Production of Bioethanol from Cassava stem var.

YTP1
by Consortia-mediated Bioprocessing

Presented By Under the guidance of

Ms.S.Kavitha Mr. S. Sivamani
Roll no:13MBT06 Assistant Professor (SRG)

Department of biotechnology

Affiliated to Anna University

Chennai-25

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 INTRODUCTION
Bioethanol is one of the widely used liquid biofuels. It does not
cause any environmental hazards. It can be produced from different
types of raw materials like starchy, sugary, lignocellulosic and algal
biomass.
Lignocellulosic feedstock is considered as an attractive raw
material not only for the liquid transportation fuel but also for the
production of biochemicals and precursors.
Cassava stem, one of the important agro-residues in India, is rich
in carbohydrate (cellulose and hemicellulose). Hence, it can be used
for bioethanol production.
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 OBJECTIVES

 To characterize stem of cassava var. YTP1 for its biochemical

parameters
 To formulate and optimize the production medium for
maximum growth of Cellulomonas fimi MTCC24 and
maximum cellulase activity
 To produce ethanol by consortia mediated bioprocessing.

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 OVERVIEW OF WORK

Cassava stem Biochemical characterization

Formulation and optimization of

production medium

Production of ethanol by
consortia mediated
bioprocessing (CMP)

Ethanol

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 REVIEW OF LITERATURE

 Han et al., (2011) revealed Bioethanol production from optimized

pretreatment of cassava stem.
 Magesh et al.,(2011) studied Simultaneous saccharification and
fermentation of cassava stem var. 226 white rose to ethanol by cellulase
enzyme and Saccharomyces cerevisiae.
 Sovorawet and Kongkiattikajorn (2012) compared Bioethanol Production
from Cassava Peels by Monoculture and Co-Culture of Yeast
 Nuwamanya et al., (2012) produced Bioethanol from non-food parts of
cassava (Manihot esculenta crantz).
 Ali et al., (2013) investigated Cassava pulp as a biofuel feedstock of an
enzymatic hydrolysis process.a
02/10/2024 5
 MATERIALS AND METHODS

1. Sample collection
2. Biochemical characterization of cassava stem
Cellulose, hemicellulose, starch, soluble sugars,
pectin,protein, nitrogen, lignin, lipids, moisture,
crude fibre and ash were estimated
biochemically using standard operating methods.

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 MATERIALS AND METHODS

3. Formulation and optimization of production

medium for maximum cellulase activity:
One-factor-at-a-time (OFAT) method to study the effect of
carbon sources, nitrogen sources and minerals

Placket-Burman design (PBD)

Box-Behnken design (BBD)

Genetic algorithm (GA)

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 Contd..
4. Optimization of non-nutritional factors for maximum
ethanol production by CMP
One-factor-at-a-time (OFAT) method to study the effect of:

pH

Temperature

Inoculam size

Agitation speed

Substrate concentration
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 Results
 From OFAT method 11 medium components were screened
based on the cellulase activiy.
 Among 11 factors, pretreated cassava stem, malt extract,
Ammonium chloride and sodium chloride into the culture
medium were selected due to significant positive effect on
cellulase yield.
 From Box - Behnken design, the media formulations were
optimized having the factors such as pretreated cassava stem
3.07g/L, malt extract 2.99 g/L, Ammonium chloride 2.81g/L
and NaCl 2.49g/L.
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Figure 1.Biochemical characterization of stem of cassava var.YTP1

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 One-factor-at-a-time method
Figure 2.Effect of different concentration of PCS

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 Effect of nitrogen sources

Figure 3.Organic nitrogen sources Figure 4.Inorganic nitrogen sources

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 Effect of minerals

Figure 5.Effect of macronutrients Figure 6.Effect of micronutrients

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 Table 1.Media components and their levels used in
PBD
Low (-1) High (+1)
Media components
(g/L) (g/L)
A Pretreated cassava stem (PCS) 3 7
B Malt extract 1 3
C Ammonium chloride 1 3
D Ammonium nitrate 1 3
E Ammonium phosphate 1 3
F CaCl2 1 3
G MgSO4.7H2O 1 3
H NaCl 1 3
I MnSO4 0.05 0.15
J FeSO4 0.05 0.15
K Na2MoO4 0.05 0.15
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 Table 2.Plackett- Burman design for screening of
factors
for
A cellulase
B C Dproduction
E F G H I J K Cellulase activity
(IU/ml)
1 +1 -1 +1 -1 +1 -1 +1 -1 +1 -1 +1 3.64±0.764

