hydrolysis of sugarcane bagasse (saccharum officinarum l...

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


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.


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.


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


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

MV

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4.003

4.677

4.940 5.6

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6.609

7.954 9.5

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Pra-perlakuan Hidrolisis asam Asam - enzim

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4.44

9 5.02

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6.03

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6.61

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812

7.97

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8.91

7 9.53

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63

11.5

2511

.961

Fermentasi oleh S. cerevisiae

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4.017

5.030

5.534

6.390

6.610

10.51

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Fermentasi oleh P. stipitis

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4.448

4.680 5.0

15

6.035

6.628

8.090

9.531

10.57

1

11.52

8

11.96

4

Fermentasi oleh Z. mobilis


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

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20,00

25,00

30,00

Dosis 1/3 Dosis 2/3 Dosis 3/3


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.

0,005,00

10,0015,0020,0025,0030,0035,00

Pra perlakuan Hidrolisis asam

Hidrolisis enzimatik

2,32

27,6532,00

Konsentrasi gula pereduksi (g/L)

Tahapan Perlakuan


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


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)

Figure 6. Production of ethanol from sugarcane bagasse hydrolysates by (a) Z. mobilis (B) S. D1/P3GI cerevisiae, (c) P. stipitis CBS 5773.


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.

Reference Apriyantono, A., Dedi Fardiaz, Ni Luh Puspitasari, Sedarnawati, & Slamet Budiyanto. 1989.

Analisis Pangan. Pusat Antar Universitas Pangan dan Gizi. Institut Pertanian Bogor.

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. Jakarta.

Caputi Jr. Arthur, Masao Ueda, & Thomas Brown. 1968. Spectrophotometric Determination of Ethanol in Wine. The American Society for Enology and Viticulture, 19:3:160-165.

Fardiaz, S. 1988. Fisiologi Fermentasi. Pusat Antar Universitas Insitut Pertanian Bogor bekerjasama dengan Lembaga Sumberdaya Informasi-IPB.

Fengel, D. 1995. Kayu. Kimia, Ultrastruktur, Reaksi-Reaksi. Penerbit Gadjah Mada University Press. Yogyakarta.

Gaur. 2006. Process Optimation For The production Of Etanol Via Fermentation. Department Of Biotechnology And Anvironmental Science Thapar Institute Of Engg & Technology.

Gunasekaran, P., T. Karunakaran, & M. Kasthuribai. 1986. Fermentation Pattern of Zymomonas mobilis Strains On Different Substrates—a Comparative Study. J. Biosci Volume 10. No. 2 pp. 181-186.

Gurav, M.S., & G.S. Geeta. 2007. Effectivenes of Fungal Pretreatment of Agro Residues on Etanol Production by Yeast and Zymomonas mobilis. Karnataka J. Agric.Sci, 20(2) : 301-304.

Indriani dan Sumiarsih. 1992. Analisis Serat Bagas. http://bioindustri.blogspot.com/ 2008/04/ampas-tebu.html. Diakses tanggal 22 Juli 2009.

Inoue, H., Sutipa, T., Akira, K., & Shinichi Y. 2006. Biomass Technology Research Center. Natural Institute of Advanced Industrial Science and Technology. AIST, http://unit.aist.go.jp/btrc/ci/

Judoamidjojo, M., A. A. Darwis, & E. G. Sa’id. 1992. Teknologi Fermentasi. Penerbit Rajawali. Jakarta.


Kompala, D.S. 2001. Maximizing Ethanol Production by Engineered Pentose-Fermenting Zymomonas mobilis. Department of Chemical Engineering University of Colorado. Boulder.

Lee, Y. J. 2005. Oxidation Of Sugarcane Bagasse Using A Combination Of Hypochlorite And Peroxide. A Thesis Submitted to The Graduate Faculty of the Louisiana State University.

Morohoshi, N. 1991. Journal of Wood Chemistry and Technology (4): 111–128. Murni, R., Suparjo, Akmal, & BL. Ginting. 2008. Buku Ajar Teknologi Pemanfaatan untuk

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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