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 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-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, ligno-cellulose and hemiligno-cellulose. Lignoligno-cellulose 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).
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
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
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
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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4 9.50
7
10
.6
50
Pra-perlakuan Hidrolisis asam Asam - enzim
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7 9.53
0 10 .5 63 11 .5 25 11 .9 61
Fermentasi oleh S. cerevisiae
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Fermentasi oleh P. stipitis
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4. 01 6 4. 44 8 4. 68
05.01
5 6. 03 5 6. 62 8 8. 09 0 9. 53 1 10 .5 71 11 .5 28 11 .9 64
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.
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 reducing sugar monomers. According Taherzadeh (2008), lignin is a complex compound that is bound to one another
0,00 5,00 10,00 15,00 20,00 25,00 30,00
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.
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% glucose, 56.91% silosa, and 19.31% arabinose.
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Pra perlakuan Hidrolisis
asam
Hidrolisis enzim at ik 2,32
27,65
32,00
Konsentrasi gula pereduksi
(g/L)
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).
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).
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
(a) (b) (c)
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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