Hydrolysis Of Sugarcane Bagasse (Saccharum Officinarum L.) By Acid-enzyme Combinations And Further Fermentation Of The Hydrolysate By Pichia Stipitis, Saccharomyces Cerevisiae, And Zymomonas Mobilis.
Hydrolysis of Sugarcane Bagasse (Saccharum officinarum L.) by Acid-Enzyme
Combinations and Further Fermentation of The Hydrolysate
by Pichia stipitis, Saccharomyces cerevisiae, and Zymomonas mobilis
RATU SAFITRI *, IN-IN HANIDAH**, TOTO SUBROTO*
ABSTRACT
Sugarcane bagasse (Saccharum officinarum L.) is a readily available waste product of cane-sugar
processing. The major components of bagasse are cellulose and hemicellulose. The objective of the research was
to produce bio-ethanol using bagasse as raw materials, involving optimization of pretreatments, sulphuric acid
hydrolysis, enzyme hydrolysis using respectively cellulose and hemicellulase; and fermentation of the
hydrolysate by three types of microorganisms, P. stipitis CBS 5773, or S. cerevisiae D1/P3GI, or Z. mobilis
0056 FNCC respectively. The experiment employed descriptive analyses in triplicates. The results were as
follows: Pretreatments of 1 : 10 (w/v) sugarcane bagasse of 30 mesh particle size required a thermal process of
30 minutes at 120oC; the sulphuric acid hydrolysis was best by using a 2% (w/w) sulphuric acid solution and
heating for 60 minutes at 120oC; the enzymatic hydrolysis was the best when using hemicellulase at a
concentration of 0,001 g/g, followed by cellulase hydrolysis at a concentration of 0,083 µL/g. This enzymatic
hidrolyses was able to hydrolyze 63,52% of the bagasse lignocellulose, producing a hydrolysate containing
32,00 g/L reducing sugars, mainly glucose, xylose, and arabinose. Effectiveness of Z. mobilis 0056 FNCC was
highest for producing bio-ethanol, 18,99 g/L within 3 hours. S. cerevisiae D1/P3GI produced 17,05 g/L bioethanol within 12 hours, and P. stipitis CBS 5773 13,03 g/L bio-ethanol within 24 hours respectively.
Keywords : bagasse, P. stipitis, S. cerevisiae, Z. mobilis, ethanol
INTRODUCTION
Sugarcane bagasse (Saccharum officinarum) is a byproduct of the extraction process
or milking cane liquid. From one mill generated bagasse approximately 35-40% of the weight
of the milled sugar cane (Winston & Sumiarsih, 1992). Bagasse contains most of lignocellulose are composed of lignin, cellulose and hemicellulose. Lignocellulose is a substrate
that can be renewed, most are not used and is available in abundance (Taherzadeh and
Karimi, 2007). Pretreatment physical, chemical, and biological can produce fuel ethanol from
lignocellulosic (Taherzadeh and Karimi, 2008). Physical and chemical methods can be used
to separate cellulose from lignin layer by minimizing the particle size and swelling of
cellulose particles through the pre-treatment (Roehr, 2001). In this study, reduction of size in
sugar cane bagasse done on size 30 mesh (0.595 mm).
Chemical hydrolysis with sulfuric acid can be performed on ebrlignoselulosa
materials with specific time and temperature can be produced four main components namely
carbohydrate polymers (cellulose, hemicellulose), lignin, extractive materials, and ash.
Further elaborate polysaccharide polymers into a single sugar monomers (Morohoshi, 1991),
so the enzyme is more easily hydrolyze these compounds into monomers more simple sugars
(Taherzadeh and Karimi, 2007). Enzyme hydrolysis of sugarcane bagasse can be done with
Inter national Seminar Biotechnology
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the addition of cellulase enzymes, and aimed hemiselulase hydrolyze cellulose and
hemicellulose into sugar monomers.
Using a combination of acid hydrolysis and enzyme more effectively and efficiently
produce DE value of about 65% (Langlois & Dale (1940) in Tjokroadikoesoemo, 1986). he
last stage is fermentation with culture P. stipitis, S. cerevisiae, and Z. mobilis. Fermentation is
the anaerobic catabolic process with the help of enzymes microorganisms without the
electron transfer chain and organic compounds serve as electron acceptor (Campbell et al.,
1999). Sugar cane bagasse hydrolysates containing C-5 and C-6. P. stipitis and Candida
shehateae silosa and able to ferment hexoses with the result that relatively high, low ethanol
tolerant, and can produce ethanol concentrations above 30-35 g / l so that it can inhibit other
reactions. According Rouhollah (2007), S. cerevisiae has the ability to ferment glucose,
maltose, and aerobic trehalosa to ethanol. However, S. cerevisiae is not able to survive on
high concentrations of ethanol produced during fermentation. In addition, S. cerevisiae is not
able to ferment silosa because they do not have silosa silitol reductase and dehydrogenase
(Gaur, 2006). Z. mobilis is a bacterium that has the properties is more tolerant of ethanol with
the concentration levels of 2.5-15% for plasma membrane structure containing a compound
hopanoid and sterols (Gunasekaran and Raj, 2005). Z. mobilis is more tolerant of compounds
in the hydrolyzate inhibitors, a higher ethanol production, fermentation at low pH, grown on
high sugar concentrations (Kompala et al., 2001).
In this study, sugar cane bagasse through the stages of size reduction, pre-treatment, a
combination of acid hydrolysis - are expected to aimlessly enzymes capable hydrolyzate
sugar fermented by P. stipitis, S. cerevisiae, and Z. mobilis to ethanol.
RESEARCH
MATERIALS AND METHODS.
Preparation of hydrolysates Sugarcane bagasse
Sugarcane bagasse (Saccharum officinarum) were obtained from plantations of PT
Persada Stone. RNI (Rajawali Nusantara Indonesia) is located in Cirebon, then conducted a
physical treatment through penggililingan until sugar cane bagasse obtained powder size of
30 mesh, dried in an oven at a temperature of 80oC for 10 minutes. Flour sugar cane bagasse
as dried sugar cane bagasse suspension made with water at a ratio of 1: 20; 1: 13.3; and 1: 10
(w / v), each in 100 ml aquades. Further heating (steam) at a temperature of 120oC for 30 and
45 minutes at a pressure of 1 atm using the autoclave. Sugarcane bagasse suspension with
the optimum concentration (suspension I) inserted into the erlenmeyer, 250 ml, was added a
solution of H2SO4 as much as 1%, 1.5%, 2% (w / w) of the weight of sugar cane bagasse.
