Journal of Biotechnology 129 (2007) 415–420

An novel immobilization method of Saccharomyces cerevisiae

to sorghum bagasse for ethanol production
Jianliang Yu, Xu Zhang, Tianwei Tan ∗
Beijing Key Lab of Bioprocess, College of Biology Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China
Received 19 September 2006; received in revised form 19 December 2006; accepted 2 January 2007

Abstract
Natural sorghum bagasse without any treatment was used to immobilize Saccharomyces cerevisiae at 0.6 ± 0.2 g dry cell weight (DCW)/g
dry sorghum bagasse weight (DSW) through solid-state or semi-solid state incubation. The scanning electron microscopy (SEM) of the carriers
revealed the friendship between yeast cells and sorghum bagasse are adsorption and embedding. The ethanol productivity of the immobilized cells
was 2.24 times higher than the free cells. In repeated batch fermentation with an initial sugar concentration of 200 g/L, nearly 100% total sugar was
consumed after 16 h. The ethanol yield and productivity were 4.9 g/g consumed sugar on average and 5.72 g/(L h), respectively. The immobilized
cell reactor was operated over a period of 20 days without breakage of the carriers, while the free cell concentration in the effluent remained less
than 5 g/L thoughout the fermentation. The maximum ethanol productivity of 16.68 g/(L h) appeared at the dilution rate of 0.3 h−1 .
© 2007 Elsevier B.V. All rights reserved.

Keywords: Immobilization; Sorghum bagasse; Ethanol; Repeated batch fermentation; Continuous fermentation

1. Introduction
Immobilized cell technology has been suggested as an
effective means for improving ethanol fermentation. The immo-
bilization of cells leads to higher cell densities with consequent
increases in reaction rates and productivity. As a result, shorter
residence time and smaller reactor size can be employed.
Ethanol production by immobilized yeast cells has been
extensively investigated during the last few decades. The most
widely used immobilization methods are based on cell entrap-
ment in gels, such as carrageen and Ca alginate (Luong, 1985;
Godia et al., 1987; Hamamchi and Ryu, 1987). Other meth-
ods are based on passive adhesion to the surfaces, such as glass
beads, stainless steel wire spheres (Van Haecht et al., 1985; Vega
et al., 1987; Sho et al., 2001). For the former, the main draw-
back of these systems is the instability of Ca-alginate against
phosphates and the disruption of gel particles due to CO2 evolu-
tion during fermentation. For the second one, as the cells attach
to the carriers’ surface by electrostatic interactions or covalent
binding of the cells, the two major drawbacks are the limitation
∗ Corresponding author.
E-mail address: twtan@mail.buct.edu.cn (T. Tan).
of the biomass loading by the carriers surface, and the effect of
various factors that can cause cell desorption thereby limiting
the operational stability.
Cells have been immobilized on a variety of natural and syn-
thetic supports. One of the extensively used classes of natural
support is lignocellulosic materials. Some lignocellulosic sup-
ports investigated include sawdust, wood chips/shavings, rice
husk, and straw (Shukla et al., 1988; Das et al., 1993; Maryse
and Zdravko, 1996). However, there are a few drawbacks, for
example, some of these supports have non-uniform structures
which are often present in a particulate, powder or chip form.
The preparation of stable beds with such materials is difficult
and pressure drops or floatation is often observed especially
when viscose and high density substrates are employed. In these
literatures, the immobilization methods are all based on the
recirculation of the concentrated yeast suspension through the
reactor.
Sorghum is a natural economical lignocellulosic material
which grows well in different parts of the world and is pro-
duced in large quantities in most Asian countries, including
China where it is used extensively in ethanol industry (Woods,
2000; FAO, 2002). Sorghum bagasse is highly fibrous with a
good water retention capacity. The advantages of the Sorghum
bagasse as yeast cells carrier are: (1) the surface of the Sorghum

0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2007.01.039
416 J. Yu et al. / Journal of Biotechnology 129 (2007) 415–420

Fig. 1. Scanning electron micrograph of sorghum bagasse and immobilized yeast cells which were embedded by the sweet sorghum bagasse: (a) portrait slice of the
sorghum bagasse; (b) the surface of the stalk cell walls; (c) immobilized cells adhere to the surface of the stalk cell walls; (d) large amount of cells; (e) landscape
orientation slice of the sorghum bagasse with immobilized cells embedded in the cavum of the stalk cells; (f) portrait slice of the sorghum bagasse with immobilized
cells embedded in the cavum of the stalk cells.

