Effect of physical parameters such as initial pH, agitation (rpm), and temperature (°C) for cellulase production from
Cellulases have versatile applications in textile, laundry, pulp and paper, fruit juice extraction, and animal feed additives [
The traditional “one-variable-at-a-time approach” for optimization disregards the complex interactions among various components. Statistically based experimental designs such as Placket-Burman design and response surface methodology (RSM) can be effectively used to study the effects of factors and to search for optimum levels of parameters for desired response [
Simultaneous saccharification and fermentation (SSF) process combines enzymatic hydrolysis of cellulose with subsequent fermentation of reducing sugar (glucose) to ethanol [
In the present study, the physical process parameters such as initial pH, temperature, and agitation speed of the culture were optimized by central composite design technique using multiresponse analysis to enhance the alkaline CMCase activity from newly isolated
Carboxymethylcellulose (CMC) was purchased from Sigma-Aldrich (St. Louis, USA). All other chemicals and reagents of analytical grade like potassium dichromate (K2Cr2O7), glucose, yeast extract, and peptone used in the study were procured from Merck and Himedia laboratories (India). Lignocellulosic biomass wild grass (
CCD showing experimental and regression model predicted CMCase activity (U/mL) and cell growth (g/L).
Run no. | pH | Temp (°C) | Agitation (rpm) | CMCase activity (U/mL) | Cell growth (g/L) | ||
---|---|---|---|---|---|---|---|
Measured | Predicted | Measured | Predicted | ||||
1 | 0 (7.0) | 0 (37) | 0 (180) |
|
0.437 |
|
3.18 |
2 | 1 (7.6) | −1 (33) | 1 (215) |
|
0.335 |
|
2.39 |
3 | −1 (6.4) | 1 (41) | −1 (144) |
|
0.512 |
|
1.85 |
4 |
|
0 (37) | 0 (180) |
|
0.503 |
|
2.53 |
5 | 0 (7.0) | 0 (37) | − |
|
0.490 |
|
2.14 |
6 |
|
0 (37) | 0 (180) |
|
0.360 |
|
2.10 |
7 | 0 (7.0) | 0 (37) | 0 (180) |
|
0.437 |
|
3.18 |
8 | 0 (7.0) | 0 (37) | 0 (180) |
|
0.437 |
|
3.18 |
9 | 0 (7.0) | 0 (37) | 0 (180) |
|
0.437 |
|
3.18 |
10 | 1 (7.6) | 1 (41) | −1 (144) |
|
0.538 |
|
2.06 |
11 | 1 (7.6) | 1 (41) | 1 (215) |
|
0.374 |
|
2.93 |
12 | 0 (7.0) | 0 (37) | 0 (180) |
|
0.437 |
|
3.18 |
13 | −1 (6.4) | −1 (33) | −1 (144) |
|
0.202 |
|
1.75 |
14 | 0 (7.0) | 0 (37) | 0 (180) |
|
0.437 |
|
3.18 |
15 | 1 (7.6) | −1 (33) | −1 (144) |
|
0.325 |
|
2.01 |
16 | −1 (6.4) | 1 (41) | 1 (215) |
|
0.327 |
|
2.67 |
17 | 0 (7.0) | 0 (37) |
|
|
0.344 |
|
3.15 |
18 | 0 (7.0) |
|
0 (180) |
|
0.365 |
|
2.13 |
19 | 0 (7.0) | − |
0 (180) |
|
0.071 |
|
1.58 |
20 | −1 (6.4) | −1 (33) | 1 (215) |
|
0.192 |
|
2.08 |
Values are mean ± SE (
In order to determine the best set of culture conditions to obtain maximum cellulase activity by
Multiple response or desirability function is an analysis in which a number of responses (output variables) are measured simultaneously for each setting of a group of parameters (input variables) and is also called multiresponse analysis [
It was reported earlier that although higher cell growth was achieved at favorable agitation speed, pH, and temperature, at the higher biomass concentration, the cellulase activity is inhibited [
In this study, enzyme activity was given higher weight of 2 : 1 ratio as compared to cell growth. The overall desirability function (
