Manganese peroxidase (MnP) is a ligninolytic enzyme that is involved in the removal of lignin from the cell wall of plants. This removal facilitates the access of hydrolytic enzymes to the carbohydrate polymers that are hydrolyzed to simple sugars, which allows the subsequent fermentation to obtain bioproducts, such as ethanol. In this work, response surface methodology (RSM) was employed to optimize the culture conditions on unexpensive substrate for MnP secretion by Trametes villosa. Three independent variables were evaluated ( i.e., temperature, moisture content and pH). The crude extract containing MnP was used in the delignification experiment and it caused a reduction in lignin content for all residues tested: 35.05 ± 1.45 (%) for the sugar cane bagasse; 63.11 ± 0.06 (%) for the sisal fiber and 39.61 ± 0.39 (%) for the coconut shell, under the reaction conditions tested after 4 hours of fermentation. The preliminary results exhibited the potential application of this enzyme in the removal of lignin from plant residues. However, the conditions should be evaluated and optimized for each residue type.
Conversion of agricultural wastes to ethanol presents an important opportunity to reduce energy costs. Compared to other forms of biofuels (biogas, biodiesel) delignification of lignocellulosic biomass becomes important because they are plentiful and inexpensive materials [
Lignocellulose is a structure composed of lignin, cellulose and hemicellulose and represents the most abundant renewable organic resource from the soil. When bound to cellulose and hemicellulose, lignin forms a barrier that reduces the degradation of lignocellulosic materials and complicates the industrial use of plant polysaccharides in paper and biofuel production, among other processes [
The biological process involved in the conversion of lignocellulosic residues to high value products requires the following: 1) a delignification pretreatment (i.e., mechanical, chemical or biological) to release cellulose and hemicellulose from their complex, 2) the depolymerization or hydrolysis of carbohydrate polymers to produce metabolizable molecules (i.e., free sugars: hexoses and pentoses), 3) the use of these molecules to support microbial growth or obtain chemical products, and 4) the separation and purification of the obtained products. The delignification process of lignocellulosic feedstocks is considered the most difficult step and is the limiting step in the conversion [
The biological delignification process is primarily performed by basidiomycete fungi: a large group with over 30,000 species that includes brown and white rot fungi. The later are organisms capable of efficiently degrading and mineralizing lignin to CO2 and water, which is termed “enzymatic combustion” [
Mn-dependent peroxidase (or manganese peroxidases―MnP) (EC 1.11.1.13) are extracellular glycoproteins that contain a heme prosthetic group and show molecular weight ranging from 45 to 47 KDa. Currently, MnP is considered the key enzyme in lignin degradation. The secretion of this enzyme depends on environmental factors and is strongly regulated by nutrients availability [
Ligninolytic systems have been extensively studied in recent years, and researchers agree that the type and composition of the substrate appears to determine the amount of enzymes produced by basidiomycetes. Additionally, the temperature, pH, carbon source and nitrogen affect the growth of the fungus and ligninolytic activity [
According to Arantes et al. [
This study was performed to optimize the production of MnP by the basidiomycete fungus Trametes villosa. The subsequent application of the enzyme extract was to determine its potential for the delignification of sugarcane bagasse, coconut shell and sisal fibers, which are abundant agricultural wastes in Northeastern Brazil.
Ten unidentified fungi isolates were tested for their ability to decolorize Remazol Brillinat Blue R (RBBR) dye in Petri dishes containing agar and the dye, according to methodology described by Palimeri et al. [
The fungal strain used was Trametes villosa (Sw.) Kreisel CCMB 651 deposited in the Culture Collection of Microorganisms of Bahia―CCMB, Feira de Santana, BA, Brazil (http://www.uefs.br/ccmb) [
The medium used for the activation and growth of the isolate consisted of wheat grain containing calcium carbonate and was incubated at 28˚C in a biological oxygen demand (BOD) incubator for seven days. The plates were stored at 4˚C and the subcultures were performed every three months in duplicate.
The experiments were based on a central composite rotatable design using Statistica (6.0) software, in which three independent variables (i.e., temperature, moisture content and pH) were evaluated at five levels according to
The fixed conditions for all assays were as follows: 250 mL conical flasks containing 20 g of sugarcane bagasse (chopped, yielding little cubes of 0.5 cm side), an inoculum of five grains of wheat showing mycelium with seven days of growth and a cultivation time of 14 days. The initial moisture of the bagasse was adjusted for 50% by drying in a ventilated oven and the final moisture used in the assays (
After the culture period, an aqueous extraction of the enzymes was performed from the fermented biomass, i.e., 50 mL of cold distilled water was added to the medium and the mixture remained in an ice bath for one hour with occasional stirring. The content was then filtered through gauze and centrifuged at 4˚C and 5000 × g for 10 minutes. The supernatants were separated into aliquots, frozen and stored for the determination of enzyme activity.