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Furthermore, each diautoclave at a temperature of 100oC, 110 oC; 120oC, for 60, 90 minutes,
so that obtained suspensions II.
Sugarcane bagasse suspension with the optimum concentration of acid hydrolysis
(suspension II) was followed by cooling to 25oC temperature and setting the pH to 6.0 by
using HCl and NaOH 1 N. Hemiselulase enzyme is then added as much as 0.00033 g / g
(dosis1 / 3), 0.00067 g / g (dosis2 / 3), and 0.001 g / g (dosis3 / 3) of the weight of enzyme / g
substrate, then incubated at 55oC temperature for 4.5 hours with agitation speed of 150 rpm.
In this phase III obtained suspension.
Suspension III hydrolysates were then followed
cooling to 25oC temperature and pH to 4.8 with the settings using a solution of HCl and
NaOH 1 N. Cellulase enzyme is then added as much as 0.277 mL / g (dosis1 / 3), 0.553 mL /
g (dosis2 / 3), and 0.83 mL / g (dosis3 / 3) of the volume of enzyme / g substrate, and enzyme
as much as 0.123 mL amiloglukosidase / g (dosis1 / 3), 0.247 mL / g (dosis2 / 3), and 0.37
mL / g (dosis3 / 3) of the volume of enzyme / g substrate. Hydrolysates incubated at 60oC for
48 hours with agitation speed of 130 rpm. At this stage sugar cane bagasse hydrolyzate
obtained.
Microorganisms and growth media.
The microorganism used was S. D1/P3GI cerevisiae, P. stipitis CBS 5773, and Z.
mobilis FNCC 0056. Starter media for culturing yeast S. cerevisiae, and P. stipitis is YEPD
agar containing per liter: 3 g yeast extract, 10 g Peptone, 20 g dextrose, 15 g agar-agar, and 1
L aquades (Gaur, 2006). As for the culture of Z. Zymomonas mobilis used medium
containing per liter: 10 g yeast extract, 10 g Peptone, 20 g glucose, 15 g agar-agar, and 1 L
aquades (Atlas, 1993). Rejuvenation and multiplication in italics for by taking a loop of pure
culture medium and then inoculated on slanted for pH 7 aseptically. Subsequently incubated
at 30oC for 24 hours. Making the starter is done by adding sterile physiological NaCl
solution into pure culture for italics, stirring until the suspension is obtained which is
measured on the spectrophotometer absorbance value equivalent to McFarland 3 at λ 600 nm.
The suspension will be used is taken 1 ml to the amount calculated using the method
of TPC. Each culture was grown on media adaptation by taking a 3% suspension (v / v) pure
culture into 250 ml erlenmeyer containing 100 ml of media adaptation (YEPD broth pH 7),
incubated aerobically at 30oC using a shaker with agitation speed 100 rpm, incubation time
for P. stipitis for 39 hours, S. cerevisiae for 18 hours, and Z. mobilis for 9 hours.
Fermentation
Sugarcane bagasse hydrolyzate having the highest DE value is used as a fermentation
substrate. Previously sugar hydrolyzate is filtered using filter paper no.4 Waltman.
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Hydrolyzate plus add-fermentation medium containing (/ L): 4 g yeast extract, 2 g KH2PO4,
3 g (NH4) 2SO4, 1 g MgSO4.7H2O, and 3.6 g peptone (Sanchez et al., 2002). Then by
controlling the pH to 7, then sterilized in an autoclave at a temperature of 121oC for 15
minutes. Addition of inoculum in the hydrolysates was 10% (v / v). Each inoculum P.
stipitis, S. cerevisiae, and Z. mobilis put into 100 ml erlenmeyer containing 27 ml
fermentation substrate. Then incubated at 30oC, for 84 hours for P. stipitis, S. cerevisiae and
21
hours
to
Z.
mobilis,
pH
7, and
agitation
speed
of
150
rpm
.
Analytical method.
Changes in pH with a pH meter. Total reducing sugars with the DNS (Apriyantono, et al.,
1989), type of sugar at the end of each stage with the HPLC column HPX-87H AMINEX.
Ethanol bichromate oxidation method (Caputi et al., 1968). Type of organic acids formed
during the final stage of fermentation by HPLC column HPX-87H AMINEX .
RESULTS AND DISCUSSION.
Concentration, temperature, and hydrolysis incubation on Sugar Cane Bagas
hydrolysates by H2SO4.
The process of hydrolysis in this study used a solution of concentrated H2SO4. Data
calculated concentration of reducing sugars obtained by acid hydrolysis stage is presented in
Figure 1.
25
20
15
10
5
0
Remark:
a.
b.
c.
d.
e.
f.
1%, 100oC, 60 menit
1%, 100oC, 90 menit
1%, 110oC, 60 menit
1%, 110oC, 90 menit
1%, 120oC, 60 menit
1%, 120oC, 90 menit
g.
h.
i.
j.
k.
l.
1,5%, 100oC, 60 menit
1,5%, 100oC, 90 menit
1,5%, 110oC, 60 menit
1,5%, 110oC, 90 menit
1,5%, 120oC, 60 menit
1,5%, 120oC, 90 menit
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m.
n.
o.
p.
q.
r.
2%, 100oC, 60 menit
2%, 100oC, 90 menit
2%, 110oC, 60 menit
2%, 110oC, 90 menit
2%, 120oC, 60 menit
2%, 120oC, 90 menit
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Figure 1. Effect of sulfuric acid concentration, temperature, and time of heating steam to the
reducing sugar concentration (g / L)
Treatment with H2SO4 concentration of 2% (w / w), heating temperature of 120oC for 60
minutes produced the highest reducing sugar concentration of 27.65 g / L with a DE value of
54.88%. This means that 45.12% of reducing sugar is still not hydrolyzed. At this stage
produced the type of reducing sugar glucose, silosa, and arabinose. These results prove that
the use of higher H2SO4 concentration can reduce the length of hydrolysis, whereas the use
of a lower hydrolysis temperatures require longer hydrolysis time. In research Guraf & Geeta
(2007), ethanol production through the pre-treatment with the help of fungi Phanerochaete
chrysosporium and Pleurotus sp concentration sugar produced from sugarcane bagasse by
2.54 mg / g.
