RESEARCH ARTICLE | JUNE 14 2016

Bioethanol fermentation from sugarcane bagasse using ragi

tape 
Endang Sutariningsih Soetarto; Riana Nindita Putri

AIP Conf. Proc. 1744, 020020 (2016)

https://doi.org/10.1063/1.4953494

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 Bioethanol Fermentation from Sugarcane
Bagasse Using Ragi Tape
Endang Sutariningsih Soetarto 1, a) and Riana Nindita Putri1
1)
Laboratory of Microbiology, Faculty of Biology, Universitas Gadjah Mada,
Jl.Teknika Selatan, Yogyakarta 55281, Indonesia
a)
Corresponding author: annisah-endang@ugm.ac.id

Abstract. The purpose of the research was to use sugarcane bagasse as a fermentation substrate for bioethanol production
and to determine the effect of yeast types from traditional inoculum for tapay making (ragi) on ethanol yield. Sugarcane
bagasse was composed of lignocellulosic materials and collected from a sugar factory in Yogyakarta. Yeasts from ragi
were used to break down the cellulose containing in sugarcane bagasse. Dilute alkali hydrolysis of sugarcane bagasse was
performed to obtain sugarcane bagasse cellulosic hydrolysate. This hydrolysate was supplemented with nutrient
formulations to prepare the fermentation medium to the yeast activity. Saccharomyces cerevisieae pombe SPJ, strain
YRT-01, strain YRT-02 and YRT-03 (isolated from Ragi) were used in fermentations carried out using batch culture

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system in Erlenmeyer flasks incubated in a rotator shaker (100 rpm for 72 h) at room temperature. Yeast activity was
determined based on their growth (OD660 nm) and reducing sugar concentration (A540 nm – 575nm) with xylose or glucose as
standard. The ethanol concentration was determined by Conway method. The results showed that bagasse hidrolyzate
contains (6.9 to 8.0) g · L–1 of xylose and glucose. The highest ethanol concentration was obtained 1.387 % by strain
YRT-03 during 4 d incubation, indicating that yeast strain YRT-03 was the promising yeast for sugarcane bagasse
fermentation to produce ethanol under temperature at 32 oC and pH 5.5. This strain resembles to Saccharomyces
cerevisiae pombe.

Keywords: Bioethanol, Saccharomyces sp., sugarcane bagasse, xylan, xylose, yeast.

INTRODUCTION
Biological conversion of cellulosic materials and industrial residues into fuels could be a promising and
environmentally friendly fuel alternative to currently expensive petroleum derivative. Due to the depleting amount
of fossil fuels, alternative energy sources need to be renewable, sustainable, efficient, cost-effective and safe. A
process design for conversion of cellulosic materials to ethanol estimated that cost of ethanol production by this
process was slightly more expensive than ethanol derived from petroleum. Lignocelluloses technology has
stimulated a study of the microbial hydrolysis mechanism of cellulosic materials, particularly hemicelluloses.
Hemicelluloses are one of the most abundant polysaccharides derived from plant constituents and agricultural waste.
The solid residual fraction from the sugarcane stem milling was called bagasse produced more than million tons [1].
Sugarcane bagasse contains glucan (42.7 %), xylan (21.0 %) and arabinan (0.6 %). Xylan binds to cellulose, lignin
and other polysaccharides [2, 3]. Sugarcane hemicellulose represents about one-third of the carbohydrate fraction
available in bagasse [4]. Dry sugarcane bagasse contains up to 32 % with D-xylose as the major pentose [5, 6]. This
bagasse may serve as an excellent raw material for generation ethanol production due to the presence of high
amount glucose and xylose [5, 7]. Originally the utilization of xylose for ethanol production presented a problem
because xylose was nonfermentable sugar which is not fermented by yeasts, particularly native Saccharomyces
cerevisiae Hansen, 1883 [8]. However, there are some yeasts able to ferment xylose to ethanol or other products,
such as Pachysolen tannophilus Boidin and Adzet, 1957 and Pichia stipitis Pignal, 1967 [9, 10, 5], Candida
tropicalis (Castellani) Berkhout, 1923 and Candida shehatae Kurtzman, 1990 [11, 5, 12], Scheffersomyces shehatae

