Bioprocess Technology to Produce Bioethanol from Cassava by Co-Culture

Trichoderma viride, Aspergillus niger and Saccharomyces cerevisiae

I Wayan Arnata 1) Dwi Setyaningsih2), Nur Richana3)

1)
Agroindustrial Technology, Udayana University 2) Agroindustrial Technology, Bogor Agricultural University
3) Research Institute for Food Crops Biotechnology, Indonesia

ABSTRACT
Cassava (Manihot utilisima) is one of raw material to produce bioethanol that
contain starch and fiber. This study aimed at increasing the yield of ethanol
concentration from cassava by making bioprocess alternative through coculturing T.
viride, A. niger and S. cerevisiae. The experiments were performed in bacth system with
two alternatives production process, that is separate hydrolysis fermentation (SHF) and
simultaneous saccarification fermentation (SSF).The control process was using
enzyme hydrolisate and the addition of monoculture S. cerevisiae. Result of this
research, with coculturing T. viride, A. niger and S. cerevisiae in the fermentation
process of the enzyme hydrolisate and SSF for 4 days, ethanol concentration increased
from 5.36 ± 0.63 % (w/v) in the control to 7.41 ± 1.79 % (w/v) or an increase of
38.29 % compared to that of monoculture S. cerevisiae, while gradually addition of
coculture in the fermentation process was only able to increase the ethanol
concentration of 5.36 ± 0.63 % (w/v) in the control to 6.38 ± 0.83 % (w/v) or increasing
19.057 % compared to that of monoculture S. cerevisiae.

Keywords: Bioprocess, Bioethanol, Cassava, Co-culture

INTRODUCTION
Considerable attention has been given to the production of ethanol from various
sugary substrates such as mollases, starchy material like corn, sweet potato, cassava and
cellulolytic materials, because of increasing demand for ethanol which is considered to
be an alternative energy source. Cassava has potential of becoming a useful energy crop.
Cassava has high carbohydrate content about 32-35 % (wb), able growth on marginal
land area and arrange harvesting time.
Casssava usually as a bioethanol material only use starch component, whereas,
cassava not only starch content but also cellulosic content that it has potential material
as a sugar sources. Most production process concerned with the conversion of starchy
materials into ethanol have three steps, liquification of starch, enzymatic
saccharification and fermentation of sugar to etanol. Utilization of comercial enzyme is
an expensive process for the production of alchohol from starchy material.
Cellulases are a group of hydrolytic enzymes capable of degrading cellulose to
the smaller glucose units. These enzymes are produced by fungi and bacteria.
Trichoderma viride is a filamentous soil fungus known to be an effective produce
cellulolytic enzyme. It produces cellulase by secreting an enzyme system capable to
degrade crystalline cellulose which consists of several cellobiohydrolases,
endoglucanases and ß-glucosidases. Aspergillus niger is able to produce glucoamylase
that it is useful to degrade starch become glucose.
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Starch fraction in cassava is widely used in bioethanol produce. Most often, the
enzymatic hydrolysis of starch carried out with the enzyme α-amilase and
amiloglucosidase, whereas, the fiber fraction that contain cellulose and hemicellulose is
unable to hydrolyzed by amylolytic enzyme. Cellulose is glucose homopolymer and
hemicellulose contain heteropolymer such as glucose, galactose, arabinose and xylose
(Mohamed and Duarteb 2003).
Enzymatic hydrolysis with mono-culture S.cerevisiae was conducted in this
research and compared with separate hydrolysis fermentation (SHF) and simultaneous
saccharification fermentation (SSF) process with co-culture T. viride, A. niger and S.
cerevisiae. The substrate was pretreated and then subsequently hydrolyzed. More
recent applications integrated the saccharification and fermentation steps to improve
production economics. Simultaneous saccharification and fermentation combined the
cellulase enzymes and fermenting microbes in one vessel. This enabled a one-step
process of sugar production and fermentation into ethanol. The disadvantage, however,
was that the cellulase enzyme and fermentation organism had to operate under the same
conditions, decreasing the sugar and ethanol yields.
The aimed of this research was to increasing the yield of ethanol concentration
from cassava by making bioprocess alternative through coculturing T. viride, A. niger
and S. cerevisiae.

