Bioresource Technology 101 (2010) 31263131

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Fuel ethanol production from granular corn starch using Saccharomyces cerevisiae
in a long term repeated SSF process with full stillage recycling
Wojciech Biaas *, Daria Szymanowska, Wodzimierz Grajek
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48 St., 60-637 Poznan, Poland

a r t i c l e i n f o a b s t r a c t

Article history: A major problem with fermentative ethanol production is the formation of large amounts of numerous
Received 22 October 2009 organic pollutants. In an industrial distillery, stillage, fermenter and condenser cooling water are the
Received in revised form 19 December 2009 main sources of wastewater. However, the selection of a proper technology makes it possible to almost
Accepted 21 December 2009
completely avoid emissions of such kind of wastewater to the environment. This study examines the
Available online 12 January 2010
effect of stillage recirculation on fuel ethanol production. It is based on the use of Saccharomyces cerevisiae
and a granular starch hydrolyzing enzyme in a simultaneous saccharication and fermentation process
Keywords:
using a native starch obtained from corn our. It was shown that the yield of the ethanol production
Ethanol
Simultaneous saccharication and
was not inuenced by the recycled stillage, a mean yield being 83.38% of the theoretical value. No signif-
fermentation icant trend for change in the ethanol concentration or in the residual starch was observed during any par-
Stillage ticular run, even after the 75% of fresh water was replaced with stillage. Thus, by applying this new clean
Recycling technology it is possible to signicantly reduce the rate of water consumption and in this way the pro-
duction of by-products such as stillage.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Fuel ethanol can be manufactured from sucrose or from starchy
and lignocellulosic biomass. Over the last decade, there has been
substantial research on the production of ethanol from lignocellu-
losics. Although lignocellulose is inexpensive, it is difcult to con-
vert to fermentable sugars. Cellulose hydrolysis is much slower
than the enzymatic degradation of other biopolymers. For exam-
ple, the hydrolysis rate of starch by amylases is 100 times faster
than the hydrolysis rate of cellulose by cellulases under industrial
processing conditions (Sanchez and Cardona, 2008). For this rea-
son, starch is still the main feedstock for ethanol production in
the United States, Canada, and Europe. However, due to the high
energy consumption during the mashing process as well as the
large amounts of amylolytic enzymes which must be added,
namely, glucoamylase and a-amylase, the current production cost
of traditional starch substrate processing for ethanol remains high.
According to Lim et al. (2003) the energy demand of the conven-
tional cooking process is equivalent to 3040% of the fuel value
of the ethanol produced. Besides the energy consumption for mash
heating and ethanol distillation, a major problem with fermenta-
tive ethanol production is the formation of large amounts of
numerous organic pollutants. In an industrial distillery, stillage,
fermenter and condenser cooling water and fermenter wastewater
* Corresponding author. Tel.: +48 61 8466025; fax: +48 61 8466003.
E-mail address: wbialas@up.poznan.pl (W. Biaas).
are the main sources of wastewater (Pant and Adholeya, 2007).
According to Prasad et al. (2007) up to 20 l of efuents may be gen-
erated for each liter of ethanol produced. Hence to compete with
the existing fossil fuel industry and become commercially viable,
the overall cost must be reduced.
There are several possible ways to approach this problem.
The design of a novel zero-discharge, non-cooking fermentation
system for direct production of ethanol from raw starch seems
to be the best way to minimize energy costs, as well as the
formation of pollutants. However, the complete hydrolysis of
highly concentrated raw starch mashes requires at least two
types of enzymes capable of digesting raw starch granules:
1.4-a-D-glucan glucanohydrolase (a-amylase, EC 3.2.1.1) and
1.4-a-D-glucan glucohydrolase (glucoamylase, EC 3.2.1.3). Nor-
mally, the action of hydrolytic enzymes on native starch gran-
ules is not very effective because the granules are very
resistant to amylolytic digestion and a long hydrolysis period
is required for degrading the starch (Sarikaya et al., 2000). Re-
cently, highly active enzyme systems with raw starch hydrolytic
capabilities were created by using biological combinatorial pro-
cesses, which employed naturally occurring amylases from
Aspergillus kawachi and glucoamylase from Aspergillus niger
(STARGEN 001, Genecor International, Palo Alto, CA). Accord-
ing to Shariffa et al. (2009) these have the potential to overcome
the limitations of the natural set of enzymes which are currently
utilized. The application of tailored granular starch hydrolyzing
enzymes eliminates the cost-intensive liquefaction cooking step,