Towards the sustainable use of biodiversity in a changing environment: From basic to applied research
AIP Conf. Proc. 1744, 020020-1–020020-7; doi: 10.1063/1.4953494
Published by AIP Publishing. 978-0-7354-1401-3/$30.00

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Urbina, 2012 [13]. It is now considered possible to hydrolyze xylan enzymatically to simple reducing sugar, and use
it to produce ethanol [14]. Xylose is not fermented by S. cerevisiae. Alternatively, a process is possible whereby the
enzymatical hydrolysis to xylose is coupled with yeast fermentation of xylose to ethanol.
Some different strategies have been envisioned to convert these polysaccharides into fermentable sugars. One of
them, the hemicellulose (xylan) fraction can be hydrolyzed with dilute acids [15]. Dilute acid hydrolysis is an
efficient process for the hemicellulose depolymerization into variety of priority pentose sugars such as arabinose and
mainly xylose. This xylose and arabinose can be fermented to produce ethanol, although some other byproducts
such as furans, 5-hydroxymethylfurfurals, phenolics and weak acids considering inhibitors to microbial metabolism.
There is an important consideration when taking account of the fact that conversion of xylan hydrolysates to ethanol
may be difficult in the high salt concentrations which result from acid hydrolysis since yeast growth is inhibited by
high salt concentration [16]. Therefore, it is necessary to reduce the concentration of these inhibitors before using
the hemicellulosic hydrolysate into ethanol via microbial fermentation.
Biomass energy has developed involving the activity of microbes to convert biomass into biofuels (ethanol or
biogas) and biodiesel [17, 18]. Bioethanol is simply ethanol as a renewable energy source made by fermenting the
sugar and starch components of the plant [19]. Bioethanol is an alternative fuel with the oxygen content is high
enough (35 %) with an octane rating of more than 118 and removing the CO gas is low (19 % to 25 %), so that the
nature of the fuel being environmentally friendly [20]. In the current study, traditional inocula of tapey called ragi
was used for the production of ethanol from sugarcane bagasse hydrolyzate and yeast isolates of the spirit plant of
Madukismo used as a model of ethanol-producing fermentative yeast. The performance of a novel xylose-
fermenting yeast, Saccharomyces cerevisieae pombe SPJ, Candida shehatae strain YRT-01, strain YRT-02 and
YRT-03 (isolated from Ragi), was evaluated under batch fermentation conditions using sugarcane bagasse
hemicellulosic hydrolysate as a carbon source.
The aim of this work was to use sugarcane bagasse as fermentation substrate for bioethanol production and to
determine the effect of yeast types from traditional inoculum for tapay making (ragi) on ethanol yield; and to
investigate acid hydrolysis of sugarcane bagasse to obtain the hydrolysate containing high fermentable sugar and

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low inhibitor concentrations for use as a substrate for bioconversion under batch fermentation by yeast from
traditional inocula.

MATERIALS AND METHODS

Preparation of Hemicellulose Hydrolysate

Powdered Sugarcane bagasse mill was provided by Madukismo sugarcane industry located in Yogyakarta. The
powdered bagasse was dried with oven dried (at 80 °C) until the dry weight was constant and then was sieved the 30
mesh to obtain fine particles (less than 5 mm). The particles were kept at a low temperature until use.

Lignin Extraction (Delignification Process)

The extraction was performed at room temperature for 24 h using 10 % NaOH solution under a liquor/solid ratio
of 100 mL liquor · 5 g–1 dry weight of sugarcane bagasse [21]. The lignin content in the soluble fraction was
measured by spectrophotometrically (absorbance of 649 nm), filtered and measured pH then neutralized using 6 N
H2SO4.

Acid Hydrolysis
After the lignin extraction, the remaining solid fraction was hydrolysed. The solid fraction was washed twice
with distilled water and dried at 70 °C for 12 h or until dry weight was constant. The hemicellulosic hydrolysate was
prepared in hydrolysis vessel (100 L) using 98 % H2SO4. All conditions were carried out using an LSR of 15 mL
liquor · g–1 dry weight of sugarcane bagasse and then was autoclaved for 20 min at 121 °C. The hydrolysate was
separated from solid material via filtration. The hydrolysate was then detoxified following the methodology
established by Chandel et al. [22].