MATERIALS AND METHODS

Raw material
Fresh cassava was collected from Sukabumi, West Java. It was sun dried and
milled to uniform size (40 mesh). S. cerevisiae, A. niger and T. viride was collected
from microbiology laboratorium PAU IPB. Cassava flour was analysed to determine
chemical compotition before it is used to fermentation process. Analysed that is
moisture, fat, proteine, carbohydrate and crude fiber contents.

Inoculum culture A. niger, T. viride and S. cerevisiae

The strains A. niger and T. viride were used in this work. Stock cultures were
maintained on potato dextrose agar (PDA) and cultivation for 5 days at 30 oC. Properly
sporulated cultured were used for inoculation. The strain S. cerevisiae were maintained
on potato dextrose agar (PDA). The medium composition for cultivation was as follow:
yeast extract 5 g/L, malt 5 g/L, glocose 10 g/L and peptone 5 g/L. Cultivation was
conducted in 250 ml Erlenmeyer flask for 48 hours at 30 oC and agitation 125 rpm.

Hydrolysis and fermentation

Before fermentation process, cassava flour was hydrolysed through liquification
process with added α-amilase. Condition of hydrolyze and fermentation for SSF, SHF
and control is presented in Table 1. Fermentation process was conducted in batch
system on 1000 ml Erlenmeyer flask with 500 ml working volume, 96 hours at 30 oC.
Sample were withdrawn after 0, 6, 12, 18, 24, 36, 48, 72, 96 hours and analysed to
change of total sugar and pH. The ethanol concentration was analised at the end of time
fermentation process. Fermentation and substrate effisiensi and yield was measured
according to formulation :
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Ethanol actual
Fermentation effisiensi = x 100 %
Ethanol teoritic

Volume ethanol actual

Yield (% v/b) = x 100 %
Weight of cassava flour

So - S
Substrate effisiensi (ds/s) = x 100 %
So

Table 1 Condition of hydrolyze and fermentation process on SHF, SSF dan control.
Treatment Liquification Saccharification Fermentation
SSF Material α -amilase T. viride, S..cerevisiae A.niger
Concentration 1 ml/kg 1 : 1 :1 (10 % respectively)
Temperature 90 oC 28-30 oC
pH 4,8 5,0
Time 1h 96 h
SHF Material α -amilase T. viride, A.niger S.cerevisiae
Concentration 1 ml/kg 1 : 1 (10 % respectively) 10 %
Temperature 90 oC 28-30 oC 28-30 oC
pH 4,8 5,0 5,0
Time 1h 48 h 96 jam
Control Material α-amilase AMG S.cerevisiae
Concentration 1 ml/kg 1,2/kg , 10 % 10 %
Temperature 90 oC 50 oC 28-30 oC
pH 4,8 4,8 5,0
Time 1h 48 h 96 jam

Analitical methods
Total sugars was assays by the phenol-sulfuric acid method. The ph of substrate
assays by pH-meter. Ethanol concentration was analysed by gas chromatography (GC)

RESULT AND DISCUSSION

Characteristic of cassava flour

Proximate analyse showed that cassava flour contents 8.65 ± 0.10 % total
moisture, 2.55 ± 0.14 % dust, 6.54 ± 0.02 % fat, 1.81 ± 0.03 % proteine, 2.69 ± 0.04 %
crude fiber and 62.54 ± 0.00 % starch. Crude fiber content 69.98 % hemicellulose and
13,44 cellulose. The moisture content of the substrate has a major impact on how long it
can keep in the storage and still remains nutritions. Padonou et al. (2005) was reported
that cassava flour content 0,56 % fat (wb) and Richana et al. (1990) reported that
carbohydrate content on cassava flour about 82.30 ± 0,31 % (wb)
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Ethanol production

Figure 1 demonstrated the decrease in total sugar concentration and pH during

SSF process. Total sugar concentration decrease from 340,29 ± 10,49 g/L to 24,81 ±
7,09 g/L after 96 h, whereas pH decrease from 5,01 ± 0,01 to 3,93 ± 0,10. Total sugar
concentration rapidly decrease between 18 h and 24 h of fermentation time.
Pattern change of total sugar & pH on SSF process

6,00 400

Total sugar
4,00 300

(g/L)
pH

200
2,00 100
0,00 0
0 6 12 18 24 36 48 72 96
Time (hour)
pH Total sugar

Figure 1. Pattern channge of total sugar concentration and pH during SSF process.