2 +1 +1 -1 +1 -1 +1 -1 +1 -1 +1 -1 10.31±0.63

3 -1 +1 +1 -1 +1 -1 +1 -1 +1 -1 +1 9.801±0.74

4 +1 -1 +1 +1 -1 +1 -1 +1 -1 +1 -1 5.064±0.458

5 -1 +1 -1 +1 +1 -1 +1 -1 +1 -1 +1 7.9±0.37

6 +1 -1 +1 -1 +1 +1 -1 +1 -1 +1 -1 5.792±0.201

7 -1 +1 -1 +1 -1 +1 +1 -1 +1 -1 +1 9.691±0.27

8 +1 -1 +1 -1 +1 -1 +1 +1 -1 +1 -1 3.49±0.179

9 -1 +1 -1 +1 -1 +1 -1 +1 +1 -1 +1 9.654±0.58

10 +1 -1 +1 -1 +1 -1 +1 -1 +1 +1 -1 4.189±0.87

11 -1 +1 -1 +1 -1 +1 -1 +1 -1 +1 +1 9.181±0.581

12 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 3.57±0.664

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 Table 3.ANOVA table for PBD
Co-
Source df efficient Seq SS Adj SS Adj MS F P
Main 11 0.049 0.0238 0.0238 0.0022 * *
A 1 0.024 0.001 0.0013 0.0013 * *
B 1 0.077 0.004 0.0014 0.0143 * *
C 1 0.047 0.001 0.005 0.0053 * *
D 1 -0.025 0.00075 0.0015 0.0015 * *
E 1 -0.045 0.0083 0.0047 0.0048 * *
F 1 -0.022 0.001 0.0011 0.0011 * *
G 1 -0.024 0.0008 0.0013 0.0014 * *
H 1 -0.037 0.00086 0.00321 0.0032 * *
I 1 -0.027 0.002 0.0017 0.00176 * *
J 1 0.014 0.0001 0.00046 0.00046 * *
K 1 0.023 0.001 0.00131 0.00131 * *

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 Box- Benhken design
Table 4.Ranges of variables used in RSM
Levels(g/L)
Variables Code
-1 0 +1
Pretreated
X1 3 5 7
cassava stem
Malt extract X2 1 2 3
Ammonium
X3 1 2 3
chloride
Sodium
X4 1 2 3
chloride

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Table 5. Box-Behnken design of factors in coded levels with cellulase activities
as responses
Run X1 X2 X3 X4 Cellulase activity (IU/ml)
Experimental Predicted
1 7 1 2 2 2.26 1.99
2 5 2 3 3 5.54 5.49
3 7 3 2 2 4.88 4.46
4 3 1 2 2 1.38 1.73
5 5 2 2 2 2.33 2.78
6 5 2 2 2 3.42 2.78
7 5 2 2 2 2.26 2.78
8 5 2 2 2 2.62 2.78
9 5 2 2 2 3.28 2.78
10 5 2 2 2 5.54 5.73
11 3 3 2 2 4.37 4.35
12 5 2 1 1 6.12 5.55
13 5 3 3 2 3.79 3.36
14 3 2 2 1 4.17 3.49
15 3 2 2 3 5.61 5.23
16 5 3 1 2 2.33 2.28
17 5 1 1 2 2.26 2.02
18 5 1 3 2 3.72 3.89
19 3 2 3 2 2.84 3.24
20 3 2 1 2 3.01 2.55
21 5 2 1 3 1.09 1.48
22 5 2 3 1 4.15 3.96
23 7 2 2 3 1.82 1.88
24 7 2 2 1 2.26 2.71
25 5 1 2 3 4.72 5.65
26 5 3 2 3 2.84 3.36
27 7 2 1 2 4.59 4.84
28 5 3 2 1 2.48 2.77
29 7 2 3 2 1.55 1.31
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Table 6.ANOVA table for Box-Benhken design
p-value (Prob >
Source Sum of squares Df Mean square F-value
F)
Model 49.22233 14 3.51588 10.0652 < 0.0001
A 0.753868 1 0.753868 2.15816 0.1639
B 31.41308 1 31.41308 89.92879 < 0.0001
C 0.003282 1 0.003282 0.009396 0.9242
D 3.664132 1 3.664132 10.48961 0.0059
AB 0.585395 1 0.585395 1.675857 0.2164
AC 0.383626 1 0.383626 1.098238 0.3124
AD 0.947972 1 0.947972 2.713838 0.1217
BC 0.084955 1 0.084955 0.243208 0.6295
BD 0.084956 1 0.084956 0.243211 0.6295
CD 8.436912 1 8.436912 24.15304 0.0002
A^2 0.095081 1 0.095081 0.272197 0.6100
B^2 2.136961 1 2.136961 6.117653 0.0268
C^2 1.10394 1 1.10394 3.160338 0.0972
D^2 0.470503 1 0.470503 1.346947 0.2652
Residual 4.890348 14 0.349311
Lack of Fit 3.728585 10 0.372859 1.283768 0.4363
Pure Error 1.161763 4 0.290441
Cor
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Figure 7.Three-dimentional response surface plot for
cellulase production (interactive effects of variables)

7(a) 7(b)

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 7(d)
7(e)

02/10/2024 7(f) 21
Table 8.Optimized medium composition & Cellulase
activity as response

Medium Composition (g/L) Cellulase activity

components (IU/ml)
Pretreated cassava 3.07
stem
Malt extract 2.99
4.69±0.0072
Ammonium 2.81
chloride
Sodium chloride 2.49