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 Microorganisms and Growth Conditions
Saccharomyces cerevisieae pombe SPJ (strain for spirit production), strain YRT-01, strain YRT-02 and YRT-03
(isolated from ragi) were used in fermentations. They were grown separately in Peptone Glucose yeast extract
(PGY) medium with the composition of (g · L–1): a 10-Peptic digest of animal tissue, 50-Yeast extract, 20-Dextrose,
15-Agar. Final pH (at 25 °C): 6.0 ± 0.2. PGY (G9663 Sigma): Suspend 50 g of PGY powder in 1 000 mL of
distilled water. Heat boiling dissolved the medium completely. Sterilization was conducted by autoclaving at 121 °C
for 15 min.

Preparation of Fermentation Medium and Conditions

The hydrolysate from sugarcane bagasse with and without detoxification (550 mL) was added to PGY medium
with a final volume of 1 L, used as a fermentation medium for ethanol production, then plus 0.25 g Ammonium
Hydrogen Phosphate. A mixture of 1 N HCl added to achieve pH 5 to pH 6. The mixture was divided into four (500
mL) Durant bottles and four (50 mL) flasks for inocula (fermentation starter). Ethanol fermentation was carried out
in a batch system in vessel glass laboratory scale with a working volume of 500 mL at room temperature. The
culture was agitated at 100 rpm (1 rpm = 1/60 Hz).The pH of the culture was kept at 6.0 by the addition of 2.5 M
NaOH.

Determination of Cell Growth, Sugar, and Product Concentrations

Yeast growth was monitored spectrophotometrically at 562 nm (Shimadzu, Japan) and converted to biomass
concentration from a standard calibration curve. Xylose, glucose, and ethanol were determined. The total reducing
sugar was measured by the dinitrosalicylic acid (DNS) method [23]. Ethanol concentrations were determined by
Conway micro diffusion analysis and Redox titration. Ethanol is oxidized to ethanoic acid when ethanol reacts with

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an excess of potassium dichromate solution (0.05 N) and then determined by adding potassium iodide (50 % KI)
solution. Potassium iodide reacts with potassium dichromate and iodine. Then the iodine is treated with a standard
solution of Sodium Thiosulphate (0.1 N). The titration reading is used to calculate the ethanol content after
fermentation. The Conway unit center was placed 1 mL potassium dichromate and the sample was placed in the
center. A Conway unit was used as a blank and in that unit 1 mL distilled water was used as a sample.

RESULT AND DISCUSSION

The first steps in the preparation of hemicellulose were carried out as previously reported [5], by alkali treatment
and subsequent solvent precipitation. A further step, consisting of treatment with sulphuric acid was added to
oxidize the alkali-lignin residue of the hemicelluloses. The total yield of hemicelluloses in the experiments was
approximately 28.5 %. There was no detectable reducing sugar. Analysis by Thin Layer Chromatography (TLC),
total acid hydrolysis of the hemicelluloses revealed intermediate products and the highest spot was xylose (Fig. 1).
The hydrolysate from sugarcane bagasse revealed that the reducing sugar released during that 24 hours
incubation equivalent to 10.21 g · L–1. Only a minor further release of reducing sugar to 10.42 g · L–1 occurred when
the incubation period was extended to 48 h. The results were nearly the same with previous experiment [5].
Sugarcane bagasse hydrolysate contained glucose and xylose or other reducing sugar (Table 1). The products were
mostly xylose as reducing sugar quite high (6.9 g · L–1) that stimulates the growth of three isolates (strain YRT-01,
YRT-02 dan YRT-03) to ferment xylose into ethanol.

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 Xylose
Arabinose

Glucose

FIGURE 1. Thin Layer Chromatogram (TLC) of acid bagasse hydrolysate.