HPLC:
240.00
300.00
220.00
4.003
18.00
4.017
3.938
20.00
200.00
16.00
180.00
250.00
14.00
160.00
200.00
140.00
120.00
100.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00
9.507
10.650
5.624
6.609
4.677
4.940
20.00
0.00
1.00
15.00
7.954
7.956
9.538
6.636
6.047
40.00
0.00
0.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.0
Minutes
Pra-perlakuan
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.00
14.00
15.0
Asam - enzim
Hidrolisis asam
280.00
240.00
260.00
240.00
220.00
220.00
200.00
200.00
200.00
180.00
180.00
180.00
160.00
160.00
140.00
140.00
100.00
40.00
20.00
5.00
6.00
0.00
11.528
11.505
6.390
6.610
10.517
60.00
10.571
0.00
5.534
5.030
20.00
11.964
80.00
40.00
11.961
11.525
10.563
9.530
7.974
8.917
20.00
120.00
60.00
6.034
4.449
40.00
6.619
6.812
60.00
140.00
8.090
80.00
9.531
80.00
160.00
6.035
100.00
6.628
100.00
5.022
120.00
MV
MV
220.00
120.00
4.016
240.00
4.017
260.00
4.015
260.00
4.448
4.680
5.015
280.00
MV
5.643
50.00
9.542
7.954
2.00
4.443
4.739
4.965
60.00
6.625
4.00
80.00
100.00
4.757
4.960
6.00
6.022
150.00
6.179
8.00
MV
10.00
MV
5.644
MV
12.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
Minutes
14.00
15.0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.0
1.00
2.00
3.00
4.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
Minutes
Fermentasi oleh S. cerevisiae
Fermentasi oleh P. stipitis
Fermentasi oleh Z. mobilis
This study proves that the hydrolysis of lignocellulosic material with sulfuric acid is more
effective than the use of fungi and is able to degrade lignin by 31.98% and the rest are still
bound to each other with the structure of cellulose and hemicellulose. According to Inoue
(2006), acid hydrolysis is very important to the efficiency of enzyme hydrolysis stage.
Cellulose is naturally bound by the hemicellulose and protected by lignin, which causes the
biomass of this structure is difficult to dihirolisis. The purpose of the acid hydrolysis is to
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open the lignocellulosic structure for cellulose becomes more accessible to enzymes that
break down polysaccharides into monomeric sugar polymers. Because one component of
hemicellulose (glucose) has the nature of cellulose, the hemicellulose degraded earlier than
the cellulose. The combination of acid hydrolysis - Enzymes Enzymes in Various Doses
Hydrolyzate acid hydrolysis results with the addition of H2SO4 2% (w / w), temperature of
120oC for 60 minutes heating was continued for the enzyme hydrolysis step. This study used
enzyme hemiselulase and continued with the enzyme cellulase. In this stage has to be
optimized hemiselulase and cellulase enzyme dosage. From the data calculation result
obtained by reducing sugar concentration of reducing sugar concentration (g / L) of enzyme
dosage on enzyme hydrolysis.
30,00
25,00
20,00
15,00
10,00
5,00
0,00
Dosis 1/ 3
Dosis 2/ 3
Dosis 3/ 3
Figure 2. Effect of enzyme dosage on the reducing sugar concentration (g / L).
Based on the results shown in Figure 4, the highest sugar concentration obtained by
the treatment and the addition of cellulase enzyme dosage hemiselulase 3 / 3, amounting to
32.00 g / L with a DE value of 63.52%. Hydrolyzed sugar polymer that has not caused the
persistence of 24.23% lignin in sugarcane bagasse acid hydrolysis results, so the enzyme
cellulase
and
hemiselulase
difficult
to
degrade
into
reducin
g
sugar
monomers.
According Taherzadeh (2008), lignin is a complex compound that is bound to one another
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with cellulose and hemicellulose. This bond causes enzyme is difficult to degrade cellulose
and hemicellulose into sugar monomers reduction. Degradation of lignin in berlignoselulosa
material will affect the amount of biodegradable cellulose into reducing sugars. The higher
the lignin is degraded, the more easily break down cellulose to form reducing sugar during
hydrolysis enzyme (Harlina, 2002). The result of HPLC analysis showed that the enzyme
hydrolysis step using enzyme dosage hemiselulase 3 / 3 on sugarcane bagasse can degrade
hemicellulose into sugar monomers silosa and arabinose. While the enzyme hydrolysis using
cellulase enzyme dosage of 3 / 3 on sugarcane bagasse able to degrade cellulose to glucose
sugar monomers. The figure below shows the increase in reducing sugar concentration (g / L)
at any given stage of treatment.
)L
/g
(
32,00
27,65
35,00
30,00
25,00
20,00
15,00
10,00
5,00
0,00
2,32
is
k
u
d
e
rp
a
lg
tn
o
K
Pra perlakuan
Hidrolisis
asam
Hidrolisis
enzim at ik
Tahapan Perlakuan
Figure 3. Effect of treatment of phase reducing sugar concentration (g / L)
Fermentation
The microorganism used in fermentation of sugarcane bagasse hydrolysates include: P.
stipitis CBS 5773, S. D1/P3GI cerevisiae, and Z. mobilis FNCC 0056. Stage of fermentation
was conducted to determine the ability of each microorganism in ferment the sugar cane
bagasse hydrolyzate to ethanol.
Based on the results shown in Figure 6, the adaptation phase S. cereviseae D1/P3GI,
P. stipitis CBS 5773, and Z. mobilis FNCC happen until 0056 at the 3rd fermentation.
Adaptation phase is a phase of adjustment to new environment, where there is synthesis of
new enzyme according to the media. In this phase does not increase the number of cells but
the cell volume increases. This proves that the three microorganisms capable of using
reducing sugar from sugarcane bagasse hydrolyzate as a nutritional source of growth.
Based on the results of HPLC analysis of hydrolysed cane is used for the fermentation
process
containing
23.78%
Inter national Seminar Biotechnology
glucose,
56.91%
silosa, and
19.31%
arabinose.
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During the ongoing growth of microorganisms, pH control is imperative to maintain the
optimum pH. Decrease in pH value is very real in the first 12 hours of fermentation either by
culture of S. cereviseae D1/P3GI, P. stipitis CBS 5773, and Z. mobilis FNCC 0056. At this
stage, ethanol was formed accompanied with the decrease in pH due to formation of organic
acids. The trend of pH change caused by ammonia in the fermentation media used yeast cells
as a source of nitrogen is converted into NH4 +. NH4 + molecules will merge into the cell as
R-NH 3. In this process H + stays in the media, so that more biomass and increasing time of
fermentation causes H + ions are more and more in the substrate which causes the lower the
pH of the media (Judoamidjojo & Saeed, 1993; Fardiaz, 1988).
