Process Biochemistry 34 (1999) 115 – 119

Ethanol production in solid substrate fermentation using

thermotolerant yeast
N. Kiran Sree a, M. Sridhar a, L. Venkateswar Rao a,*, Ashok Pandey b
a
Department of Microbiology, Osmania Uni6ersity, Hyderabad 500 007, A.P, India
b
Regional Research Laboratory, CSIR, Tri6andrum 695019, Kerala, India

Received 4 November 1997; received in revised form 1 May 1998; accepted 3 May 1998

Abstract

A novel solid substrate fermentation system was used to produce fuel ethanol from sweet sorghum and sweet potato using a
thermotolerant Saccharomyces cere6isiae strain (VS3) and a local isolate of amylolytic Bacilllus sps. (VB9). The process was
carried out on a laboratory scale using broth cultures. Alcohol produced was estimated by gas chromatography after an
incubation time of 72 h at 37 and 42°C. More ethanol was produced in co-culture with a mixed substrate than with the
thermotolerant yeast (VS3) alone. The maximum amount of ethanol produced in co-culture with a mixed substrate was 5 g/100
g of substrate at 37°C and 3·5 g/100 g of substrate at 42°C. © 1999 Published by Elsevier Science Ltd. All rights reserved.

Keywords: Thermotolerant yeasts; Saccharomyces cere6isiae; Solid substrate fermentation; Ethanol; Co-culture; Amylolytic Bacillus

Nomenclature decreases sharply because of greater ethanol inhibition

EC Ethanol concentration in g/100 g substrate
TY (VS3) thermotolerant yeast
AB (VB9) amylolytic Bacillus
SSF Solid substrate fermentation
1. Introduction
There are several potential benefits of using thermo-
tolerant yeasts for use in the production of industrial
alcohol. Cooling costs during the process of ethanol
fermentation are expensive; hence, by using thermotol-
erant yeasts cooling and distillation costs can he re-
duced [1]. In addition, they have faster fermentation
rates. Thermotolerant yeasts are especially attractive in
tropical countries like India, where the temperatures are
high [2]. However, there are only a limited number of
reports on the successful selection and isolation of
yeasts capable of growth or fermentation at or above
40°C [3,4]. As the temperature increases productivity
* To whom all correspondance should be addressed. Tel. 040-
7018951 extn: 246; fax: 91-040-7019020.
[5]. Due to the increasing costs of substrates such as
molasses and increasing demand for fuel ethanol, there
is a need to search for cheaper substrates and to
develop an efficient and less expensive technology so
that product can be made available more cheaply.
Industrial alcohol is produced from various substrates
like molasses, sweet sorghum, maize starch, sugarcane,
sugar beet, tapioca etc. Of these, sweet sorghum is a
significant potential substrate for ethanol production
because of the following advantages [6]: (1) higher
ethanol productivity, (2) good fermentable sugar con-
tent. (3) adaptability to various climatic conditions and
therefore ability to grow throughout the year, (4) re-
quires less fertilizers and water, and (5) total energy
required for production and harvesting of sweet sor-
ghum is less than sugar crops. Although ethanol is
produced by the process of submerged fermentation in
various parts of the world, there is tremendous scope
for solid substrate fermentation (SSF) because of the
following potential advantages [7–9]: (1) less require-
ment of water (especially attractive in summer months
when water is scarce), (2) smaller volumes of the fer-
mentation mash, (3) less physical energy requirement.
(4) smaller capital investment, fewer operating costs
and lower space requirements, (5) reduced reactor vol-

0032-9592/99/$ - see front matter © 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 9 8 ) 0 0 0 7 4 - 0
116 N. Kiran Sree et al. / Process Biochemistry 34 (1999) 115–119