0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.12.090
 W. Biaas et al. / Bioresource Technology 101 (2010) 31263131
and thus reduces the energy input per unit of ethanol produced.
In addition to the advantages already mentioned, another attrac-
tive feature of this enzymatic complex is that it can be used to
break down the starch molecules in very concentrated mashes,
in which the dry solid content oscillates in the range of 12
38% w/w. Unfortunately, when starch is used as a raw material,
the amylases are strongly inhibited by hydrolysis products, such
as glucose. This inconvenience can be overcome by a simulta-
neous saccharication and fermentation process (SSF), which
combines enzymatic hydrolysis with fermentation. Furthermore,
the SSF process also reduces osmotic stress since the yeast cells
are exposed to a relatively lower sugar concentration, thereby
offering the possibility of higher production rates. An additional
advantage of SSF is that a multistage process for the conversion
of the starch into ethanol is carried out in one bioreactor, which
provides not only a reduction in the overall fermentation time
but also a reduction in the investment and operational costs
(Kobayashi et al., 1998).
The use of a zero-discharge fermentation system means that the
solid-containing whole stillage is separated using a decanter cen-
trifuge. Afterwards, the solids phase is dried in a rotary dryer, to
produce DDGS (distillers dry grain solids), a valuable co-product
used for animal feed, whereas the liquid phase is evaporated in a
double effect evaporator. The condensed water from the evapora-
tors is then recycled into the liquefaction stage, while the residue
from the bottom of the rectication column and a fraction of the
thin stillage are recycled to the fermentation system (Quintero
et al., 2008). The reduction of the amount of fresh water used in
this system is a considerable improvement over the norm. The per-
formance of this kind of clean technology for alcohol-from-starch
production has also been described by Kim et al. (1997). They
found that even after recycling eight times, the average production
yield (8.8%) was quite similar to the conventional process (9.0%).
However, the use of the zero-discharge system caused the fermen-
tation time to increase from 60 h to over 90 h. This result indicates
that the performance of such a system is dependent on a number of
parameters, such as the composition of the feed and the number of
times the liquid is recycled. Until now, investigations have, for the
most part, been limited to the ethanol fermentation of gelatinized
starch, in accordance with conventional technology. This raises the
crucial question of how a fermentation system based on the SSF of
native corn starch, and associated with the full utilization of the
stillage, could be operated indenitely.
In this study, we focused on investigating the complete utiliza-
tion of the stillage in the repeated simultaneous saccharication
and fermentation of native corn starch for the production of fuel
ethanol, and aimed at constructing an effective and simple ethanol
production system with an extremely long term stability and zero
wastewater discharge.
2. Methods
2.1. Microorganism
Freeze-dried distillers yeast, Ethanol Red (Saccharomyces cere-
visiae), obtained from Lesaffre Company (Marcq en Baroeul, France)
was used in this study, for the production of ethanol from corn
mashes. The number of living cells at packing was >2.0  1010/g,
as dened by the manufacturer.
2.2. Starch material
Commercially available corn our (BIO CORN, Ziebice, Poland)
was used as a raw material for fermentation. It had a median diam-
eter of 250 lm, and contained 84% starch.
3127
2.3. Enzymes
A mixture of granular starch hydrolyzing enzymes, containing
A. kawachi a-amylase expressed in Trichoderma reesei, and a gluco-
amylase from A. niger were employed in this study (STARGEN
001, Genecor International, Palo Alto, CA). The enzymatic activity
of this set of enzymes was P456 GSHU/g (Granular Starch Hydro-
lyzing Units), as dened by Genencore International. In addition,
fungal acid protease GC 106 (A. niger), also obtained from Genen-