Based on the results of HPLC, the final stage of fermentation produced lactic acid
and acetic acid from fermentation using a culture of S. cereviseae D1/P3GI and acetic acid
from fermentation culture by Z. 0056 FNCC mobilis produced. Concentration of ethanol
produced in line with the growth of each culture and the amount of reducing sugar consumed.
In this study, Z. 0056 FNCC mobilis able to ferment sugar into ethanol hydrolyzate highest P.
stipitis CBS 5773 and S. cerevisiae D1/P3GI with a shorter fermentation time, which is 3
hours produced ethanol at 18.99 g / L.
The results of this study reinforced with literature that states that Z. mobilis is able to
change the mix of sugar into ethanol is greater 92% - 94% compared to the yeast which only
reached 88% -90% (Silalahi, 1987). It is possible or ethanol fermentation by Z. mobilis, the
conversion of glucose into two molecules of ethanol produced one molecule of ATP. The low
energy produced by ATP resulting in cell mass of the resulting low and high ethanol
produced (Jeffries, 2005).
Based on research data presented in Figure 7, the cell growth of S. cerevisiae
D1/P3GI in the first 12 hours of fermentation is constantly increasing. This means that S.
D1/P3GI cerevisiae capable of using sugar cane bagasse hydrolysates as a source of nutrition.
This can be seen from the decrease in the amount of sugar in the hydrolyzate peredukasi
accompanied with the production of ethanol amounted to 17.05 g / L. While the research
Rouhollah et al. (2007), fermentation using microorganisms Fermentative S. cerevisiae in
sugar mixture to produce ethanol 0.32 g / L per gram of glucose or by 62% with ethanol
concentrations of 14.25 g / L and ethanol productivity was 0.88 g / L / hour in 48 hours
fermentation time. This proves that S. cerevisiae D1/P3GI able to ferment sugar cane bagasse
hydrolysates with a shorter time which is 12 hours with levels of ethanol produced 17.00% g
/ L. Okur & Nurdan (2006) conducted research on the production of ethanol from waste
sunflower seed using P.stipitis NRRL Y-124 with condition 6 of the initial pH of hydrolyzate
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fermentation and the fermentation process lasts for 55 hours produced ethanol concentration
of 9.66 g / L. While the fermentation of sugar mixed media using P. CCUG18492 stipitis
produces ethanol 0.40 g / L per gram silosa or by 78% and ethanol concentration of 30.23 g /
L with productivity of ethanol is 0.95 g / L / hour in 72 hours fermentation time (Rouhollah et
al., 2007) . While in this study, P. stipitis CBS 5773 is able to ferment sugar cane bagasse
hydrolyzate to ethanol as much as 13.03 g / L in 24 hours fermentation time. Overall, both P.
stipitis CBS 5773, S. D1/P3GI cerevisiae, and Z. 0056 FNCC mobilis each use sugar cane
bagasse hydrolyzate in an optimal as a source of nutrition in the period 0-24 hours coupled
with the optimal ethanol production. Starting at the 24th production of ethanol from each
microorganism has decreased. This happens because the availability of sugar in the
hydrolyzate under 10 g / L or less than 1%. Meanwhile, according Budiyanto (2003) in a
Putri (2008), the optimum concentration of sugar to produce the optimum alcohol content is
14% - 28% (w / v).
(a)
(b)
(c)
Figure 6. Production of ethanol from sugarcane bagasse hydrolysates by (a) Z.
mobilis (B) S. D1/P3GI cerevisiae, (c) P. stipitis CBS 5773.
From the research results obtained can be concluded :
1. Hydrolysis by sulfuric acid with a concentration of 2% (w / w), steam heating temperature
120oC for 60 minutes could be able to hydrolyze lignocellulosic sugars into simple
monomers with reducing sugar concentration of 27.65 g / L with a DE value of 54.88%
2. The best hydrolysis by enzymes in the enzyme dosage hemiselulase 0.001 g / g (1500
units), followed by the cellulase enzyme dosage 0.83 mL / g (0.6970 units). Both enzymes
are able to hydrolyze sugar cane bagasse by 63.52% with a reducing sugar content in
sugarcane bagasse hydrolyzate of 32.00 g / L.
3. Z. 0056 FNCC mobilis able to ferment sugar cane bagasse hydrolyzate to ethanol by 18.99
g / L with the fermentation time of 3 hours, S. D1/P3GI cerevisiae, is able to ferment the
sugar cane bagasse hydrolyzat to ethanol amounted to 17.05 g / L with the fermentation time
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of 12 hours, whereas P. stipitis CBS 5773 is able to ferment sugar cane bagasse hydrolyzate
to ethanol at 13.03 g / L in 24 hours.
ACKNOWLEDGMENTS
Upon completion of this research I would like to thank:
1. Department of Agriculture which has helped fund this research through KKP3T.
2 Program. Ministry of National Education has helped fund education and research through
the Competitive Scholarship Program and P3SWOT.
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Pakan. Laboratorium Makanan Ternak Fakultas Peternakan. Universitas Jambi.
Okur,Telli & Nurdan Eken S. 2006. Etanol Production from Sunflower Seed Hull
Hydrolysate by Pichia stipitis under Uncontrolled pH Conditions in a Bioreactor. Turkish
J. Eng. Env. Sci. (30): 317 – 322.
Putri, L.S. & Dede Sukandar. 2008. Konversi Pati Ganyong (Canna edulis Ker.)
Menjadi
Bioetanol
melalui Hidrolisis Asam dan Fermentasi. BIODIVERSITAS. Volume 9,
Nomor 2: 112-116.
Roehr, M. 2001. The Biotechnology of Etanol. The Biotechnology of Etanol: Classical and
Future Applications. ISBN: 3-527-30199-2. WILEY-VCH Verlag GmbH, Weinheim.
Rouhollah, H., N.Iraj, E.Giti, & A.Sorah. 2007. Mixed sugar fermentation by Pichia stipitis,
Sacharomyces cerevisiae, and an isolated xylose fermenting Kluyveromyces marxianus
and their cocultures. African Journal of Biotechnology. 6 (9) :1110-1114.
