Energy & Environmental Science

Pretreatment optimization using Box Behnken design and

process validation for bioethanol production

Journal: Energy & Environmental Science

Manuscript ID EE-ART-04-2017-000984

Article Type: Paper

Date Submitted by the Author: 11-Apr-2017

Complete List of Authors: Selvaraju, Sivamani; Kumaraguru College of Technology, Biotechnology

Chandrasekaran, Arun Pandian; Kumaraguru College of Technology,
Biotechnology
Baskar, Rajoo; Kongu Engineering College, Food Technology
Page 1 of 20 Energy & Environmental Science

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 Energy & Environmental Science Page 2 of 20

Pretreatment optimization using Box Behnken design and process validation

for bioethanol production

Sivamani Selvarajua*, Arun Pandian Chandrasekarana, Baskar Rajoob

a
Chemical Engineering Laboratory, Department of Biotechnology, Kumaraguru College of

Technology, Chinnavedampatti, Coimbatore-641 049, Tamil Nadu, India

b
Department of Chemical Engineering, Kongu Engineering College, Perundurai, Erode-638

052, Tamil Nadu, India

* Correspondence should be addressed to:

Email: sivamani.s.bt@kct.ac.in

Mobile: +91 9486159667

Fax: +91 4221669406

Page 3 of 20 Energy & Environmental Science

Abstract

Cassava stem is generally considered as an agricultural waste and was found to contain 25%

and 40% of glucan and xylan respectively on a dry solid basis. Bioethanol is produced from

cassava stem by dilute acid pretreatment followed by alkali delignification, cellulase

digestion and fermentation by Fusarium oxysporum are reported. Dilute acid pretreatment

was optimized by response surface methodology based on Box-Behnken design. The

optimum xylose concentration obtained by the experiment was 8.79 g/L and the model was

8.64 g/L at solid to liquid ratio of 0.20 g/mL, dilute sulfuric acid concentration of 1.88% v/v

and reaction time of 68 min. After alkali delignification and cellulase hydrolysis of pretreated

cassava stem, the glucose content of 2.62 g/L was obtained and the bioethanol yield of 1.13

g/L by F. oxysporum was achieved. The above results indicated that the cassava stem can be

used as a potential substrate for bioethanol production.

Keywords: Cassava stem, Bioethanol, Dilute acid pretreatment, Delignification, Cellulase,

Fusarium oxysporum,
 Energy & Environmental Science Page 4 of 20

1. Introduction

Fossil fuels account for over 80.3% of the primary energy consumed and 57.7% of that

amount has been utilized in the transport and power sectors around the globe 1. For the past

few years, there has been an increasing interest among researchers to find an alternative

proposal for the fossil fuels to meet the future needs. Bioethanol has many advantages over

fossil fuels and helps in making a smoother transition from a petroleum-based product to a

bio-based sustainable and environment friendly fuel 2,3. Most of the industries are looking for

renewable energies to replace petroleum-based feedstocks. It is mainly because of the less

CO2 emissions and it is readily absorbed by the plants through photosynthesis. Thus, overall

emission of such gases will be considerably reduced and results in the reduction of global

warming.

Bioethanol production is generally obtained from agricultural products such as starchy

biomass, starch hydrolysis and sugar fermentation or by direct-fermentation of sugars from

molasses or sugar beet. Initially, bioethanol was produced from starch or sugar-based sources

which are referred as first generation of biofuels. Second generation biofuels are derived from

non-food plant sources like woody biomass and plant debris which are known as

lignocellulose materials. These non-edible based biofuel production are being developed

rapidly so that large-scale production will hit the market soon 4. The production of bioethanol

from the conversion of lignocellulosic biomass has received the considerable attention

because of its potential for producing a low cost fuel 5.

Previously, we successfully characterized the cassava stem and unraveled its potential role

for the production of bioethanol by combined acid pretreatment 6. Thus, in this study, cassava

stems were used as a substrate for the bioethanol production. Cassava (Manihot esculenta
Page 5 of 20 Energy & Environmental Science

Crantz) is the most important root crop in the tropics and ranks third after rice and corn as
7,8
calorie source for human consumption . Traditionally, cassava is propagated vegetatively

using 15-30 cm cassava stem with 7-8 viable nodes. Rapid propagation techniques are

already available; one stem cutting will yield 10 planting materials within a time span of 10-

12 months 9. The above ground biomass of cassava, includes stem, is not utilized for practical

purposes 10, other than being a primary source of planting material. The average composition

of cassava stem is hemicellulose 12% (xylan), cellulose 21% (glucan), lignin 23%, crude

protein 2.7% and starch 8.4% 11. It is noteworthy that the pretreatment involves the removal

of hemicellulose from lignocellulosic complex matrix. This paper focuses only on the

optimization of the pretreatment process of cassava stem by autoclaving using response

surface methodology (RSM).