umes, easier product recovery, less liquid water to be
disposed of and hence less pollution problems. There
are some reports of ethanol production using SSF, but
few reports are available with thermotolerant yeasts in
these systems. In this investigation the use and suitabil-
ity of thermotolerant yeasts for SSF to produce ethanol
with different substrates were investigated. The sub-
strates used include sweet potato, sweet sorghum and
molasses in various combinations with different particle
sizes both in co-culture and with thermotolerant yeasts
alone at temperatures of 37° and 42°C.
2. Materials and methods
Dried sweet sorghum grains were ground to three
particle sizes using a food processor (60 – 100 m, 120–
160 m and 500–600 m). Fresh sweet potatoes were made
into one particle size (120 – 160 m) and dried in an oven
at 80°C for 2 h. About 100 g of the above substrates of
different particle sizes were taken into 250 ml conical
flasks with and without molasses and autoclaved at 10
lbs for 15 min.
2.1. Inoculum preparation
Thermotolerant yeast isolate (VS3) was isolated in
this laboratory from soil samples collected from hot
regions of Kothagudem Thermal Power Plant located
in Paloncha, Khammam District, A.P. The yeast was
identified as Saccharomyces cere6isiae and was main-
tained on YEPD medium containing 1% yeast extract,
2% peptone, 2% dextrose at pH 5·5. Inocula were
grown in 50 ml YEPD in 100 ml flasks. The amylolytic
Bacillus used in the present study was a local isolate
(VB9) isolated in this laboratory. This was identified as
Bacillus sps and was maintained on 2% starch agar and
incubated into 50 ml starch broth containing 2% starch,
1% beef extract, 1% peptone and 0·5 g NaCl, pH 7·2 in
150 ml conical flasks. The inoculated flasks were incu-
bated on a laboratory shaker incubator at 30°C for 24
h at 200 rpm. After incubation 50 ml culture was
transfered to sterile tubes and centrifuged for 15 min at
20°C and 300 rpm (5 g). After the first centrifugation
the supernatant was discarded and the yeast and bacte-
rial pellets suspended in 0·1 M phosphate buffer. This
process was repeated. After the second centrifugation
the supernatent was discarded and the cells collected
were resuspended in 0·1 M phosphate buffer. The ab-
sorbance of yeast culture was adjusted to 1·6 at 600 nm
which is equivalent to 1·62× 102 cells and the ab-
sorbance of the bacterial culture was adjusted to 1·4 at
600 nm which is equivalent to 1×1012 bacilli. The
numbers of both bacterial and yeast cultures were
obtained by plating tests.
2.2. Fermentation studies
Eight groups of the following experiments were car-
ried out in 250 ml conical flasks with different sub-
strates, quantities and particle sizes. Details of the
experiments are shown in the Table 1. 100 g of sub-
strate was placed in 250 ml and 6% inoculum added. In
co-culture flasks 3% of TY and 3% AB was added. In
mixed substrate flasks 50 g of each substrate was added.
No additional nutrients were added to the flasks. The
moisture content of 70% was maintained by adding
autoclaved distilled water. After inoculation the con-
tents were mixed thoroughly and one set of flasks was
incubated at 37°C and another set at 42°C. After an
incubation time of 72 h, ethanol was recovered by
adding 50 ml of autoclaved distilled water and the
contents after thorough shaking were centrifuged at
3000 rpm (500 g) for 20 min at 20°C to separate solids
and cells from the fermented broth. The supernatent
was filtered through moistened Whatman no. 1 filter
paper to obtain a clear solution. The ethanol content
was analysed by gas chromatography (CIC pro-86
packed with Chromosorb 101, 80–100 mesh size). The
chromatogram was run at 160°C oven temperature and

Table 1
SSF experiments with different substrates for ethanol production using thermotolerant yeast and Bacillus sps

S. no. Substrate Particle size Inoculum

1. Sweet sorghum (1) 60–100 m 6% VS3

(2) 120–160 m
(3) 500–600 m
2. Sweet sorghum (1) 60–100 m 3%VS3 & 3%VB9
(2) 120–160 m
(3) 500–600 m
3. Sweet potato 120–160 m 6%VS3
4. Sweet potato 120–160 m 3%VS3 & 3%VB9
5. Sweet potato & sweet sorghum 120–160 m of sweet potato & 60–100 m of sweet sorghum 6% VS3
6. Sweet potato & sweet sorghum 120–160 m of sweet potato & 60–100 m of sweet sorghum 3%VS3 & 3%VB9
7. Sweet sorghum & molasses 60–100 m of sweet sorghum 6%VS3
8. Sweet sorghum & molasses 60–100 m of sweet sorghum 3%VS3 & 3%VB9
 N. Kiran Sree et al. / Process Biochemistry 34 (1999) 115–119 117

Fig. 1. Influence of particle size in sweet sorghum for ethanol

production by solid substrate fermentation.

170°C injection temperature using N2 as a carrier gas

and H2 as a flaming gas.