bagasse as represented in Fig. 1(a) is much more porous than
the straw and wood chips are, which facilitates the transmis-
sion of substrates and products between carriers and medium.
(2) As we can see from Fig. 1(b), there are lots of pores among
the sorghum cells which make the sorghum bagasse integrative.
This makes the transmission of substrates and products among
the bagasse cells more easily. Both of them can solve the prob-
lem of mass transfer which occurs in the systems of calcium
alginate and straw. The present paper describes the possibil-
ity of using sorghum bagasse as a support; delineates a novel
and simple technique for the rapid and strong immobilization
of cells in sorghum bagasse and also discusses the application
of sorghum bagasse containing the yeast cells for the ethanol
fermentation.
2. Materials and methods
2.1. Microorganism and mediums
The laboratory mutant strain of baker yeast 3013 was used
throughout the experiments. The yeast strain was maintained in
MY medium (in g/L): glucose, 20; yeast extract, 3; polypeptone,
5; malt extract, 3; agar, 20. In all cases, cultures were maintained
at 30 ◦ C for 24 h and then stored at 4 ◦ C. Subculturing was done
in every 2 months. The composition of the pre-culture medium
for yeast was (in g/L): glucose, 15; sucrose, 15; yeast extract, 3;
polypeptone, 5; malt extract, 3. The cells were cultivated in this
medium in a controlled environment shaker at 28 ◦ C for 20 h in
order to obtain high cell density. At the end of incubation period,
 J. Yu et al. / Journal of Biotechnology 129 (2007) 415–420
cells were centrifuged aseptically and resuspended in fresh pre-
culture medium to be used as inoculum. The compositions of the
fermentation media (in g/L) were: (a) glucose, 70; sucrose, 80;
polypeptone, 50; MgSO4 , 10; KH2 PO3 , 0.5; (b) glucose, 100;
sucrose, 100; polypeptone, 80; MgSO4 , 12; KH2 PO3 , 0.6. All
the mediums were adjusted to pH 5.5 and autoclaved at 116 ◦ C
for 20 min before use.
2.2. Reactor and immobilization
The reactor was composed of two water-jacketed borosili-
cate glass tapered column (top, 3 cm i.d.; bottom, 3 cm i.d.; and
height, 30 cm) equipped with a stainless steel wire mesh at the
bottom of the reactor. The carriers were aseptically transferred
to the sterilized reactor. The carriers volume was about 50% of
the working reactor volume of 200 mL.
After the skin and the outside fiber were removed, sorghum
was chopped into small particles using a food processor. The
chopped sorghum was then dried, and approximately 50 mL
moisture was vaporized from 100 g raw sorghum. Sorghum
bagasse which was obtained after drying was sieved to remove
fine and larger particles. A 25 g sorghum bagasse was autoclaved
at 116 ◦ C for 20 min and then mixed with 50 mL fresh pre-culture
medium of which the cell concentration was 2 × 108 mL in flask.
Twenty-four hours later, the sorghum bagasse prepared above
was combined with 50 mL medium (a) and replaced by a fresh
lot of 50 mL medium (a) after 24 h. The sorghum bagasse was
then put into the reactor which had been autoclaved at 121 ◦ C
for 20 min.
2.3. Analytical methods
The moisture of the sorghum bagasse was obtained after dry-
ing at 60 ◦ C for 24 h. Glucose was determined enzymatically
with a glucose oxidase-chromogen reagent (Shandong Univer-
sity). Sorghum sucrose was hydrolysed in 1.2N HCl for 7 min
at 60 ◦ C and neutralized with 1N NaOH prior to its determi-
nation by the method of glucose and translated into sucrose.