In order to validate the model, experiments were performed in triplicate in a batch shake flask and 2 L stirred tank fermentor (Applicon, model Bio Console ADI 1025) using optimal levels of culture conditions (pH 7.2, 39°C, and 121 rpm) and optimized medium [
20 mL of 0.5 M NaOH was added to one gram of the powdered wild grass in a 250 mL Erlenmeyer flask [
One gram of substrate was incubated with 8 mL of concentrated phosphoric acid at 50°C at 120 rpm for one hour. The slurry was then poured in to 24 mL of chilled acetone and thoroughly mixed. The mixture was then centrifuged at 8,000 g for 10 minutes. The pellet was collected and centrifuged in distilled water for five minutes thrice. The pH was adjusted between 5 and 6 using NaOH during the third wash [
One gram of the pretreated wild grass was taken in a 250 mL flask to which 100 mL of sodium phosphate buffer (pH 6.0, 20 mM) containing yeast extract (0.1%, w/v) and peptone (0.1%, w/v) was added. Then, 1 mL of isolated
Cell growth was determined by measuring absorbance at optical density of 600 nm using a UV-visible spectrophotometer (Perkin Elmer, Model lambda-45) and the absorbance values were expressed as dry cell weight using a calibration curve of optical density (
The assay of cellulase was carried out in 100
For ethanol content estimation, dichromate method was used where ethanol produced was converted to acid by reaction with dichromate [
For maximizing CMCase activity, the levels of the three important factors, pH, agitation speed (rpm), and temperature (°C), were varied using the central composite design of experiment. Table
(a) Analysis of variance (ANOVA) for alkaline CMCase activity (U/mL) in the optimization study, (b) analysis of variance (ANOVA) for cell growth (g/L) of
Source | df | SS | Adj MS |
|
|
|
---|---|---|---|---|---|---|
Regression | 9 | 0.261363 | 0.029040 | 21.18 | 0.000 | 95.02 |
Linear | 3 | 0.154385 | 0.051462 | 37.54 | 0.000 | |
Square | 3 | 0.086877 | 0.028959 | 21.13 | 0.000 | |
Interaction | 3 | 0.020102 | 0.006701 | 4.89 | 0.024 | |
Residual error | 10 | 0.013708 | 0.001371 | |||
Pure error | 5 | 0.000404 | 0.000081 | |||
Total | 19 | 0.275071 |
Source | df | SS | Adj MS |
|
|
|
---|---|---|---|---|---|---|
Regression | 9 | 6.30943 | 0.70105 | 37.48 | 0.000 | 97.12 |
Linear | 3 | 1.82418 | 0.60806 | 32.51 | 0.000 | |
Square | 3 | 4.36800 | 1.45600 | 77.85 | 0.000 | |
Interaction | 3 | 0.11725 | 0.03908 | 2.09 | 0.165 | |
Residual error | 10 | 0.18702 | 0.01870 | |||
Pure error | 5 | 0.00508 | 0.00102 | |||
Total | 19 | 6.49645 |
df: degrees of freedom; SS: sum of squares; MS: mean sum of squares.
Further, to determine significance of the regression coefficients in the two models, the results were subjected to Student’s
Result of Student’s
Term | CMCase activity (U/mL) | Cell growth (g/L) | ||
---|---|---|---|---|
|
|
|
| |
Constant | 28.927 | 0.000 | 57.032 | 0.000 |
pH ( |
4.229 | 0.002 | 3.518 | 0.006 |
Temperature (°C) ( |
8.712 | 0.000 | 4.355 | 0.001 |
Agitation (rpm) ( |
−4.341 | 0.001 | 8.136 | 0.000 |
( |
−0.192 | 0.852 | −8.511 | 0.000 |
( |
−7.938 | 0.000 | −13.026 | 0.000 |
( |
−0.728 | 0.483 | −5.272 | 0.000 |
|
−1.848 | 0.094 | −0.207 | 0.840 |
|
0.387 | 0.707 | 0.259 | 0.801 |
|
−3.332 | 0.008 | 2.482 | 0.032 |
In order to determine the optimal levels of the variables for maximum CMCase activity, three dimensional response surface plots as shown in Figure
Three dimensional response surface plots for cellulase production showing the interaction effect between (a) temperature and agitation, (b) pH and temperature, and (c) pH and agitation.