Sànchez, S., V. Bravo, E. Castro, A.J. Moya, & F. Camacho. 2002. Fermentation of Mixtures
of D-glucose and D-xylose by Candida shehatae, Pichia stipitis or Pachysolen
tannophilus to Produce Etanol. J.Chem.Technol.Biotechnol, 77 : 641-648.
Silalahi, T. D.1987. Isolasi Bakteri Z. mobilis dari Nira Aren (Arenga pinnata) dan Menguji
Kemampuannya dalam Pembuatan Alkohol dari Tetes. ITB. Bandung.
Taherzadeh, M.J. & K. Karimi. 2007. Acid Based Hydrolysis Processes For Etanol From
Lignocellulose Materials : A Review. Bio Resources, 2(3) : 472-499.
Taherzadeh, M.J. & K. Karimi. 2008. Pretreatment of Lignocellulosic Wastes to Improve
Etanol and Biogas Production: A Review. International Journal of Molecular Sciences, 9:
1621-1651.
Tjokroadikoesoemo, P. S. 1986. HFS dan Industri Ubi Kayu Lainnya. Penerbit Gramedia.
Jakarta.
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Combinations and Further Fermentation of The Hydrolysate
by Pichia stipitis, Saccharomyces cerevisiae, and Zymomonas mobilis
RATU SAFITRI *, IN-IN HANIDAH**, TOTO SUBROTO*
ABSTRACT
Sugarcane bagasse (Saccharum officinarum L.) is a readily available waste product of cane-sugar
processing. The major components of bagasse are cellulose and hemicellulose. The objective of the research was
to produce bio-ethanol using bagasse as raw materials, involving optimization of pretreatments, sulphuric acid
hydrolysis, enzyme hydrolysis using respectively cellulose and hemicellulase; and fermentation of the
hydrolysate by three types of microorganisms, P. stipitis CBS 5773, or S. cerevisiae D1/P3GI, or Z. mobilis
0056 FNCC respectively. The experiment employed descriptive analyses in triplicates. The results were as
follows: Pretreatments of 1 : 10 (w/v) sugarcane bagasse of 30 mesh particle size required a thermal process of
30 minutes at 120oC; the sulphuric acid hydrolysis was best by using a 2% (w/w) sulphuric acid solution and
heating for 60 minutes at 120oC; the enzymatic hydrolysis was the best when using hemicellulase at a
concentration of 0,001 g/g, followed by cellulase hydrolysis at a concentration of 0,083 µL/g. This enzymatic
hidrolyses was able to hydrolyze 63,52% of the bagasse lignocellulose, producing a hydrolysate containing
32,00 g/L reducing sugars, mainly glucose, xylose, and arabinose. Effectiveness of Z. mobilis 0056 FNCC was
highest for producing bio-ethanol, 18,99 g/L within 3 hours. S. cerevisiae D1/P3GI produced 17,05 g/L bioethanol within 12 hours, and P. stipitis CBS 5773 13,03 g/L bio-ethanol within 24 hours respectively.
Keywords : bagasse, P. stipitis, S. cerevisiae, Z. mobilis, ethanol
INTRODUCTION
Sugarcane bagasse (Saccharum officinarum) is a byproduct of the extraction process
or milking cane liquid. From one mill generated bagasse approximately 35-40% of the weight
of the milled sugar cane (Winston & Sumiarsih, 1992). Bagasse contains most of lignocellulose are composed of lignin, cellulose and hemicellulose. Lignocellulose is a substrate
that can be renewed, most are not used and is available in abundance (Taherzadeh and
Karimi, 2007). Pretreatment physical, chemical, and biological can produce fuel ethanol from
lignocellulosic (Taherzadeh and Karimi, 2008). Physical and chemical methods can be used
to separate cellulose from lignin layer by minimizing the particle size and swelling of
cellulose particles through the pre-treatment (Roehr, 2001). In this study, reduction of size in
sugar cane bagasse done on size 30 mesh (0.595 mm).
Chemical hydrolysis with sulfuric acid can be performed on ebrlignoselulosa
materials with specific time and temperature can be produced four main components namely
carbohydrate polymers (cellulose, hemicellulose), lignin, extractive materials, and ash.
Further elaborate polysaccharide polymers into a single sugar monomers (Morohoshi, 1991),
so the enzyme is more easily hydrolyze these compounds into monomers more simple sugars
(Taherzadeh and Karimi, 2007). Enzyme hydrolysis of sugarcane bagasse can be done with
Inter national Seminar Biotechnology
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the addition of cellulase enzymes, and aimed hemiselulase hydrolyze cellulose and
hemicellulose into sugar monomers.
Using a combination of acid hydrolysis and enzyme more effectively and efficiently
produce DE value of about 65% (Langlois & Dale (1940) in Tjokroadikoesoemo, 1986). he
last stage is fermentation with culture P. stipitis, S. cerevisiae, and Z. mobilis. Fermentation is
the anaerobic catabolic process with the help of enzymes microorganisms without the
electron transfer chain and organic compounds serve as electron acceptor (Campbell et al.,
1999). Sugar cane bagasse hydrolysates containing C-5 and C-6. P. stipitis and Candida
shehateae silosa and able to ferment hexoses with the result that relatively high, low ethanol
tolerant, and can produce ethanol concentrations above 30-35 g / l so that it can inhibit other
reactions. According Rouhollah (2007), S. cerevisiae has the ability to ferment glucose,
maltose, and aerobic trehalosa to ethanol. However, S. cerevisiae is not able to survive on
high concentrations of ethanol produced during fermentation. In addition, S. cerevisiae is not
able to ferment silosa because they do not have silosa silitol reductase and dehydrogenase
(Gaur, 2006). Z. mobilis is a bacterium that has the properties is more tolerant of ethanol with
the concentration levels of 2.5-15% for plasma membrane structure containing a compound
hopanoid and sterols (Gunasekaran and Raj, 2005). Z. mobilis is more tolerant of compounds
in the hydrolyzate inhibitors, a higher ethanol production, fermentation at low pH, grown on
high sugar concentrations (Kompala et al., 2001).
In this study, sugar cane bagasse through the stages of size reduction, pre-treatment, a
combination of acid hydrolysis - are expected to aimlessly enzymes capable hydrolyzate
sugar fermented by P. stipitis, S. cerevisiae, and Z. mobilis to ethanol.
RESEARCH
MATERIALS AND METHODS.
