In the present investigation,
Lignocellulosic biomass, for its large quantities and relatively low cost, is regarded as the potential renewable energy resource for cost-effective bioethanol production. Lignocellulosic bioethanol production involves three major steps, including pretreatment, enzymatic hydrolysis, and fermentation. Among all these steps, efficient hydrolysis of cellulose component of lignocellulosic biomass is the key step for cost-effective bioethanol production. Cellulase is the key enzyme for hydrolysis of cellulose.
Lignin is key barrier which restricts the access of cellulase to cellulose. Laccase, a copper containing oxidase enzyme, can remove lignin effectively and increase the accessibility of cellulase to cellulose at mild operating conditions and minimal byproduct formation.
In the present study, we investigated the possibility of enzymatic hydrolysis of laccase-pretreated
Laccase and cellulase were produced from
Enzymatic pretreatment of
For enzymatic hydrolysis, pretreated sample of
Response Surface Methodology- (RSM-) based three level Central Composite Design (CCD) was employed for optimization of enzymatic pretreatment and saccharification of
Experimental design (conditions and responses) for enzymatic pretreatment of
Run order | Delignification (%) | ||||||
Experimental | Predicted | ||||||
(1) | −1 (6.5) | −1 (35) | −1 (2) | −1 (400) | +1 (8) | 75.33 | 73.807 |
(2) | +1 (7.5) | −1 (35) | −1 (2) | −1 (400) | −1 (6) | 62.41 | 63.282 |
(3) | −1 (6.5) | +1 (45) | −1 (2) | −1 (400) | −1 (4) | 43.36 | 41.834 |
(4) | +1 (7.5) | +1 (45) | −1 (2) | −1 (400) | +1 (8) | 45.13 | 44.381 |
(5) | –1 (6.5) | –1 (35) | +1 (6) | −1 (400) | −1 (4) | 82.64 | 82.938 |
(6) | +1 (7.5) | −1 (35) | +1 (6) | −1 (400) | +1 (8) | 38.7 | 39.775 |
(7) | –1 (6.5) | +1 (45) | +1 (6) | −1 (400) | +1 (8) | 41.73 | 40.843 |
(8) | +1 (7.5) | +1 (45) | +1 (6) | +1 (600) | −1 (4) | 51.78 | 52.852 |
(9) | +1 (7.5) | −1 (35) | −1 (2) | +1 (600) | +1 (8) | 63.29 | 64.066 |
(10) | −1 (6.5) | +1 (45) | −1 (2) | +1 (600) | +1 (8) | 55.53 | 54.345 |
(11) | +1 (7.5) | +1 (45) | −1 (2) | +1 (600) | −1 (4) | 50.69 | 50.392 |
(12) | −1 (6.5) | −1 (35) | +1 (6) | +1 (600) | +1 (8) | 35.6 | 36.239 |
(13) | +1 (7.5) | −1 (35) | +1 (6) | +1 (600) | −1 (4) | 83.1 | 84.626 |
(14) | −1 (6.5) | +1 (45) | +1 (6) | +1 (600) | −1 (4) | 80.19 | 79.318 |
(15) | +1 (7.5) | +1 (45) | +1 (6) | +1 (600) | +1 (8) | 62.34 | 62.68 |
(16) | −1 (6.5) | 0 (40) | 0 (4) | 0 (500) | 0 (6) | 68.2 | 73.255 |
(17) | +1 (7.5) | 0 (40) | 0 (4) | 0 (500) | 0 (6) | 71.09 | 66.477 |
(18) | 0 (7) | −1 (35) | 0 (4) | 0 (500) | 0 (6) | 82.37 | 78.707 |
(19) | 0 (7) | +1 (45) | 0 (4) | 0 (500) | 0 (6) | 73.41 | 77.515 |
(20) | 0 (7) | 0 (40) | −1 (2) | 0 (500) | 0 (6) | 63.11 | 66.744 |
(21) | 0 (7) | 0 (40) | +1 (6) | 0 (500) | 0 (6) | 81.9 | 78.709 |
(22) | 0 (7) | 0 (40) | 0 (4) | −1 (400) | 0 (6) | 63.4 | 65.84 |
(23) | 0 (7) | 0 (40) | 0 (4) | +1 (600) | 0 (6) | 80.2 | 78.203 |