Fig. 2. Influence of co-culture on solid substrate fermentation for

3. Results and discussion ethanol production using substrates of different particle sizes.

In SSF productivity depends on a number of factors

including particle size, moisture content, size of the
inoculum, nature of the substrate, temperature etc.
Since ethanol fermentation is anaerobic, the particle
size of the substrates affects the process significantly.
As the particle size increased, ethanol production de-
creased (Fig. 1). More ethanol was therefore produced
with smaller particle sizes (60 – 100 m) [9] due to closer
packing densities of substrate and reduction of void
space between particles. This tends to decrease the area
for heat transfer and gas exchange with the surrounding
atmosphere, as a result of which oxygen diffusion will
be decreased by generating anaerobic conditions
quickly for ethanol fermentation. The moisture content
of 70% proved to be optimal for ethanol production [7].
Sweet sorghum usually contains sufficient sugar content
in addition to starch for the production of ethanol by
TY alone [6]. Results on production of ethanol by SSF
with individual substrates using different particle sizes
are shown in Table 2. These results indicate that more
ethanol (3·7 g/100 g of substrate) was produced with
sweet sorghum of particle size (60–100 m) in co-culture
at 37°C than with TY alone (2·6 EC). This is because
Saccharomyces cere6isiae is non-amylolytic and the AB
converted the starch to sugars, thereby increasing the
concentration of total reducing sugars for the produc-
tion of ethanol. An increase in temperature from 37° to
42°C resulted in the production of a smaller concentra-
tion of ethanol. This increase in temperature resulted in
enzyme destruction cell membrane damage and inacti-
vation of yeast cells which resulted in decreased
biomass [10]. More ethanol was produced at 37°C from
sweet sorghum of particle size (60–100 m) which was
3·7 EC in co-culture and 2·6 EC with TY and at 42°C
less ethanol was produced 2·6 EC in co-culture and 1·4

Table 2
Production of ethanol by SSF with individual substrates using different particle sizes

S. no. Substrate Ethanol at 37°C g/100g substrate Ethanol at 42°C g/100g substrate

1. Sweet sorghum* (60–100 m) 3·7 2·6

2. Sweet sorghum† (60–100 m) 2·6 1·4
3. Sweet sorghum* (120–160 m) 3·0 1·6
4. Sweet sorghum† (120–160 m) 2·0 1·20
5. Sweet sorghum* (500–600 m) 1·4 0·8
6. Sweet sorghum† (500–600 m) 0·61 0·35
7. Sweet potato* (120–160 m) 3·0 1·2
8. Sweet potato† (120–160 m) 1·6 0·7

* Co-culture
†
VS3 only
118 N. Kiran Sree et al. / Process Biochemistry 34 (1999) 115–119

Sweet potatoes usually contain 3–3·5% free sugars in

Fig. 3. Influence of mixed substrates in solid substrate fermentation
for production of ethanol.
addition to the starch content [11]. The concentration
of ethanol produced from sweet potato was less than
that obtained from sweet sorghum (3 EC and 1·2 EC at
37°C and 42°C in co-culture). With TY alone the
concentration of ethanol was also lower, 1·6 EC and 0·7
EC at 37°C and 42°C, respectively. The above results
indicate that sweet sorghum was a better substrate for
SSF than sweet potato.
The influence of mixed substrates on ethanol produc-
tion by SSF process is shown in Fig. 3. More ethanol
was produced with mixed substrates than with individ-
ual substrates. These results (Table 3) indicate that
more ethanol was produced with sweet sorghum and
sweet potato in co-culture, 5 EC and 3·5 EC; with TY
alone, 3·6 EC and 1·7 EC was produced at 37°C and
42°C, respectively. These results indicate that mixed
substrate with a co-culture was a more effective SSF
process for the production of ethanol.
Several investigators were of the opinion that SSF is
more suitable and economical than conventional sub-
merged fermentation in the production of a variety of
fermentation products [12,13]. The present results on
ethanol production using SSF clearly support their view
and also suggest that thermotolerant yeast can be used
to produce more ethanol using SSF.

EC with TY. With an increase in particle size from

60 – 100 m to 120–160 m the concentration of ethanol
produced was less (3 EC and 1·6 EC in co-culture at
37°C, respectively). With TY alone the ethanol pro-
duced decreased from 3 EC to 2 EC and 1·6 EC to 1·2
EC at 37°C and 42°C, respectively (Fig. 2). The concen-
tration of ethanol produced with particle size 500–600
m was much less, 1·4 EC and 0·8 EC in co-culture at 37
and 42°C, respectively, and with TY alone only 0·61
EC and 0·35 EC of ethanol was produced at 37 and
42°C, respectively. The results shown in Table 2 clearly
indicate that as the particle size increased less ethanol
was produced.
4. Conclusions
Ethanol may be produced effectively in SSF using a
co-culture of Saccharomyces cere6isiae and a starch
degrading Bacillus sp.
Acknowledgements
We are grateful to the University Grants Commis-
sion, New Delhi and the Department of Biotechnology,
New Delhi for grant in aid during this investigation.

Table 3
Ethanol production by SSF using mixed substrates

S. no. Substrate Ethanol at 37°C (g/100g substrate) Ethanol at 42°C (g/100g substrate)

1. Sweet sorghum* (60–100 m)

Sweet potato (120–160 m) 5·0 3·5
2. Sweet sorghum† (60–100 m)
Sweet potato (120–160 m) 3·6 1·7
3. Sweet sorghum* (60–100 m) & molasses 1·8 0·8
4. Sweet sorghum† (60–100 m) & molasses 0·9 0·4

* Co-culture
†
VS3 only
 N. Kiran Sree et al. / Process Biochemistry 34 (1999) 115–119
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