The hydrolysate from sugarcane bagasse revealed that the reducing sugar released during that 24 h incubation
equivalent to 10.21 g · L–1. Only a minor further release of reducing sugar to 10.42 g · L–1 occurred when the
incubation period was extended to 48 h. The results were nearly the same as previous experiment [5]. Sugarcane
bagasse hydrolysate contained glucose and xylose or other reducing sugar (Table 1). The products were mostly
xylose as reducing sugar quite high (6.9 g · L–1) that stimulates the growth of three isolates (strain YRT-01, YRT-02,
dan YRT-03) to ferment xylose into ethanol.
Yeast isolates (strain YRT-01, YRT-02 and YRT-03) fermented bagasse hydrolysate containing xylose into
ethanol in higher amount than those produced by Schizosaccharomyces pombe Lindner, 1893, strain YPG-01.
Previous experiment had shown that some yeast e.g. Candida tropicalis (Castellani) Berkhout, 1923 and
Pachysolen tannophilus Boidin and Adzet, 1957 produced ethanol from xylose under aeration conditions [5].
TABLE 1. Composition of sugarcane bagasse hydrolysate by acid (reducing sugar (g · L–1)

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Bagasse Hydrolysate
Analyses Bagasse Fluid
0 24 48
Reducing sugar 1.80 2.02 15.21 10.42
Xylose 0.68 0.68 6.90 5.25
Glucose 0.90 1.02 8.00 5.00

Xylose Fermentation to Ethanol

All isolates showed their abilities to produce ethanol from D-xylose. Production of ethanol was influenced by
concentration of xylose. In the experiment, bagasse hydrolysate media containing 8.365 % of reducing sugar was
used as a fermentation substrate. The four yeast strains were able to utilize xylose (Fig. 2). Figure 2 showed that all
isolates produced the highest ethanol concentration around (0.441 to 1.387) % after 96 h incubation from (0.088 to
0.100) g · L–1 reducing sugar. Decreasing the amount of reducing sugar caused by the conversion of sugar into
ethanol by yeast. The maximum level of ethanol recorded in culture of YRT-03 was 1.387 %. In neither case was
sugar completely depleted until 5 d (120 h); presumably, sugar and ethanol were utilized in the later stages of
fermentation. However, colonies of strain YRT-03 grew slowly on bagasse hydrolysate (Table 2). Optimal acidity
(pH) for bagasse hydrolyzate fermentation by yeast was pH 5.0 to pH 6.0 [24, 25]. Three isolates actively fermented
bagasse hydrolyzate occurred at pH 5.0 and pH decreased during fermentation due to the accumulation of
fermentation products.
Culture growth rate affected the speed of product formation. Ethanol formation process occurred and lasted at
the end of exponential growth phase generated an extracellular product. The relationship between the cell growth
and ethanol production runs in parallel, but inversely proportional to the reducing sugars in the medium [26]. Figure
2 showed ethanol production increased along with the growth of biomass accompanied by decreasing sugar levels
due to activities of microbes in the fermentation medium.

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 (h) (h)
a. b.

(h)
(h)
c. dd.

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FIGURE 2. Yeast isolates activity: a). YRT-01; b). YRT-02; c). YRT-03; and d). YPG-01 growth (OD660 nm) on 8.365 %
sugarcane bagasse hydrolysate after 120 h incubation, at room temperatures.
TABLE 2. Comparison of bagasse hydrolysate fermentation by YRT-01, YRT-02, YRT-03 after 120 h incubation, at room
temperatures
Cultures Time of Fermentation (h) Growth (OD660 nm) pH Reducing Sugar (g · L-1) Ethanol (g · L-1)
YRT-01 0 0.013 5 0.227 0.002
24 0.074 5 0.221 0.157
48 0.162 5 0.204 0.249
72 0.182 4 0.181 0.284
96 0.330 4 0.168 0.366
120 0.352 4 0.139 0.411
YRT-02 0 0.013 5 0.253 0.001
24 0.338 5 0.113 0.222
48 0.129 5 0.123 0.386
72 0.181 4 0.112 0.405
96 0.201 4 0.100 0.578
120 0.229 4 0.088 0.623
YRT-03 0 0.025 5 0.240 0.014
24 0.106 5 0.026 0.735
48 0.376 4 0.013 0.903
72 0.402 4 0.013 1.195
96 0.404 3 0.002 1.387
120 0.366 3 0.020 1.105
YPG-01 0 0.010 5 0.230 0.012
24 0.104 5 0.033 0.777
48 0.236 4 0.019 0.894
72 0.288 4 0.017 1.057
96 0.310 4 0.006 1.188
120 0.293 3 0.003 1.206