Figure 2 demonstrated the decrease in total sugar concentration and pH during

SHF process. Total sugar concentration decrease from 335,76 ± 12,85 g/L to 32,05 ±
4,51 % g/L after 96 h, whereas pH decrease from 5,10 ± 0,10 to 4,12 ± 0,12.. Total
sugar concentration rapidly decrease between 18 h and 24 h of fermentation time.
Pattern change of total sugar & pH on SHF process

6,00 400
Total sugar

4,00 300
(g/L)
pH

200
2,00
100
0,00 0
0 6 12 18 24 36 48 72 96
Time (hour)

pH Total sugar

Figure 2. Pattern channge of total sugar concentration and pH during SHF process.
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Pattern change of total sugar & pH on control process

6,00 500

Total sugar
400
4,00

(g/L)
300
pH

2,00 200
100
0,00 0
0 6 12 18 24 36 48 72 96
Time (hour)

pH Total sugar

Figure 3. Pattern channge of total sugar concentration and pH during control process.

Figure 3 demonstrated the decrease in total sugar concentration and pH during

control process. Total sugar concentration decrease from 376,91 ± 15,17 g/L menjadi
24,11 ± 1,42 g/L after 96 h, whereas pH decrease from 4,91 ± 0,11 menjadi 3,07 ± 0,59.
Total sugar concentration sharp decrease between 12 h and 36 h of fermentation time.
During fermentation process was accured decrease total sugar concentration that
it was followed by decrease pH of substrate. It indicated that the substrate
accommodated the microorganism growth and produce ethanol. The decrease pH of
substrate might due to the ionitation of NH4SO4 that is used as nitrogen sources on
fermentation process. Saccharomyces cerevisiae used NH3 as a nitrogen sources and
liberated H+ into substrate solution. Accumulation of H+ given occaasion to decrease
substrate solution.
The culture T. viride, A. niger and S. cerevisiae in SSF process able to increase
ethanol concentration from 5.36 ± 0.63 % (w/v) in the control to 7.41 ± 1.79 % (w/v) or
an increase of 38.292 % compared to that of monoculture S. cerevisiae, whereas, the
SHF process was only able to increase the ethanol concentration of 5.36 ± 0.63 % (w/v)
in the control to 6.38 ± 0.83 % (w/v) or increasing 19.057 % compared to that of
monoculture S. cerevisiae (Figure 3).
EtOH concentration (% w /v)
Percentage (%)

10
8
6
4
2
0
SSF SHF Control
Treatments

Figure 3. Ethanol concentration for each treatment production process

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Table 2 Fermentation parameters of ethanol production from cassava for each treatment
process
Glucose consume Substrate effisiensi Fermnetation Yield
Treatment
(g/L) (%) effisiensi (%) (% v/b)
SSF 315,48 92,71 46,05 32.764
SHF 303,71 90,45 41,18 28.209
Control 349,59 92,75 30,05 20.051
* Control was used mono-culture S. cerevisiae

The maximum ethanol yield obtained at SSF process was 32.76 % (w/v),
corresponding 46.05 % of theoretical yield. The SSF process gave more ethanol than
SHF and control process. It might have been due to the substrate was converted directly
by amylolytic fungus A. niger dan cellulolytic fungus T. viride to glucose, and
simultaneously fermented by S. cerevisiae to ethanol. The result of effisiensi substrate,
fermentation and yield during production process for each treatment process represented
at Table 2. Nurdyastuti (2005) reported that the conversion of casssava starch to
ethanol was given about 16,67 % yield. Ethanol production by co-culture amylolytic
yeast and S. cerevisiae in starch substrate was produced ethanol concentration 6.0 %
(w/v), corresponding 93 % of theoritical yield. Suresh et al. (1998) was reported that
SSF can be conducted efficiently by using a co-culture of amilolytic A. niger and a non-
amylolytic sugar fermenter S. cerevisiae.

CONCLUSION

The culture T. viride, A. niger and S. cerevisiae in SSF process able to increase
ethanol concentration 38.292 %, whereas, the SHF process was only able to increase the
ethanol concentration 19.057 % compared to that of monoculture S. cerevisiae.

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