Preparation of hydrolysates Sugarcane bagasse
Sugarcane bagasse (Saccharum officinarum) were obtained from plantations of PT
Persada Stone. RNI (Rajawali Nusantara Indonesia) is located in Cirebon, then conducted a
physical treatment through penggililingan until sugar cane bagasse obtained powder size of
30 mesh, dried in an oven at a temperature of 80oC for 10 minutes. Flour sugar cane bagasse
as dried sugar cane bagasse suspension made with water at a ratio of 1: 20; 1: 13.3; and 1: 10
(w / v), each in 100 ml aquades. Further heating (steam) at a temperature of 120oC for 30 and
45 minutes at a pressure of 1 atm using the autoclave. Sugarcane bagasse suspension with
the optimum concentration (suspension I) inserted into the erlenmeyer, 250 ml, was added a
solution of H2SO4 as much as 1%, 1.5%, 2% (w / w) of the weight of sugar cane bagasse.
Inter national Seminar Biotechnology
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Furthermore, each diautoclave at a temperature of 100oC, 110 oC; 120oC, for 60, 90 minutes,
so that obtained suspensions II.
Sugarcane bagasse suspension with the optimum concentration of acid hydrolysis
(suspension II) was followed by cooling to 25oC temperature and setting the pH to 6.0 by
using HCl and NaOH 1 N. Hemiselulase enzyme is then added as much as 0.00033 g / g
(dosis1 / 3), 0.00067 g / g (dosis2 / 3), and 0.001 g / g (dosis3 / 3) of the weight of enzyme / g
substrate, then incubated at 55oC temperature for 4.5 hours with agitation speed of 150 rpm.
In this phase III obtained suspension.
Suspension III hydrolysates were then followed
cooling to 25oC temperature and pH to 4.8 with the settings using a solution of HCl and
NaOH 1 N. Cellulase enzyme is then added as much as 0.277 mL / g (dosis1 / 3), 0.553 mL /
g (dosis2 / 3), and 0.83 mL / g (dosis3 / 3) of the volume of enzyme / g substrate, and enzyme
as much as 0.123 mL amiloglukosidase / g (dosis1 / 3), 0.247 mL / g (dosis2 / 3), and 0.37
mL / g (dosis3 / 3) of the volume of enzyme / g substrate. Hydrolysates incubated at 60oC for
48 hours with agitation speed of 130 rpm. At this stage sugar cane bagasse hydrolyzate
obtained.
Microorganisms and growth media.
The microorganism used was S. D1/P3GI cerevisiae, P. stipitis CBS 5773, and Z.
mobilis FNCC 0056. Starter media for culturing yeast S. cerevisiae, and P. stipitis is YEPD
agar containing per liter: 3 g yeast extract, 10 g Peptone, 20 g dextrose, 15 g agar-agar, and 1
L aquades (Gaur, 2006). As for the culture of Z. Zymomonas mobilis used medium
containing per liter: 10 g yeast extract, 10 g Peptone, 20 g glucose, 15 g agar-agar, and 1 L
aquades (Atlas, 1993). Rejuvenation and multiplication in italics for by taking a loop of pure
culture medium and then inoculated on slanted for pH 7 aseptically. Subsequently incubated
at 30oC for 24 hours. Making the starter is done by adding sterile physiological NaCl
solution into pure culture for italics, stirring until the suspension is obtained which is
measured on the spectrophotometer absorbance value equivalent to McFarland 3 at λ 600 nm.
The suspension will be used is taken 1 ml to the amount calculated using the method
of TPC. Each culture was grown on media adaptation by taking a 3% suspension (v / v) pure
culture into 250 ml erlenmeyer containing 100 ml of media adaptation (YEPD broth pH 7),
incubated aerobically at 30oC using a shaker with agitation speed 100 rpm, incubation time
for P. stipitis for 39 hours, S. cerevisiae for 18 hours, and Z. mobilis for 9 hours.
Fermentation
Sugarcane bagasse hydrolyzate having the highest DE value is used as a fermentation
substrate. Previously sugar hydrolyzate is filtered using filter paper no.4 Waltman.
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Hydrolyzate plus add-fermentation medium containing (/ L): 4 g yeast extract, 2 g KH2PO4,
3 g (NH4) 2SO4, 1 g MgSO4.7H2O, and 3.6 g peptone (Sanchez et al., 2002). Then by
controlling the pH to 7, then sterilized in an autoclave at a temperature of 121oC for 15
minutes. Addition of inoculum in the hydrolysates was 10% (v / v). Each inoculum P.
stipitis, S. cerevisiae, and Z. mobilis put into 100 ml erlenmeyer containing 27 ml
fermentation substrate. Then incubated at 30oC, for 84 hours for P. stipitis, S. cerevisiae and
21
hours
to
Z.
mobilis,
pH
7, and
agitation
speed
of
150
rpm
.
Analytical method.
Changes in pH with a pH meter. Total reducing sugars with the DNS (Apriyantono, et al.,
1989), type of sugar at the end of each stage with the HPLC column HPX-87H AMINEX.
Ethanol bichromate oxidation method (Caputi et al., 1968). Type of organic acids formed
during the final stage of fermentation by HPLC column HPX-87H AMINEX .
RESULTS AND DISCUSSION.
Concentration, temperature, and hydrolysis incubation on Sugar Cane Bagas
hydrolysates by H2SO4.
The process of hydrolysis in this study used a solution of concentrated H2SO4. Data
calculated concentration of reducing sugars obtained by acid hydrolysis stage is presented in
Figure 1.
25
20
15
10
5
0
Remark:
a.
b.
c.
d.
e.
f.
1%, 100oC, 60 menit
1%, 100oC, 90 menit
1%, 110oC, 60 menit
1%, 110oC, 90 menit
1%, 120oC, 60 menit
1%, 120oC, 90 menit
g.
h.
i.
j.
k.
l.
1,5%, 100oC, 60 menit
1,5%, 100oC, 90 menit
1,5%, 110oC, 60 menit
1,5%, 110oC, 90 menit
1,5%, 120oC, 60 menit
1,5%, 120oC, 90 menit
Inter national Seminar Biotechnology
m.
n.
o.
p.
q.
r.