The MnP activity was determined by the oxidation of phenol red in the presence of hydrogen peroxide according to the modified methodology of Kuwahara et al. [
The activity of MnP (U/L) was calculated using Equation (1), as described by Menezes, Silva and Durrant [
where DAbs is the difference between the absorbance of the boiled extract and non-boiled extract (i.e., the blank and test) at 0 and 5 minutes; e is the extinction coefficient of oxidized phenol red at 610 nm = 4460 L, m−1・cm−1 ;
Variable | Code | Level | ||||
---|---|---|---|---|---|---|
−a (−1.68)* | −1 | 0 | +1 | +a (+1.68) | ||
Temperature (˚C) | X1 | 20 | 23 | 28 | 33 | 36 |
Moisture content (%) | X2 | 70 | 74 | 80 | 86 | 90 |
pH | X3 | 3.0 | 4.62 | 7.0 | 9.38 | 11.0 |
*Value of α for experiments with three independent variables.
R is the aliquot of crude enzyme extract (mL); and t is the reaction time (min).
The specific activity was calculated from the quotient of enzyme activity by the protein concentration determined by the Lowry method [
According to the results obtained after the statistical evaluation of the optimization experiments, another experiment was set up to verify the influence of incubation time, applying the optimized conditions, on the secretion of MnP. The enzyme activity was measured at 5, 10, 15, 20, 25 and 30 days of fungus growth in conical flasks (250 mL) containing 20 g of sugarcane bagasse, at 80% moisture content and pH 9.4, incubated at 20˚C in a BOD. The cultivation was conducted in duplicate and the enzyme activity was evaluated in triplicate. The crude protein content of the enzyme extract was determined by the Lowry method [
After defining the optimal culture conditions that favored the secretion of MnP, the crude enzymatic extract was produced and applied for the delignification of sugarcane bagasse, coconut shell and sisal fiber. The reaction medium consisted of 2 g of plant residue, 5 mL of enzyme extract (specific activity: 0.236 U/mg) and 15 mL of sodium succinate buffer (0.2 M, pH 4.5) containing manganese sulfate (2.0 mM), bovine albumin (0.5%), hydrogen peroxide (2 mM) and sodium lactate (0.25 M). The medium was incubated in 250 mL conical flasks at 30˚C and 150 rpm. The reaction times were 4, 12 and 72 hours, and the treated residues were washed with distilled water and frozen for later determination of lignin content. A blank without the enzyme was also perfomed and provided the initial lignin content. All tests were performed in triplicate, and the percentage of lignin removed was determined by Equation (2):
The lignin content of the samples was determined by the Van Soest method [
The crucibles containing the filtered residue from the acid detergent fiber were supplemented with 72% sulfuric acid and subjected to vacuum filtration. The residues were dried in an oven at 100˚C for 8 hours and incinerated in the oven at 550˚C for 3 hours. The lignin content was calculated by the weight loss after incineration.
The software Statistica (6.0) was applied to construct the table of analysis of variance (ANOVA), response surfaces, contour plots and the predictive model.
According to Elisashvili and Kachlishvili [
In this work, specific activity was evaluated as the response to the optimization of MnP using the software Statistica (6.0) (
Trials 9 and 4 showed greater crude (117.327 U/L) and specific activities (0.167 U/mg), respectively. Trial 9 presented a greater crude activity but a lower specific activity which can be explained by the high protein content of the extract, that was presumably caused by the medium pH (equal to 7) that most likely favored the production of other proteins.
The response surface plot in
Assay | Temperature (˚C) | Moisture content (%) | pH | Crude activity (U/L)* | Protein content (mg/L)* | Specific activity (U/mg) |
---|---|---|---|---|---|---|
1 | 23 | 74 | 4.60 | 75.09 | 3825.28 | 0.020 |
2 | 23 | 74 | 9.38 | 112.70 | 865.14 | 0.130 |
3 | 23 | 86 | 4.60 | 0.00 | 2422.96 | 0.000 |
4 | 23 | 86 | 9.38 | 89.50 | 534.35 | 0.167 |
5 | 33 | 74 | 4.60 | 0.00 | 4100.93 | 0.000 |
6 | 33 | 74 | 9.38 | 71.68 | 1413.63 | 0.051 |
7 | 33 | 86 | 4.60 | 0.00 | 2921.97 | 0.000 |
8 | 33 | 86 | 9.38 | 31.96 | 638.96 | 0.050 |
9 | 20 | 80 | 7.00 | 117.33 | 825.56 | 0.142 |
10 | 36 | 80 | 7.00 | 0.08 | 1761.38 | 0.000 |
11 | 28 | 70 | 7.00 | 67.00 | 3303.65 | 0.020 |
12 | 28 | 90 | 7.00 | 0.00 | 404.30 | 0.000 |
13 | 28 | 80 | 3.00 | 0.00 | 3812.55 | 0.000 |
14 | 28 | 80 | 11.00 | 1.93 | 1526.72 | 0.001 |
15 | 28 | 80 | 7.00 | 87.28 | 747.81 | 0.117 |
16 | 28 | 80 | 7.00 | 71.91 | 770.43 | 0.093 |
17 | 28 | 80 | 7.00 | 82.30 | 675.71 | 0.122 |
18 | 28 | 80 | 7.00 | 81.18 | 722.36 | 0.112 |
*An average absorbance of the three repetitions was used in the calculations.