RSM is a commonly used method to evaluate the optimal conditions and also an efficient

statistical technique for optimization of multiple variables with minimum number of

experiments. This method has been successfully applied to optimize alcoholic fermentation
1215
process . Box Behnken (BBD) is a three-level factorial design, which allows the

estimation and interpretation of interactions between variables at a time during the

optimization process 16.

An effective process of converting lignocelluloses to bioethanol uses one or two organisms to

carry out simultaneous hydrolysis and fermentation (SHF). Previously, it was reported that

the F. oxysporum has the ability to convert cellulose to ethanol directly with promising yields
17,18
. Xylanase produced by F.oxysporum have also been characterized and moreover it

produces enough -glucosidase activity to prevent cellobiose inhibition during hydrolysis

19,20
. Thus, we utilized F. oxysporum for the fermentation of pretreated substrate for the
 Energy & Environmental Science Page 6 of 20

production of bioethanol. The overall process of the bioethanol production is depicted in Fig.

1. The objectives of this study are: (i) to evaluate the suitability of cassava stem for the

bioethanol production by SHF; (ii) to optimize the process parameters of dilute acid

pretreatment by BBD; and (iii) to develop a mathematical model to predict xylose recovery

from cassava stem based on BBD.

2. Materials and Methods

2.1 Collection and preparation of cassava stem

Cassava stem samples were collected from agricultural fields of Namakkal district, Tamil

Nadu. The collected material was dried in hot air oven at 105C until constant weight is

obtained. The material was grounded and sieved to obtain particle size of 212 m. Finally,

the material was stored in sealed plastic bags and placed in incubator maintained at 4C until

use. All chemicals used in this study were of analytical grade and procured from Sigma

Aldrich.

2.2 Inoculum preparation

For 100 mL of potato sucrose broth, 20 g of scrubbed potato and 2 g of sucrose were used.

Duplicate sample of inoculum medium was prepared in 500 mL Erlenmeyer flasks (pH 6.5)

and autoclaved at 121C for 15 minutes after closing the flask with cotton plug. The flasks

were placed in rotary shaker with 120 rpm at 300.5C.

2.3 Dilute acid pretreatment and delignification

The sulfuric acid of 0.3-2.7% (v/v) concentration at solid to liquid ratio of 0.05-0.20 g/mL

was used to pretreat 1 g sample of cassava stem. The treatments were performed at 121C for

a reaction time of 40-120 min and the coded values were noted in Table 1. The pretreatment
Page 7 of 20 Energy & Environmental Science

conditions of dilute acid concentration, solid to liquid ratio and reaction time were optimized

by using RSM as described in Table 2. The acid pretreated sample was washed with distilled

water to remove excess acid and it solubilizes the sugars present in the solution. Then, the

sample was dried in hot air oven at 105C. The filtrate of each sample was centrifuged at

10,000 rpm for 10 minutes and the sample was used for xylose analysis and was analyzed by
21
dinitrosalicylic acid (DNS) assay . Finally, the acid pretreated sample was delignified by

soaking in 1.5% (w/v) sodium hydroxide solution (1 g/10 mL) for 3 h.

2.4 Enzymatic hydrolysis and fermentation

The Cellulase from Trichoderma reesei was purchased from Sigma Aldrich and its enzymatic

activity was measured according to the mentioned protocol 22. The activity of cellulase was

determined to be 72 FPU/mL. The enzymatic hydrolysis was carried out in 250 mL reagent

bottles containing 1 g of substrate and 100 mL of 50 mM acetate buffer (pH 5). Cellulase

concentration of 0.5 mL/g substrate was loaded in each sample bottle without shaking at

50C for 30 minutes. Then, the bottle was heated in boiling water bath for 10 minutes and

centrifuged at 10,000 rpm for 5 minutes after cooling. The total reducing sugars

concentration was determined by 3, 5 dinitrosalicylic acid assay 21. Fermentation was carried

out in duplicate by adding 10% (v/v) of F. oxysporum inoculum in 250 mL Erlenmeyer flask.

After fermentation at 30C for 72 h, the sample was collected for ethanol determination.