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 The highest ethanol produced from sugarcane bagasse hydrolysate performed by strain YRT-03 (1.387 %) up to
4 d incubation. Previous research showed that most Candida sp. was capable of fermenting xylose under anaerobic
conditions, even though ethanol product was limited. Under anaerobic conditions xylose largely converted into
xylitol and only small fractions converted to ethanol [11, 5]. D-xylose fermented into ethanol by yeast involving
xylose reductase and xylitol dehydrogenase. Yeasts were not capable of fermenting D-xylose only involving
NADH-xylose reductase other than NADPH-xylose reductase [27].
This research revealed that all yeast isolates from ragi tapey were able to ferment bagasse hydrolyzate, especially
strains YRT-03 with an ethanol concentration of 1.387 % after four day incubation. The product was higher than
those produced by Schizosaccharomyces pombe Lindner, 1893, strain YPG-01, although not significantly different.
Previous studies Candida shehatae Kurtzman, 1990 [11], Candida tropicalis (Castellani) Berkhout, 1923 and
Pachysolen tannophylus Boidin and Adzet, 1957 [5] produce more than 1 % ethanol from the medium containing
2 % xylose and xylooligomer, respectively. While Candida chilensis Pollard, 2006, Candida entomophila (Scott)
van der Walt & Klift, 1971, Candida insectamans (Scott) van der Walt & Klift, 1972, Candida succiphila Lee &
Komag, 1980 [11]; and Scheffersomyces shehatae Urbina, 2012 [13] produced only (0.1 to 1) % ethanol. Therefore,
strain YRT-03 promises yeast to produce bioethanol from hemicelluloses.
Based on its properties, strain YRT-02 resembled Saccharomyces sp. not capable of fermenting xylose into
ethanol but still produced ethanol with low concentration (Table 2). Saccharomyces cereviciae generally ferment
glucose into ethanol with 97 % efficiency [4] and no fermentation for xylose and arabinose [28]. Only strain YRT-
03 showed high and slightly higher than strain PGY-01 activity on the bagasse hydrolyzate fermentation. It was
assumed that the strain was tolerant to inhibitors [29] and influenced by substrate concentration.
Overall all three yeast isolates in this research, except strain YRT-03 was intolerant to ethanol and other
intermediate product. Fermentation products accumulation in the medium were toxic to the yeast and inhibited
xylose-reductase enzyme synthesis [30] and also resulted in cell membranes fluidity decreased [31]. Identification
result showed strain YRT-03 had some characteristics resembled Saccharomyces cerevisiae pombe but in some cell

20 September 2023 07:17:59

morphological characteristics mostly similar to Candida sehatae .

CONCLUSIONS
The use of sugarcane bagasse for fermentation substrate utilizing alternative local yeast isolates from traditional
inocula (tapay ragi) for bioethanol production is economically advantageous and has been standing out. Three yeast
strains (YRT-01, YRT-02 and YRT-03) as bioethanol producers have been successfully isolated from ragi. In order
to obtain fermentable sugars, sugarcane bagasse was hydrolyzed using acid for 24 h producing bagasse hydrolysate
that can be converted into ethanol by those yeasts. These isolates have potential for an efficient conversion of
lignocellulosic materials into products of bioethanol, especially strain YRT-3 having characters resembled
Saccharomyces cerevisiae pombe. The search for microbial strains suitable for producing enzymes with
characteristics appropriates to the bioethanol processes to which they are intended of great importance.

ACKNOWLEDGEMENT
This research was partly supplied by Sugar Cane and Methylated Spirits Factory, Madukismo, Yogyakarta,
especially for the supply of sugarcane bagasse and yeast strain, are highly acknowledged. The authors are thankful
to Fitri and Technician of Laboratory of Microbiology, Faculty of Biology, Universitas Gadjah Mada for valuable
technical assistance.

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