Residual sugars were determined using the 3,5-dinitrosalicylic
acid (DNS) method (Bernfeld, 1959; Bertolini et al., 1991).
The total biomass was determined at the end of each set of
experiments. The yeast cells were separated from the support
by washing sorghum bagasse with distilled water and centrifug-
ing the collected liquid. Cell number was determined using a
haemacytometer. The total biomass removed from the sorghum
bagasse was then dried at 80 ◦ C for 24 h and weighed (Kargi and
Curme, 1985).
The ethanol content was measured by using Shimadzu GC-
2050 gas chromatography with cbp-20 capillary column and a
flame ionization detector. The chromatogram was run at 180 ◦ C
oven temperature and 90 ◦ C injection temperature using N2 as
a carrier gas and H2 as a flaming gas.
2.4. Electron microscopic scanning
The carrier sorghum bagasse and immobilized cells were
studied with an electron microscope. The samples were soaked
417
in 3.5% glutaraldehyde for 6 h, and dried by treatment with
50, 70, 90, 95 and 100% ethanol, followed by overnight
retention of the samples in a desiccator for the removal of
moisture.
3. Results and discussion
3.1. Immobilization of yeast cells to sorghum bagasse
Almost all of the immobilization methods are based on the
recirculation of the concentrated yeast suspension through the
reactor when lignocellulosic materials act as supports (Shukla
et al., 1988; Das et al., 1993; Maryse and Zdravko, 1996). The
drawback is cell desorption, which limits the operational sta-
bility. The reason is most of the yeast cells were adsorbed to
the surface of the supports and just a few of them are embed-
ded in the inner side of the supports tightly. In this method,
the fresh pre-culture medium containing the concentrated yeast
cells was added into sorghum bagasse. This system can be cul-
tivated in the form of semi-solid state. This approach increases
the immobilized cell concentration and enhances the stability of
the fermentation system effectively.
The yeast cell concentration of different methods was com-
pared in Table 1. The maximum cell concentration of 0.61 g
DCW (dry cell weight)/g DSW (dry sorghum bagasse weight)
was obtained using the dried sorghum bagasse. Because the fresh
medium was recombined with the sorghum bagasse which was
bibulous, the medium including the yeast cells penetrated into
the porous stalk cells. The yeast cells were immobilized in the
cavum of the stalk cells firmly, and high biomass concentra-
tion was obtained. Scanning electron microscopic investigations
were carried out to elucidate the mode of immobilization of
cells in the sorghum bagasse (Fig. 1). Sorghum bagasse was
found to have a large amount of stalk cells which was vacuous
and porous. These structures might be responsible for maxi-
mum cell adsorption and good for mass transportation. From
Fig. 1(c–f), large numbers of yeast cells were observed to be
adhered on all sides of the stalk cell walls, and meanwhile the
cavum of the stalk cells were also filled with yeast cells, both
of which contributed to the maximum cell concentration and
productivity.
3.2. The kinetics of immobilization
As is shown in Fig. 2, the immobilized cell concentration
increased rapidly during the first 16 h as the sugar concentra-
tion decreased to nearly 20 g/L, then it appeared to decrease
Table 1
Effect of the pre-disposed sorghum bagasse on the immobilized cell
concentration
Pre-disposed sorghum Carriers: medium = 1 Immobilized cell
(g:mL) concentration
[g DCW/g DSW]
Dried sorghum bagasse 1:2 0.61
Fresh sorghum stalk 10:1 0.39
418 J. Yu et al. / Journal of Biotechnology 129 (2007) 415–420
Fig. 2. Effect of the cultivation time on the immobilized cell mass.
slowly. This maybe caused by the difference in sugar concen-
tration between the fermentation medium (b) and inner of the
sorghum bagasse. After the medium was replaced by fresh one,
the yeast cell concentration raised to about 0.6 g DCW/g DSW
and maintained constantly. This was regarded as the steady state
which could be used for further fermentation.