2.5 Experimental design and statistical analysis

The BBD generated 15 experiments for the optimization of the parameters (Table 1). The

selected dependent variable was xylose concentration and the independent variables were

reaction time, solid liquid ratio and dilute acid concentration. The statistical significance was

determined by the F-test and the significance of response was determined by using Students
 Energy & Environmental Science Page 8 of 20

t-test. The coefficients of the equation and analysis of variance (ANOVA) were determined

by employing Design-Expert (version 8.0.7). The response of dilute acid concentration, solid

to liquid ratio and reaction time were explained in quadratic regression model and expressed

by the second order polynomial as follow:

=  +
   +
    +

  (1)

whereas Y is the value of the response, X is the coded value of the factors; where i and j are

linear and quadratic coefficients, respectively; and b is a regression coefficient. Significance

of the design was determined by coefficient of determination R2. The response surface

equation was optimized for the maximum xylose concentration. Three dimensional surface

plots were used to determine the effect of the three parameter levels and their interactions on

the xylose concentration (Table 3).

2.6 Analytical methods

Cassava stem was characterized by adopting standard operating procedures. Cassava stem
23 23 23 24
was quantitatively analyzed for cellulose , hemicellulose , lignin , protein , moisture
26 27
(International standard: ISO 1741, 2010), total nitrogen , fiber and ash (International

standard: ISO 3593, 2010). Ethanol concentration was determined by dichromate method 29.

3. Results

3.1 Characterization of cassava stem

The average composition of cassava stem was 25.18% glucan, 40.02% xylan, 16.98% lignin,

9.36% protein, 6.72% moisture, 1.47% total nitrogen, 29.48% fiber, and 5.23% ash content. It

is noted that the cassava stem residue is rich in lignocellulose which makes it suitable for

ethanol production.
Page 9 of 20 Energy & Environmental Science

3.2 Optimization of dilute acid pretreatment by BBD

BBD was adopted to evaluate the optimum conditions of dilute acid pretreatment for the

conversion of lignocellulosic biomass to ethanol. The results of BBD experiments for

studying the effect of three independent variables were presented along with the observed and

the predicted xylose concentrations (Table 2). In this study, BBD was used to evaluate the

main and interaction effects of the factors on xylose concentration 30.

The equation that relates the xylose concentration as the dependent variable (Y, g/mL) to

other significant terms, as listed in Table 1 can be expressed as follows:

= 3.0604 + 1.6149 51.11149 + 0.22878! 0.48326 0.00928!

0.19142! 0.23579 + 197.45892  0.00127!  (2)

where Y is xylose concentration, A is dilute sulfuric acid concentration, S is solid to liquid

ratio, and T is reaction time.

ANOVA of the quadratic equation for xylose concentration was summarized in Table 3.

ANOVA of regression model demonstrates that the model is highly significant and is evident

from the Fishers F-test with low probability value [(p model > F)]. The p-value denoting the

significance of the coefficients were also important in understanding the pattern of the mutual

interactions between the variables. The goodness of the fit of the model can be checked by

the determination coefficient R2. The values of R2, predicted R2 and adjusted R2 for cassava

stem are 0.997, 0.953, and 0.992 respectively. The goodness of fit shows a high correlation

between the observed values and the predicted values. This means that the regression model

provides an excellent explanation of the relationship between the independent variables

(factors) and the response (xylose concentration). No abnormality was observed from the

diagnoses of residuals. Thus, it can be concluded that the model was statistically significant
 Energy & Environmental Science Page 10 of 20

and the interactions between the three factors were also significant (Table 3). The exception

in the interaction between dilute sulfuric acid concentration and solid to liquid ratio was non-

significant (Table 3). Table 4 summarizes regression parameters used in the xylose

concentration model. As mentioned in Table 3, linear terms (A, S and T), square terms (A2,

S2 and T2), and two-way interaction terms (AS, AT and ST) are the major factors, with p-

values under = 0.05, which is significantly affecting the xylose concentration.

The graphical representation of the regression equation (2) was presented in Fig. 2(a), 2(b)

and 2(c). The response surface model was used to predict the result by three dimensional

surface plots. Fig. 2(a) shows a plot at varying solid to liquid ratio and dilute sulfuric acid

concentration at fixed reaction time. Dilute sulfuric acid concentration has a profound effect

on xylose concentration, since dilute sulfuric acid is a catalyst. Xylose concentration is less at

low dilute sulfuric acid concentration and increases with increase in dilute sulfuric acid

concentration up to 1.88% (v/v) and then decreases.