2%, 100oC, 60 menit
2%, 100oC, 90 menit
2%, 110oC, 60 menit
2%, 110oC, 90 menit
2%, 120oC, 60 menit
2%, 120oC, 90 menit
P 190
Figure 1. Effect of sulfuric acid concentration, temperature, and time of heating steam to the
reducing sugar concentration (g / L)
Treatment with H2SO4 concentration of 2% (w / w), heating temperature of 120oC for 60
minutes produced the highest reducing sugar concentration of 27.65 g / L with a DE value of
54.88%. This means that 45.12% of reducing sugar is still not hydrolyzed. At this stage
produced the type of reducing sugar glucose, silosa, and arabinose. These results prove that
the use of higher H2SO4 concentration can reduce the length of hydrolysis, whereas the use
of a lower hydrolysis temperatures require longer hydrolysis time. In research Guraf & Geeta
(2007), ethanol production through the pre-treatment with the help of fungi Phanerochaete
chrysosporium and Pleurotus sp concentration sugar produced from sugarcane bagasse by
2.54 mg / g.
HPLC:
240.00
300.00
220.00
4.003
18.00
4.017
3.938
20.00
200.00
16.00
180.00
250.00
14.00
160.00
200.00
140.00
120.00
100.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00
9.507
10.650
5.624
6.609
4.677
4.940
20.00
0.00
1.00
15.00
7.954
7.956
9.538
6.636
6.047
40.00
0.00
0.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.0
Minutes
Pra-perlakuan
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.00
14.00
15.0
Asam - enzim
Hidrolisis asam
280.00
240.00
260.00
240.00
220.00
220.00
200.00
200.00
200.00
180.00
180.00
180.00
160.00
160.00
140.00
140.00
100.00
40.00
20.00
5.00
6.00
0.00
11.528
11.505
6.390
6.610
10.517
60.00
10.571
0.00
5.534
5.030
20.00
11.964
80.00
40.00
11.961
11.525
10.563
9.530
7.974
8.917
20.00
120.00
60.00
6.034
4.449
40.00
6.619
6.812
60.00
140.00
8.090
80.00
9.531
80.00
160.00
6.035
100.00
6.628
100.00
5.022
120.00
MV
MV
220.00
120.00
4.016
240.00
4.017
260.00
4.015
260.00
4.448
4.680
5.015
280.00
MV
5.643
50.00
9.542
7.954
2.00
4.443
4.739
4.965
60.00
6.625
4.00
80.00
100.00
4.757
4.960
6.00
6.022
150.00
6.179
8.00
MV
10.00
MV
5.644
MV
12.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
Minutes
14.00
15.0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.0
1.00
2.00
3.00
4.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
Minutes
Fermentasi oleh S. cerevisiae
Fermentasi oleh P. stipitis
Fermentasi oleh Z. mobilis
This study proves that the hydrolysis of lignocellulosic material with sulfuric acid is more
effective than the use of fungi and is able to degrade lignin by 31.98% and the rest are still
bound to each other with the structure of cellulose and hemicellulose. According to Inoue
(2006), acid hydrolysis is very important to the efficiency of enzyme hydrolysis stage.
Cellulose is naturally bound by the hemicellulose and protected by lignin, which causes the
biomass of this structure is difficult to dihirolisis. The purpose of the acid hydrolysis is to
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open the lignocellulosic structure for cellulose becomes more accessible to enzymes that
break down polysaccharides into monomeric sugar polymers. Because one component of
hemicellulose (glucose) has the nature of cellulose, the hemicellulose degraded earlier than
the cellulose. The combination of acid hydrolysis - Enzymes Enzymes in Various Doses
Hydrolyzate acid hydrolysis results with the addition of H2SO4 2% (w / w), temperature of
120oC for 60 minutes heating was continued for the enzyme hydrolysis step. This study used
enzyme hemiselulase and continued with the enzyme cellulase. In this stage has to be
optimized hemiselulase and cellulase enzyme dosage. From the data calculation result
obtained by reducing sugar concentration of reducing sugar concentration (g / L) of enzyme
dosage on enzyme hydrolysis.
30,00
25,00
20,00
15,00
10,00
5,00
0,00
Dosis 1/ 3
Dosis 2/ 3
Dosis 3/ 3
Figure 2. Effect of enzyme dosage on the reducing sugar concentration (g / L).
Based on the results shown in Figure 4, the highest sugar concentration obtained by
the treatment and the addition of cellulase enzyme dosage hemiselulase 3 / 3, amounting to
32.00 g / L with a DE value of 63.52%. Hydrolyzed sugar polymer that has not caused the
persistence of 24.23% lignin in sugarcane bagasse acid hydrolysis results, so the enzyme
cellulase
and
hemiselulase
difficult
to
degrade
into
reducin
g
sugar
monomers.
According Taherzadeh (2008), lignin is a complex compound that is bound to one another
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with cellulose and hemicellulose. This bond causes enzyme is difficult to degrade cellulose
and hemicellulose into sugar monomers reduction. Degradation of lignin in berlignoselulosa
material will affect the amount of biodegradable cellulose into reducing sugars. The higher
the lignin is degraded, the more easily break down cellulose to form reducing sugar during
hydrolysis enzyme (Harlina, 2002). The result of HPLC analysis showed that the enzyme
hydrolysis step using enzyme dosage hemiselulase 3 / 3 on sugarcane bagasse can degrade
hemicellulose into sugar monomers silosa and arabinose. While the enzyme hydrolysis using
cellulase enzyme dosage of 3 / 3 on sugarcane bagasse able to degrade cellulose to glucose
sugar monomers. The figure below shows the increase in reducing sugar concentration (g / L)
at any given stage of treatment.
)L
/g
(
32,00
27,65
35,00
30,00
25,00
20,00
15,00
10,00
5,00
0,00
2,32
is
k
u
d
e
rp
a
lg
tn
o
K
Pra perlakuan
Hidrolisis
asam
Hidrolisis
enzim at ik
Tahapan Perlakuan
Figure 3. Effect of treatment of phase reducing sugar concentration (g / L)
Fermentation
The microorganism used in fermentation of sugarcane bagasse hydrolysates include: P.
stipitis CBS 5773, S. D1/P3GI cerevisiae, and Z. mobilis FNCC 0056. Stage of fermentation
was conducted to determine the ability of each microorganism in ferment the sugar cane
bagasse hydrolyzate to ethanol.