3.3. Effect of sizes of sorghum bagasse on immobilized cell
concentration
As mentioned previously, most of the immobilized yeast cells
are embedded in the cavum of the stalk cells. If the total mass is
equal, big sorghum bagasse has more intact stalk cells than the
small ones and more yeast cells can be immobilized every unit
(Fig. 3). But as the size increases, the mass transfer at the inner
of the sorghum bagasse would become more difficult, and finally
influence the fermentation productivity. So the optimal size of
the sorghum bagasse is needed to be optimized in the further
study, size 10 mm × 10 mm × 10 mm was used temporary in the
following experiments.
Fig. 3. Effect of sizes of sorghum bagasse on immobilized cell concentration.
Fig. 4. The comparison of the fermentation kinetics between immobilized cells
system and free cells system with the initial sugar concentration of 200 g/L, (A)
free cells system, (B) immobilized cells system (carriers: medium = 1).
3.4. The comparison of batch fermentation in free cell
system and immobilized cell system
Immobilized cell technology has many advantages over con-
ventional systems. One of them is cell immobilization increases
fermentor productivity to two or three times (Najafpour et al.,
2004) by substantially increasing the population density. As is
shown in Fig. 4, experiments were carried out with the same
initial sugar concentration of 200 g/L. In the free cells system, it
took 40 h to consume 98% of the total sugar, the ethanol produc-
tivity was 2.55 g/(L h). The ethanol concentration and yield were
91.7 g/L and 0.47 g ethanol/g consumed sugar, respectively. In
sharp contrast, after 16 h, 98.7% of the total sugar was consumed
in the immobilized cells system with an ethanol productivity of
5.72 g/(L h) which was 2.24 times higher than that of the free
cells system. The ethanol concentration and yield were 92.7 g/L
and 0.48 g ethanol/g consumed sugar, respectively.
3.5. Reuse of immobilized cells in a batch process for
production of ethanol
A 50 g sorghum bagasse containing the immobilized yeast
cells was incubated in 50 mL fermentation medium (b) with
gentle shaking at room temperature. After 16 h of incubation,
all the medium was withdrawn, and the final sugar concentra-
tion and ethanol concentration were determined. The sorghum
bagasse was then retrieved and transferred to a fresh batch of
fermentation medium (b). The process was repeated. In this
system, the overall ethanol concentration remained almost con-
stant at 96 g/L during 13 repeated batch fermentations, and then
increased to 100 g/L. The immobilized and free cell concentra-
tions increased slowly with the progress of fermentation. The
immobilized cells could be reused for at least 30 days retaining
about 95% of its original activity (Fig. 5 just illustrates the first
21 bathes). This means, the immobilized Saccharomyces cere-
visiae cells in sorghum bagasse retained their activity to produce
ethanol for more than 1 month. This ability of immobilized cells
to produce ethanol for a long time yet remains to be explained.
 J. Yu et al. / Journal of Biotechnology 129 (2007) 415–420
Fig. 5. Fermentation kinetics of immobilized S. cerevisiae cells during ethanol
production in repeated fed-batch culture.
This might be due to the fact that immobilized cells contain
significantly higher percentages of saturated fatty acids com-
pared to free cells which leads to greater ethanol tolerance in the
immobilized cells, and hence greater survival and productivity
in subsequent cycles compared to free cells (Krisch and Szajani,
1997) can be observed.
Rychtera et al. (1987) reported that immobilized cells can
retain enzyme activities for a long time due to the different com-
position of cells (proteins, lipids, RNA, DNA, and inorganic
substances) compared to free cells.