Fig. 2(b) represents a plot at varying dilute sulfuric acid concentration and reaction time, at

fixed solid to liquid ratio. Xylose concentration increases with increase in reaction time up to

a certain extent (68 min) and then decreases. However, a high reaction time can adversely

affect xylose concentration due to the fact that high increase in reaction time increases the

release of inhibitors 31. Fig. 2(c) illustrates a graphical plot at varying reaction time and solid

to liquid ratio with fixed dilute sulfuric acid concentration. Solid to liquid ratio is an

important factor in pretreatment process; xylose concentration is high at two extremes of

solid to liquid ratio of 0.05 and 0.20 g/mL. Xylose concentration decreases with increase in

solid to liquid ratio and further increases in solid to liquid ratio increases xylose

concentration. The developed model was verified by performing trial under optimum

conditions. A high value of coefficient of determination (R2 = 0.997) showed that the model

was successful in predicting xylose concentration.

Page 11 of 20 Energy & Environmental Science

3.3 Enzymatic hydrolysis and ethanol fermentation

After dilute acid pretreatment under optimum conditions, the sample was delignified by

soaking in 1.5% (w/v) sodium hydroxide solution (1 g/10 mL) for 3 h. Delignification

process removed 60.59% of the lignin from the cassava stem. After delignification, the

remaining residue of cellulose present in cassava stem was hydrolyzed by cellulase. The

glucose concentration after enzymatic hydrolysis was 2.621 g/L. The concentration of ethanol

yielded after fermentation by Fusarium oxysporum was 1.126 g/L.

4. Discussion

The present study showed the significance of optimization studies to improve sugar yield for

bioethanol production. A similar research was carried out by a group on cassava stem 11. In

that work, the cassava stem was impregnated in 0.1 M sulfuric acid. They utilized the acid

treated substrate for 30 min at 135C under the pressure of 15 lb/in2 for the pretreatment. The

enzymatic solution containing cellulase, pectinase, and xylanase were employed to perform

the enzymatic hydrolysis at 50 C for 24 h. The above conditions yielded 0.57 g/g of sugar.

They also carried out separate hydrolysis and fermentation (SHF) and simultaneous

saccharification and fermentation (SSF) to obtained maximum ethanol yield. The

fermentation was carried out by S. cerevisiae KM1195 and yielded 98.43% in SHF and

95.29% in SSF 11. Han et al. performed RSM to optimize the pretreatment conditions include

time, temperature, and acid concentration for cassava stem 32. As a result, they have obtained

the optimum conditions of 10 min, 177 C and 0.14 M for time, temperature, and acid

concentration, respectively. Cellulase and -glucosidase were used as enzymes to digest

pretreated cassava stem. The resulted hydrolysate was fermented using S. cerevisiae and 7.55

g/L of final ethanol concentration was obtained. Castano et al. executed the RSM to optimize

the SSF of cassava stem. The final ethanol concentration of 1.880.04% v/v was obtained

utilizing an inoculum concentration of 1.59 g/L and an enzyme concentration of 13.3 FPU/g
 Energy & Environmental Science Page 12 of 20

33
. The current research also emphasized on the RSM to investigate the optimal conditions for

the pretreatment process. Our future work will focus on the optimization of the enzymatic

hydrolysis to abridge the utilization of commercial enzymes and reduce the overall cost of the

bioethanol production.

5. Conclusion

The overall xylose concentration was significantly improved by optimizing the pretreatment

parameters and important parameters are solid to liquid ratio, dilute sulfuric acid

concentration and reaction time. BBD was used to identify the optimal concentrations of

these parameters that can result in optimal xylose yield. The optimum xylose concentration of

8.637 g/L was obtained by solving the equation whereas the experimental value is 8.7995 g/L

for the solid to liquid ratio of 0.20 g/mL, dilute sulfuric acid concentration of 1.88% v/v and

reaction time of 68 min. The glucose content of 2.621 g/L was attained after delignification

and enzymatic hydrolysis and the ethanol conversion yield by F. oxysporum was 1.126 g/L

was achieved. Thus, the mathematical model fits well with the experiment data and

incorporating the optimized process conditions that helped us to improve the xylose

concentration from cassava stem for the production of bioethanol.

Acknowledgements
The authors gratefully acknowledge the support of the management of the Kumaraguru
College of Technology and the Department of Biotechnology for the support and guidance.
Page 13 of 20 Energy & Environmental Science

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Page 15 of 20 Energy & Environmental Science

Figure legends

Fig. 1 Overview of the bioethanol production from cassava stem. (a) Cassava stem was
collected from agricultural field and subjected to grounding followed by sieving to obtain
homogeneous size. (b) The lignocellulosic nature of cassava stem was treated with dilute acid
pretreatment to separate cellulo-lignin and hemicellulose. Then, hydrolysate was obtained by
performing delignification (using NaOH) of the samples. Finally, enzymatic hydrolysis was
done using cellulase for the production of bioethanol. (c) RSM based BBD was performed to
optimize the pretreatment conditions.