Based on the results shown in Figure 6, the adaptation phase S. cereviseae D1/P3GI,
P. stipitis CBS 5773, and Z. mobilis FNCC happen until 0056 at the 3rd fermentation.
Adaptation phase is a phase of adjustment to new environment, where there is synthesis of
new enzyme according to the media. In this phase does not increase the number of cells but
the cell volume increases. This proves that the three microorganisms capable of using
reducing sugar from sugarcane bagasse hydrolyzate as a nutritional source of growth.
Based on the results of HPLC analysis of hydrolysed cane is used for the fermentation
process
containing
23.78%
Inter national Seminar Biotechnology
glucose,
56.91%
silosa, and
19.31%
arabinose.
P 193
During the ongoing growth of microorganisms, pH control is imperative to maintain the
optimum pH. Decrease in pH value is very real in the first 12 hours of fermentation either by
culture of S. cereviseae D1/P3GI, P. stipitis CBS 5773, and Z. mobilis FNCC 0056. At this
stage, ethanol was formed accompanied with the decrease in pH due to formation of organic
acids. The trend of pH change caused by ammonia in the fermentation media used yeast cells
as a source of nitrogen is converted into NH4 +. NH4 + molecules will merge into the cell as
R-NH 3. In this process H + stays in the media, so that more biomass and increasing time of
fermentation causes H + ions are more and more in the substrate which causes the lower the
pH of the media (Judoamidjojo & Saeed, 1993; Fardiaz, 1988).
Based on the results of HPLC, the final stage of fermentation produced lactic acid
and acetic acid from fermentation using a culture of S. cereviseae D1/P3GI and acetic acid
from fermentation culture by Z. 0056 FNCC mobilis produced. Concentration of ethanol
produced in line with the growth of each culture and the amount of reducing sugar consumed.
In this study, Z. 0056 FNCC mobilis able to ferment sugar into ethanol hydrolyzate highest P.
stipitis CBS 5773 and S. cerevisiae D1/P3GI with a shorter fermentation time, which is 3
hours produced ethanol at 18.99 g / L.
The results of this study reinforced with literature that states that Z. mobilis is able to
change the mix of sugar into ethanol is greater 92% - 94% compared to the yeast which only
reached 88% -90% (Silalahi, 1987). It is possible or ethanol fermentation by Z. mobilis, the
conversion of glucose into two molecules of ethanol produced one molecule of ATP. The low
energy produced by ATP resulting in cell mass of the resulting low and high ethanol
produced (Jeffries, 2005).
Based on research data presented in Figure 7, the cell growth of S. cerevisiae
D1/P3GI in the first 12 hours of fermentation is constantly increasing. This means that S.
D1/P3GI cerevisiae capable of using sugar cane bagasse hydrolysates as a source of nutrition.
This can be seen from the decrease in the amount of sugar in the hydrolyzate peredukasi
accompanied with the production of ethanol amounted to 17.05 g / L. While the research
Rouhollah et al. (2007), fermentation using microorganisms Fermentative S. cerevisiae in
sugar mixture to produce ethanol 0.32 g / L per gram of glucose or by 62% with ethanol
concentrations of 14.25 g / L and ethanol productivity was 0.88 g / L / hour in 48 hours
fermentation time. This proves that S. cerevisiae D1/P3GI able to ferment sugar cane bagasse
hydrolysates with a shorter time which is 12 hours with levels of ethanol produced 17.00% g
/ L. Okur & Nurdan (2006) conducted research on the production of ethanol from waste
sunflower seed using P.stipitis NRRL Y-124 with condition 6 of the initial pH of hydrolyzate
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fermentation and the fermentation process lasts for 55 hours produced ethanol concentration
of 9.66 g / L. While the fermentation of sugar mixed media using P. CCUG18492 stipitis
produces ethanol 0.40 g / L per gram silosa or by 78% and ethanol concentration of 30.23 g /
L with productivity of ethanol is 0.95 g / L / hour in 72 hours fermentation time (Rouhollah et
al., 2007) . While in this study, P. stipitis CBS 5773 is able to ferment sugar cane bagasse
hydrolyzate to ethanol as much as 13.03 g / L in 24 hours fermentation time. Overall, both P.
stipitis CBS 5773, S. D1/P3GI cerevisiae, and Z. 0056 FNCC mobilis each use sugar cane
bagasse hydrolyzate in an optimal as a source of nutrition in the period 0-24 hours coupled
with the optimal ethanol production. Starting at the 24th production of ethanol from each
microorganism has decreased. This happens because the availability of sugar in the
hydrolyzate under 10 g / L or less than 1%. Meanwhile, according Budiyanto (2003) in a
Putri (2008), the optimum concentration of sugar to produce the optimum alcohol content is
14% - 28% (w / v).
(a)
(b)
(c)
Figure 6. Production of ethanol from sugarcane bagasse hydrolysates by (a) Z.
mobilis (B) S. D1/P3GI cerevisiae, (c) P. stipitis CBS 5773.
From the research results obtained can be concluded :
1. Hydrolysis by sulfuric acid with a concentration of 2% (w / w), steam heating temperature
120oC for 60 minutes could be able to hydrolyze lignocellulosic sugars into simple
monomers with reducing sugar concentration of 27.65 g / L with a DE value of 54.88%
2. The best hydrolysis by enzymes in the enzyme dosage hemiselulase 0.001 g / g (1500
units), followed by the cellulase enzyme dosage 0.83 mL / g (0.6970 units). Both enzymes
are able to hydrolyze sugar cane bagasse by 63.52% with a reducing sugar content in
sugarcane bagasse hydrolyzate of 32.00 g / L.
3. Z. 0056 FNCC mobilis able to ferment sugar cane bagasse hydrolyzate to ethanol by 18.99
g / L with the fermentation time of 3 hours, S. D1/P3GI cerevisiae, is able to ferment the
sugar cane bagasse hydrolyzat to ethanol amounted to 17.05 g / L with the fermentation time
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of 12 hours, whereas P. stipitis CBS 5773 is able to ferment sugar cane bagasse hydrolyzate
to ethanol at 13.03 g / L in 24 hours.
ACKNOWLEDGMENTS
Upon completion of this research I would like to thank:
1. Department of Agriculture which has helped fund this research through KKP3T.
2 Program. Ministry of National Education has helped fund education and research through
the Competitive Scholarship Program and P3SWOT.
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Atlas, Ronald M. 1993. Handbook of Microbiological Media. CRC Press Boca Rotan Ann
Arbor London. Tokyo.
Campbell,N.A., J.B. Reece, & L.G. Mitchell. 1999. Biolog Edisi V, Jilid 1. Erlangga.
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