(24) | 0 (7) | 0 (40) | 0 (4) | 0 (500) | −1 (4) | 45.12 | 44.92 |
(25) | 0 (7) | 0 (40) | 0 (4) | 0 (500) | +1 (8) | 80.72 | 82.234 |
(26) | 0 (7) | 0 (40) | 0 (4) | 0 (500) | 0 (6) | 82.6 | 80.736 |
(27) | 0 (7) | 0 (40) | 0 (4) | 0 (500) | 0 (6) | 74.7 | 80.736 |
(28) | 0 (7) | 0 (40) | 0 (4) | 0 (500) | 0 (6) | 80.05 | 80.736 |
(29) | 0 (7) | 0 (40) | 0 (4) | 0 (500) | 0 (6) | 81.9 | 80.736 |
(30) | 0 (7) | 0 (40) | 0 (4) | 0 (500) | 0 (6) | 83.44 | 80.736 |
(31) | 0 (7) | 0 (40) | 0 (4) | 0 (500) | 0 (6) | 84.1 | 80.736 |
(32) | 0 (7) | 0 (40) | 0 (4) | 0 (500) | 0 (6) | 81.0 | 80.736 |
Experimental design (conditions and responses) for enzymatic saccharification of pretreated
Run order | Reducing sugar (mg/g of substrate) | |||||
Experimental | Predicted | |||||
(1) | −1 (4) | −1 (40) | −1 (16) | +1 (8) | 671.21 | 671.15 |
(2) | +1 (8) | −1 (40) | −1 (16) | −1 (6) | 691.45 | 698.26 |
(3) | −1 (4) | +1 (60) | −1 (16) | −1 (4) | 621.67 | 632.60 |
(4) | +1 (8) | +1 (60) | –1 (16) | +1 (8) | 639.10 | 656.21 |
(5) | −1 (4) | −1 (40) | +1 (20) | −1 (4) | 695.47 | 683.00 |
(6) | +1 (8) | −1 (40) | +1 (20) | +1 (8) | 697.92 | 691.63 |
(7) | −1 (4) | +1 (60) | +1 (20) | +1 (8) | 700.13 | 705.95 |
(8) | +1 (8) | +1 (60) | +1 (20) | −1 (4) | 701.25 | 705.95 |
(9) | +1 (8) | –1 (40) | −1 (16) | +1 (8) | 698.32 | 693.32 |
(10) | −1 (4) | +1 (60) | −1 (16) | +1 (8) | 702.23 | 692.28 |
(11) | +1 (8) | +1 (60) | −1 (16) | −1 (4) | 657.13 | 647.90 |
(12) | −1 (4) | −1 (40) | +1 (20) | +1 (8) | 667.27 | 675.42 |
(13) | +1 (8) | −1 (40) | +1 (20) | −1 (4) | 741.71 | 750.58 |
(14) | −1 (4) | +1 (60) | +1 (20) | −1 (4) | 696.09 | 696.60 |
(15) | +1 (8) | +1 (60) | +1 (20) | +1 (8) | 660.43 | 659.35 |
(16) | −1 (4) | 0 (50) | 0 (18) | 0 (6) | 734.01 | 735.68 |
(17) | +1 (8) | 0 (50) | 0 (18) | 0 (6) | 767.35 | 751.44 |
(18) | 0 (6) | −1 (40) | 0 (18) | 0 (6) | 798.66 | 798.65 |
(19) | 0 (6) | +1 (60) | 0 (18) | 0 (6) | 801.12 | 786.90 |
(20) | 0 (6) | 0 (50) | −1 (16) | 0 (6) | 758.24 | 747.63 |
(21) | 0 (6) | 0 (50) | +1 (20) | 0 (6) | 782.41 | 778.79 |
(22) | 0 (6) | 0 (50) | 0 (18) | 0 (6) | 801.23 | 808.36 |
(23) | 0(6) | 0(50) | 0 (18) | 0(6) | 797.39 | 808.36 |
(24) | 0 (6) | 0 (50) | 0 (18) | −1 (4) | 808.56 | 805.25 |
(25) | 0 (6) | 0 (50) | 0 (18) | +1 (8) | 809.72 | 805.61 |
(26) | 0 (6) | 0 (50) | 0 (18) | 0 (6) | 801.43 | 808.36 |
(27) | 0 (6) | 0 (50) | 0 (18) | 0 (6) | 795.12 | 808.36 |
(28) | 0 (6) | 0 (50) | 0 (18) | 0 (6) | 811.06 | 808.36 |
(29) | 0 (6) | 0 (50) | 0 (18) | 0 (6) | 810.06 | 808.36 |
(30) | 0 (6) | 0 (50) | 0 (18) | 0 (6) | 802.62 | 808.36 |
(31) | 0 (6) | 0 (50) | 0 (18) | 0 (6) | 817.31 | 808.36 |
(32) | 0 (6) | 0 (50) | 0 (18) | 0 (6) | 803.08 | 808.36 |
Fourier transformed infrared spectroscopy (FTIR) was performed in both the original and pretreated samples using the KBr pellet technique. Sample spectra were obtained over the range of 400 cm−1 and 4000 cm−1 with a spectral resolution of 0.5 cm−1.