Although media composition and growth conditions strongly affect the prodution of ligninolytic enzymes [
The highest levels of specific MnP activity were achieved for the pH range between 7.0 (0) and 11 (+α) with decreased temperature levels (
Considering the analysis of response surfaces, the following conditions were established for the experiments that followed: pH 9.4, temperature 20˚C and moisture content of 80%.
The maximum MnP activity determined was of the same magnitude as that reported for many basidiomycetes. The MnP was the dominant enzyme produced by Irpex lacteus, with an average activity of 270 U/L in a study conducted by Baborová et al. [
Elisashvili et al. [
The specific MnP activity increased until the 15th day, after which it decreased until approaching zero on 30th day. The analysis of variance with the Tukey post-test for the comparison of means indicated significant differences at the 15th day compared to all others. Therefore, 15 days was considered the best time for the secretion of MnP using the optimized conditions for Trametes villosa (
Cupul et al. [
The percentage of lignin removal for plant residue after the reaction with the crude enzyme extract containing MnP is presented in
Assay | Time (days) | Specific activity (U/mg) (mean ± SD) |
---|---|---|
1 | 5 | 0.011 ± 0.008a |
2 | 10 | 0.193 ± 0.054b |
3 | 15 | 0.236 ± 0.039c |
4 | 20 | 0.195 ± 0.020b |
5 | 25 | 0.037 ± 0.001a |
6 | 30 | 0.027 ± 0.008a |
Culture conditions: 20 g of sugarcane bagasse at 80% humidity, pH 9.38 and 20˚C. The activity values are the average of three repetitions. The mean values with the same letters do not differ among themselves according to the Tukey test.
Plant residue | Lignin weight before treatment (g) | Time (hours) | Lignin weight after treatment (g) | Lignin removal (%)* |
---|---|---|---|---|
Sugarcane bagasse | 0.108 ± 0.015 | 4 | 0.070 ± 0.004 | 35.040a |
12 | 0.069 ± 0.004 | 35.680a | ||
72 | 0.067 ± 0.004 | 37.810a | ||
Coconut shell | 0.227 ± 0.014 | 4 | 0.137 ± 0.008 | 39.610b |
12 | 0.137 ± 0.008 | 39.452b | ||
72 | 0.166 ± 0.010 | 40.201b | ||
Sisal fiber | 0.054 ± 0.007 | 4 | 0.021 ± 0.003 | 63.040c |
12 | 0.023 ± 0.003 | 63.140c | ||
72 | 0.020 ± 0.003 | 63.150c |
*The mean values with the same letters do not differ among themselves according to the Tukey test.
The crude enzyme extract caused a reduction in lignin content for all residues tested: 35.05 ± 1.45 (%) for the sugar cane bagasse; 63.11 ± 0.06 (%) for the sisal fiber and 39.61 ± 0.39 (%) for the coconut shell, under the reaction conditions, after 4 hours of fermentation. No improvement was detected for longer reaction times.
There are different delignification processes applied as pretreatments for bioethanol production [
Moniruzzaman and Ono [
The results found in this work indicate that the crude enzyme extract obtained under the optimized conditions for production of MnP by Trametes villosa (Sw.) Kreisel CCMB 651 is a possible alternative to promote the lignin removal in plant wastes. The process of enzymatic delignification may lead to better results when using optimized reaction conditions, such as time, temperature, enzyme concentration, stirring, and type of residues, which can be determined in further studies.
The optimization of culture conditions on a solid substrate for MnP secretion was performed in this study. After optimization, the maximum activity was 117.327 U/L, which was approximately 20 times greater than values reported by Machado et al. (2005) for three strains of Trametes villosa grown under submerged fermentation conditions in medium containing sugarcane bagasse as the substrate. The optimal conditions for enzyme production for sugar cane bagasse were as follows: 80% moisture content at pH 9.38 incubated at 20˚C for 15 days. The results presented here show that Trametes villosa (Sw.) Kreisel CCMB 651 has a high ligninolytic potential specially because of its MnP production. Furthermore, it was possible to cultivate the fungus by use of inexpensive agroindustrial residues, such as sugarcane bagasse.
The preliminary delignification tests showed that the crude enzymatic extract containing MnP from Trametes villosa (Sw.) Kreisel CCMB 651 was capable of reducing the lignin content by 35.05 ± 1.45 (%) for the sugar cane bagasse; 63.11 ± 0.06 (%) for the sisal fiber and 39.61 ± 0.39 (%) for the coconut shell. These results are encouraging and further studies are currently being performed to optimize the reaction conditions in order to improve the lignin removal in these plant residues and others commonly found in Northeastern Brazil. It will be possible to conduct experiments of enzymatic saccharification of cellulosic material for ethanol production from the optimized conditions.
The authors would like to thank CAPES for the scholarship.