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 Genetic algorithm
Table 9.Optimum value
Medium components Composition (g/L)
Pretreated cassava stem 3.105
Malt extract 3
Ammonium chloride 2.998
Sodium chloride 2.993
Best8.fitness
Figure graph
Best fitness curve

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 Figure 9.Growth curve

0.2

0.18

0.16

0.14
Biomass (OD600)

0.12

0.1 Consortium C.fimi

0.08
Z.mobilis
0.06

0.04

0.02

0
1 2 3 4 5 6 7 8 9

Time (hr)

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 Optimization of non-nutritional factors

Figure 10. Effect of pH Figure 11.Effect of temperature

10
14

9
12
8

10 7

Ethanol production (g/L)

Ethanol production (g/L)

6
8
5

6 4

3
4
2

2
1

0
0 25 28 31 34 37
3 5 7 9 11
02/10/2024 Temperature (°C) 25
pH
 Figure 12. Effect of agitation speed Figure 13. Effect of inoculam size

12
16

10
14

12
8
Ethanol production(g/L)

Ethanol production (g/L)

10
6
8

4
6

4
2

2
0
0 25 50 75 100
0
Agitation speed (rpm) 2 4 6 8 10

02/10/2024 Inoculam size (%) 26

 Figure 14. Effect of substrate
concentration Figure 15. Effect of time interval

14
8

12
7

6 10

Ethanol production (g/L)

Ethanol production (g/L)

5 8

4
6

3
4

2
2
1

0
0 4 8 12 16 20 24
2g 4g 6g 8g 10 g
Time (hr)
Substrate concentration (g/L)
02/10/2024 27
 Discussion
 Han et al., (2011) revealed the chemical composition of the cassava
stem varies according to the growing location, season, harvesting
methods and analysis procedure. Sovorawet and Kongkiattikajorn
(2012) proposed cellulose content of cassava stalk was relatively
high compared to that of hemicellulose and cassava stalk could be a
good source of cellulose.
 The parallel study of medium optimization for cellulase production
by C.fimi NCIM-5015 reported in Ali et al., 2013.
 Magesh et al., (2011) reported the optimum values of particle size
(30 mesh), Substarte concentration(50g/L),pH (5.0) and temperature
(35°C) with the maximum ethanol concentration of 13.6 g/L.
02/10/2024 28
 Conclusion
Cassava stem contains 63% cellulose which makes it suitable
for bioethanol production.
The media components and composition were optimized at
pretreated cassava stem 3.07 g/L, malt extract 2.99 g/L,
Ammonium chloride 2.81 g/L and NaCl 2.49 g/L and
maximum cellulase activity of 5.96 IU/ml was obtained.
From consortia-mediated bioprocessing, the maximum ethanol
concentration of 12.5 g/L was achieved at 3 pH, 2% inoculum
level, 34°C, 100 rpm and 8 h incubation.
02/10/2024 29
 REFERENCES
1. Magesh A., Preetha B and Viruthagiri T (2011), ‘Simultaneous
Saccharification and Fermentation of Cassava Stem var. 226 White
Rose to Ethanol by Cellulase Enzyme and Saccharomyces cerevisiae’,
International Journal of ChemTech Research,Vol. 3, No.4, pp. 1821-
1829.
2. Nuwamanya E., ChiwonaKarltun L., Kawuki R.S and Baguma Y.
(2012), ‘BioEthanol Production from Non-Food Parts of Cassava
(Manihot esculenta Crantz)’, AMBIO, Vol. 41, pp. 262-270.
3. Oyeleke S.B., Dauda B.E.N., Oyewole O.A., Okoliegbe I.N and
Ojebode T (2012), ‘Production of Bioethanol from Cassava and Sweet
Potato Peels’, Advances in Environmental Biology, Vol.6,pp. 241-245.
4. Rattanachomsri U., Tanapongpipat S., Eurwilaichitr L and
Champreda V (2009), ‘Simultaneous non-thermal saccharification of
cassava pulp by multi-enzyme activity and ethanol fermentation by
Candida tropicalis’, Journal of Bioscience and Bioengineering, Vol.
107, pp. 488-493.
02/10/2024 30
5.Thongchul N., Navankasattusas S and Yang S (2010),
‘Production of lactic acid and ethanol by Rhizopus oryzae
integrated with cassava pulp hydrolysis’, Bioprocess and
Biosystems Engineering, Vol. 33, pp. 407-416.
6.Han M., Kim Y., Kim Y., Chung B. and Choi G. (2014),
‘Bioethanol production from optimized pretreatment of cassava
stem’, Korean Journal of Chemical Engineering, Vol. 28, No. 1,
pp. 119-125.
7.Sovorawet B. and Kongkiattikajorn J. (2012), ‘Bioproduction
of Ethanol in SHF and SSF from Cassava stalks’, KKU Research
Journal, Vol. 17, pp. 565-572.
8.Ali D., Soewarno1 N., Primarini D. and Sumaryono W.(2011),
‘Cassava pulp as a biofuel feedstock of an enzymatic hydrolysis
process’, Makara Teknologi, vol. 15(2),pp 183-192.

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 THANK YOU

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