The crystallinity of original and pretreated sample were determined by XRD1710 equipment using CoK
To analyze surface characteristics of original and pretreated samples, Scanning electron microscopic (SEM) image was taken for both original and pretreated sample of
Using the designed experimental data (Table
Whereas, using the experimental data (Table
Based on the experimental response, for enzymatic pretreatment, runs 12 and 31 had the minimum and maximum delignification, respectively, whereas, for saccharification of enzyme pretreated substrate, runs 3 and 31 had the minimum and maximum reducing sugar production, respectively. The ANOVA results of second-order response surface models for enzymatic pretreatment and saccharification of
ANOVA analysis of RSM model for enzymatic pretreatment of
Source | DFa | Seq SSb | Adj SSb | Adj MSc | ||
---|---|---|---|---|---|---|
Pretreatment of | ||||||
Regression | 20 | 7547.10 | 7547.10 | 502.796 | 22.07 | <0.001 |
Linear | 5 | 1115.70 | 1395.38 | 375.443 | 16.32 | <0.001 |
Square | 5 | 2592.68 | 3959.72 | 473.038 | 46.32 | <0.001 |
Interaction | 10 | 3838.72 | 3838.72 | 383.872 | 22.45 | <0.001 |
Residual error | 11 | 188.05 | 188.05 | 17.096 | ||
Lack-of-fit | 5 | 128.62 | 128.62 | 25.724 | 2.60 | 0.138 |
Pure error | 6 | 59.44 | 57.44 | 9.906 | ||
Total | 32 | 7735.16 | ||||
Saccharification of pretreated | ||||||
Regression | 14 | 116738 | 116737.6 | 8338.4 | 64.72 | <0.001 |
Linear | 4 | 6116 | 22570.9 | 5642.7 | 43.80 | <0.001 |
Square | 4 | 103427 | 98888.2 | 24722.0 | 191.90 | <0.001 |
Interaction | 6 | 7195 | 7194.6 | 1199.1 | 9.31 | < 0.001 |
Residual error | 17 | 2190 | 2190.1 | 128.8 | ||
Lack-of-fit | 9 | 1788 | 1788.0 | 198.7 | 3.95 | 0.033 |
Pure error | 8 | 402 | 402.1 | 50.3 | ||
Total | 32 | 118928 | ||||
aDegrees of freedom.
bSum of squares.
cMean squares.
The 3D response surface plots are generally the graphical representation of the regression equation. Figures
RSM plot showing (a) the effect of pH and liquid : solid ratio and (b) the effect of pH and incubation time on enzymatic pretreatment of
Figures
RSM plot showing (a) the effect of substrate concentration and pH and (b) substrate concentration and incubation time on saccharification of enzyme-pretreated
Similar reducing sugar yield (818.01 mg/g dry substrate) was reported by other authors at high cellulase loading [
There are so many reports on using commercial cellulase for saccharification of different lignocellulosic biomass and also further addition of several additives was required [
FTIR analysis was taken to qualitatively observe the changes of functional groups and further evaluate component modification. Significant changes in FTIR spectra could be seen after enzymatic pretreatment of
FTIR spectra of control and enzyme-pretreated
Lignocellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin. Among several effecting factors, crystallinity is believed to significantly affect enzymatic saccharification of cellulose [
XRD diagram of control and enzyme-pretreated
SEM was used to determine changes in surface structure of
SEM view of (a) control and (b) enzyme-pretreated sample of
Enzymatic pretreatment and saccharification of
The authors sincerely acknowledge Petrotech Society, New Delhi, for financial support.