Fig. 2 Surface plots showing the effect of sulfuric acid concentration, solid to liquid ratio and
time on xylose production.
 Energy & Environmental Science Page 16 of 20

Table 1. Independent variables and levels used for BBD

Levels
Variable Symbol Unit
-1 0 +1

Sulfuric acid concentration A % v/v 0.3 1.5 2.7

Solid to liquid ratio S g/mL 0.05 0.125 0.20

Time T min 40 80 120

Table 2. BBD matrix for optimization of parameters

Xylose concentration (g/L)

Std Run A (%v/v) S (g/mL) T (min)
Experimental Predicted

2 1 1.5 0.05 120 9.256 9.153

4 2 1.5 0.125 80 8.705 8.683

9 3 2.7 0.125 120 5.332 5.475

8 4 1.5 0.2 120 5.468 5.337

12 5 0.3 0.125 120 6.023 6.115

3 6 2.7 0.125 40 7.481 7.390

6 7 1.5 0.125 80 8.629 8.683

5 8 0.3 0.05 80 10.608 10.619

10 9 1.5 0.125 80 8.716 8.683

13 10 2.7 0.05 80 10.997 10.957

11 11 0.3 0.2 80 7.999 8.038

1 12 1.5 0.05 40 8.897 9.029

15 13 2.7 0.2 80 8.214 8.202

14 14 1.5 0.2 40 7.406 7.508

7 15 0.3 0.125 40 6.39 6.247

Page 17 of 20 Energy & Environmental Science

Table 3. Analysis of variance for selected BBD

Source Sum of Squares df Mean Square F value Prob > F

Model 40.14691 9 4.460768 184.2819 < 0.0001

A 0.125995 1 0.125995 5.205074 0.0714

S 14.23374 1 14.23374 588.0199 < 0.0001

T 2.096128 1 2.096128 86.59461 0.0002

AxS 0.007567 1 0.007567 0.312591 0.6002

AxT 0.793881 1 0.793881 32.79657 0.0023

SxT 1.319052 1 1.319052 54.49228 0.0007

A2 0.425686 1 0.425686 17.58579 0.0085

S2 4.555085 1 4.555085 188.1783 < 0.0001

T2 15.32511 1 15.32511 633.1061 < 0.0001

Residual 0.121031 5 0.024206

Lack of Fit 0.116542 3 0.038847 17.30914 0.0551

Pure Error 0.004489 2 0.002244

Cor Total 40.26794 14

Table 4. Regression coefficients and significance of response surface quadratic model

developed by BBD

Coefficient 95% CI 95% CI

Factor df Standard Error VIF
Estimate Low High

Intercept 8.683333 1 0.089826 8.452428 8.914239

A 0.125497 1 0.055007 -0.0159 0.266897 1

S -1.33387 1 0.055007 -1.47527 -1.19247 1

T -0.51188 1 0.055007 -0.65328 -0.37047 1

 Energy & Environmental Science Page 18 of 20

AxS -0.04349 1 0.077792 -0.24346 0.156477 1

AxT -0.4455 1 0.077792 -0.64547 -0.24553 1

SxT -0.57425 1 0.077792 -0.77422 -0.37428 1

A2 -0.33954 1 0.080968 -0.54768 -0.13141 1.011111

S2 1.110706 1 0.080968 0.902571 1.318842 1.011111

T2 -2.03729 1 0.080968 -2.24543 -1.82915 1.011111

Page 19 of 20 Energy & Environmental Science

Overview of the bioethanol production from cassava stem. (a) Cassava stem was collected from agricultural
field and subjected to grounding followed by sieving to obtain homogeneous size. (b) The lignocellulosic
nature of cassava stem was treated with dilute acid pretreatment to separate cellulo-lignin and
hemicellulose. Then, hydrolysate was obtained by performing delignification (using NaOH) of the samples.
Finally, enzymatic hydrolysis was done using cellulase for the production of bioethanol. (c) RSM based BBD
was performed to optimize the pretreatment conditions
 Energy & Environmental Science Page 20 of 20

Surface plots showing the effect of sulfuric acid concentration, solid to liquid ratio and time on xylose
production

301x167mm (96 x 96 DPI)