at 5% of significance.
Maximum enzymatic activities of the commercial cellulase on filter paper were obtained in the experiments of the central points (5, 6, and 7) with maximum value of 15.771 U/mL, at 50°C and pH 4.8, and there was no significant difference between them and Experiment 2 (40°C and pH 5.2), with value of 13.563 U/mL, as it can be observed in Table
The commercial cellulase showed greater enzymatic activity in Experiment 1 of 26.787 U/mL using corncob as substrate; however, it did not present significant difference (
Comparing the averages of maximum enzymatic activities obtained in experiments of the FFD with three substrates studied and using the commercial cellulase, through the Tukey test at 5% of significance (15.771 U/mL for the filter paper, 3.692 U/mL for eucalyptus sawdust, and 26.787 U/mL for corncob), it was verified that there was significant difference (
The commercial cellulase showed greater specificity with relation to the corncob and then with relation to the filter paper, and it was less specific for eucalyptus sawdust, showing maximum enzymatic activity value for this substrate, approximately 90% lower than the more specific substrate.
According to Sun and Cheng [
When comparing both forms of cellulase enzyme used, it is observed that the cellulolytic complex from
According to Le Ngoc Huyen et al. [
Table
Analysis of variance and effects estimated from variables pH and temperature on the enzymatic activity in the experiments of the Full Factorial Design
Substrates | Source of variation | Cellulolytic complex | Commercial enzyme | ||
---|---|---|---|---|---|
Effect estimated | Level of significance |
Effect estimated | Level of significance |
||
Filter paper |
|
−2.040 | 0.322 | −1.225 | 0.673 |
|
−0.832 | 0.663 | 2.745 | 0.373 | |
|
0.173 | 0.926 | 2.674 | 0.384 | |
|
|||||
Eucalyptus sawdust |
|
−2.250 | 0.203 | −1.972 | 0.078 |
|
0.640 | 0.676 | −0.069 | 0.932 | |
|
−1.265 | 0.429 | −0.119 | 0.883 | |
|
|||||
Corncob |
|
−1.361 | 0.005 | −12.787 | 0.059 |
|
1.153 | 0.009 | 2.098 | 0.665 | |
|
−0.432 | 0.105 | 3.578 | 0.474 |
The analysis of variance (Table
The enzymatic activity of the commercial cellulase on substrates filter paper and eucalyptus sawdust was not significantly influenced by variables pH and temperature (
The enzymes of the cellulolytic complex and the commercial cellulase present better activity and stability between 40°C and 50°C, and the enzymatic activity is not significantly influenced at pH (4.4–5.2). Considering the effect of the temperature on the enzymatic activity, the enzymes of the cellulolytic complex are less affected by the increase of temperature than the commercial cellulase enzyme. The cellulolytic complex shows high specificity for pure substrate as well as for complex substrates such as delignified corncob and eucalyptus. On the other hand, the commercial cellulase is more specific to corncob, followed by the pure substrate (filter paper). The results obtained demonstrate that fungi
The authors declare that there is no conflict of interests regarding the publication of this paper.
Financial support for this research was received from the Foundation Support Research of Rio Grande do Sul (FAPERGS, Notice PROCOREDES).