To illustrate the above mentioned interaction effect between the variables in the study, typical contour plots between temperature and agitation speed and that between pH and temperature are depicted in bottom of the response surface plots of Figures
Agitation speed is one of the important culture parameters that maintains homogenous conditions and disperses dissolved oxygen into smaller bubble thereby increasing the interfacial area and oxygen mass transfer rate for enhancing both substrate utilization and microbial activity [
Temperature is also one of the most important parameters that influences enzyme activity and is essential for a fermentation process [
The pH of the growth medium influences many enzymatic reactions by affecting the transport of chemical products and enzymes across the cell membrane [
The maximum CMCase activity obtained using the optimized physical process parameters was 0.57 U/mL which was higher than many other reported values. For example,
In order to determine the optimal levels of each variable for maximizing CMCase activity, the method of desirability function was applied. The desirability function study in this multiple response optimization method shown in Figure
Desirability function plot showing the optimum level of physical process parameters.
The CMCase activity was experimentally verified in batch shake flask and at 2 L stirred tank fermentor using optimized medium [
CMCase production at different levels of optimization.
Process conditions | Level of scale | CMCase activity (U/mL) | Enhanced production (fold) |
---|---|---|---|
Without any optimization | Shake flask | 0.07 ± 0.02 | — |
With only optimised medium | Shake flask | 0.43 ± 0.04 | 6 |
With optimised medium + optimised physical parameters | Shake flask | 0.57 ± 0.01 | 8 |
With optimised medium + optimised physical parameters | Bioreactor | 0.75 ± 0.03 | 11 |
Values are mean ± SE (
Cellulase production, cell growth, and pH profile of
The consequences of alkali (NaOH) and acid-acetone pretreatments were evaluated on wild grass (1%, w/v) subjected to simultaneous saccharification by
Bioethanol production by SSF from wild grass employing
Pretreatment | Substrate concentration (%, w/v) | Mode of SSF | SSF process parameters | ||
---|---|---|---|---|---|
Reducing sugar* (g/L) | Ethanol yield (g/g) | Ethanol titre* (g/L) | |||
Acid acetone | 1% | Shake flask | 1.10 ± 0.07 | 0.093 | 0.93 ± 0.07 |
Alkali | 1% | Shake flask | 1.25 ± 0.04 | 0.098 | 0.98 ± 0.06 |
Alkali | 1% | Bioreactor | 2.51 ± 0.03 | 0.22 | 2.23 ± 0.08 |
Alkali | 5% | Shake flask | 9.08 ± 0.06 | 0.15 | 7.56 ± 0.05 |
Alkali | 5% | Bioreactor | 13.22 ± 0.04 | 0.23 | 11.65 ± 0.04 |
Values are mean ± SE (
On the basis of ethanol titre (g/L) obtained from SSF experiments involving two pretreatments, alkali (NaOH) treatment was found to be better over acid-acetone technique. Consequently, on increasing the wild grass concentration to 5% (w/v), an ethanol concentration of 7.56 g/L was achieved from a reducing sugar content of 9.08 g/L (Table
Owing to the controlled conditions of pH and aeration in batch reactor SSF, using 1% (w/v) wild grass with same enzyme and microbial combination, an ethanol titre of 2.23 g/L was obtained from a reducing sugar concentration of 2.5 g/L. The ethanol yield (g of ethanol/g of substrate) was 0.46 (Table
Simultaneous saccharification and fermentation (SSF) profile of wild grass (
All these values of ethanol production are comparable with the other reported literature. An ethanol concentration of 0.09 g/L was obtained from 1% paper sludge waste using
The results demonstrated the effect of physical parameters such as initial pH, agitation (rpm), and temperature (°C) for cellulase production from
The research work was supported by Joint Project Grant from Department of Biotechnology, Ministry of Science and Technology, Government of India, to D. Goyal and A. Goyal.