3.6. Continuous ethanol fermentation
Continuous ethanol fermentation using a filled-bed reac-
tor mentioned above with fermentation medium (b) under the
following conditions: void volume ratio of carriers, 50%, tem-
perature, 30 ◦ C, and dilution rates, 0.1, 0.2, 0.3, 0.4 h−1 , a
start-up procedure was required in order to establish a steady
state phase. Initially, the medium was fed into the filled bed
reactor at the dilution rate of 0.1 h−1 . After 1 day, a steady state
was achieved. The average ethanol concentration of the effluent
was 9.47% (w/v). At every 5 days interval, the dilution rate was
changed into 0.2, 0.3 and 0.4 h−1 and the average ethanol con-
centration of the effluent decreased to 6.22%, 5.56% and 3.86
(w/v), respectively. As shown in Fig. 6, the system was main-
tained for 20 days and natural sorghum bagasse was not damaged
through the entire experiment. Under steady state conditions,
the biomass concentration leaving the reactor remained nearly
constant at a given dilution rate and increased slightly as the
dilution rates was raised. The biomass in the reactor increased
slightly throughout the experiments. However, it was difficult to
determine the actual biomass concentration in the reactor at all
times.
Reactor productivity (based on valid volume) and effluent
ethanol are shown as a function of dilution rate in Fig. 7. The
maximum ethanol productivity of 16.68 g/(L h) was obtained
at a dilution rate of 0.3 h−1 . However, this was at a substantial
expense of a residual glucose concentration in the effluent. Com-
plete utilization of the total sugar was obtained at a dilution rate
of 0.1 h−1 with the maximum ethanol concentration of 94.7 g/L.
419
Fig. 6. Continuous ethanol production in an immobilized cell reactor with fer-
mentation medium (b).
Fig. 7. Effect of dilution rate on reactor productivity and ethanol concentration.
Productivity began to decrease after the dilution rate reached
about 0.3 h−1 .
During the continuous ethanol fermentation, high pressure
was observed in the bioreactor which was caused by the larger
volume of CO2 production. This problem would become more
serious at higher initial sugar concentrations. This problem
could be overcome by using reactors with a special configu-
ration (Coverti et al., 1987; Hamamchi and Ryu, 1987; Qureshi
et al., 1987; Rychtera et al., 1987). Meanwhile, some of the
sorghum bagasse was covered with biofilm at the end of the
running, which might weaken the mass transfer of substrates
between sorghum bagasse and medium. This phenomenon was
not observed in the batch process because of the gentle shak-
ing, so inert gas (like CO2 ) circulation combined with air lifted
reactor could be a good solution to these problems. In the
case of immobilized cells, no contamination of the substrate
by other microorganisms occurred. This could be due to the
high amount of the inoculum dominating the existing microflora
and the ethanol produced inhibited the growth of contaminating
microorganisms.
4. Conclusions
A novel immobilization method of S. cerevisiae to sorghum
bagasse was investigated. Carriers were cultivated in the form
420 J. Yu et al. / Journal of Biotechnology 129 (2007) 415–420
of semi-solid state to achieve firm cells immobilization. This
approach increases the immobilized cell concentration and
enhances the stability of the fermentation system effectively.
Sorghum bagasse has a potential as a carrier for the whole
yeast cell immobilization using this innovative method. There
are several advantages, including low carrier cost, simplicity of
immobilization procedure, and high carrier strength and dura-
bility.
In repeated batch fermentation at an initial sugar concentra-
tion of 200 g/L, nearly 100% total sugar was consumed after 16 h
with the ethanol yield of 4.9 g/g consumed sugar on average, with
the ethanol productivity of 5.72 g/(L h). The immobilized cells
could be reused for at least 30 days retaining about 95% of its
original activity. Continuous ethanol fermentation using a filled-
bed reactor was carried out. Complete conversion of total sugar
to ethanol was obtained at a dilution rate of 0.1 h−1 and the max-
imum ethanol productivity 16.68 g/(L h) appeared at the dilution
rate of 0.3 h−1 . Continuous ethanol production was maintained
for up to 20 days. The size of the sorghum bagasse, packing
density and cell growth are required to be optimized further for
higher ethanol productivity.
Acknowledgments
This work was financially supported by Project supported by
the National Natural Science Foundation of China (Grant No.
20576013), Project supported by the National Science Found for
Distinguished Young Scholars of China (Grant No. 20325622),
973 Program (Grant No. 2003CB716002), Project supported by
the Natural Science Foundation of Beijing, China (Grant No.
07B0203), and Beijing Science and Technology projects (Grant
No. D0205004040211).
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