International Seminar Biotechnology P 187 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 120 o C; the sulphuric acid hydrolysis was best by using a 2% (w/w) sulphuric acid solution and heating for 60 minutes at 120 o C; 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 bio- ethanol 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 ligno- cellulose 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 b erlignoselulosa 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
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International Seminar Biotechnology P 187
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 bio-ethanol 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 ligno-
cellulose 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 berlignoselulosa
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
International Seminar Biotechnology P 188
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.
International Seminar Biotechnology P 189
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.
International Seminar Biotechnology P 190
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.
Remark: a. 1%, 100oC, 60 menit b. 1%, 100oC, 90 menit c. 1%, 110oC, 60 menit d. 1%, 110oC, 90 menit e. 1%, 120oC, 60 menit f. 1%, 120oC, 90 menit
g. 1,5%, 100oC, 60 menit h. 1,5%, 100oC, 90 menit i. 1,5%, 110oC, 60 menit j. 1,5%, 110oC, 90 menit k. 1,5%, 120oC, 60 menit l. 1,5%, 120oC, 90 menit
m. 2%, 100oC, 60 menit n. 2%, 100oC, 90 menit o. 2%, 110oC, 60 menit p. 2%, 110oC, 90 menit q. 2%, 120oC, 60 menit r. 2%, 120oC, 90 menit
0
5
10
15
20
25
International Seminar Biotechnology P 191
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:
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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