core International, was added to the mashes. The enzymatic activ-
ity of GC 106 was P1000 SAPU/g (Spectrophotometric Acid
Protease Units), as dened by the manufacturer.
2.4. Simultaneous saccharication and fermentation
The SSF experiments were performed simultaneously in three
batch bioreactors (BIOFLO III, New Brunswick Scientic, New
Brunswick, NJ) with a 4.0 l working volume under nonaerated con-
ditions, at 35 C, 200 rpm. A slurry of raw corn our in water (25%
w/v) was prepared and saccharication was carried out by adding
2.05 ml (per kg corn our dry matter) of GSH enzyme preparation
(STARGEN 001). The pH of the fermentation broth was measured at
each sampling and adjusted to 5.0 by the addition of either 10 wt.%
H2SO4 or 20 wt.% NaOH. In all cases, the medium was supple-
mented with the acid protease GC 106 (40 ll/kg corn our dry
matter) and chloramphenicol (50 lg/L of the fermentation med-
ium). The fermentation was started with the addition of freeze-
dried distillers yeast Ethanol Red (0.5 g/L of fermentation med-
ium). The SSF process conditions were optimized using a multifac-
torial experimental approach (Biaas et al., 2009). The time course
of each fermentation cycle was 72 h. Samples were taken and ana-
lyzed for yeast cell viability as well as for the starch, glucose, eth-
anol, acetic acid, lactic acid and glycerol concentration after
fermentation. Concentrations of metal ions (Zn, Cu, Fe, Mg, K) were
also determined. The average results of triplicate experiments are
shown on each gure.
2.5. Recycling of stillage
After the fermentation period was completed, mash containing
ethanol was pumped to a continuous distillation column (UOP3CC,
Armeld, UK). At the top of this column, operating at 78.5 C and a
reux ratio of 4:1, carbon dioxide is stripped from the ethanol solu-
tion to produce an ethanol concentration of 9395% (volume
based) in a side stream. The liquid fraction collected at the bottom
of the distillation column, was rst cooled down to 30 C and then
centrifuged at 4000g for 20 min. Supernatant was used instead of
water for the dilution of native corn starch for the next SSF run.
The recirculation of stillage was performed, with the recirculation
degree remaining constant when 75% of the fresh water was re-
placed. The procedure was repeated 20 times over a period of
60 days, using a fresh yeast culture for the inoculation of each run.
2.6. Analysis
Samples for chemical analysis were rstly centrifuged at 4000g
for 10 min at 4 C (Multifuge 3SR, Germany), ltered through a
0.22 lm membrane lter (Millex-GS, Millipore, USA), and then
analyzed on an HPLC system (Merck Hitachi, Germany). The glu-
cose, ethanol, acetic acid, lactic acid and glycerol were separated
on an Aminex HPX-87P (Bio-Rad, USA) at 30 C using a 5 mM
H2SO4 solution as the mobile phase at a ow rate of 0.6 ml/min,
and then detected with a refractive index detector (Model L-
7490, Merck Hitachi, Germany). The starch content was analyzed
according to the enzymatic method developed by Holm et al.
(1986). Concentrations of Zn, Cu, Mg and Fe were measured using
3128 W. Biaas et al. / Bioresource Technology 101 (2010) 31263131

Fig. 1. Simplied ow sheet showing the dry grind ethanol production process (1a) and SSF processing of raw starch using GSH enzymes (1b). (1a): 1 hammer mill,
2 dilution tank, 3 jet-cooker, 4 ash tank, 5 heat exchanger, 6 fermentor, 7 distillation column, 8 decanter, 9 evaporator, 10 heat exchanger. (1b): 1 hammer
mill, 2 fermentor, 3 distillation column, 4 decanter.

atomic absorption spectrometry (AAS) with the SpectrAA 220 regarded as insignicant due to their p value being greater than
apparatus (Varian, Germany). Potassium content in the solutions 0.05. The same was observed for the ethanol productivity. The cal-
was determined by ame-emission spectrometry using a ame culated average value for the parameter for the SR process was
spectrophotometer Flapho 4 (Carl Zeiss, Jena, Germany). Instru- 1.28 0.14 g/(l
h) and 1.32 0.11 g/(l
h) for the WR process,
mental parameters were adjusted according to the manufacturers respectively. At the same time the starch to ethanol conversion
recommendations. efciencies for the SR and WR systems were 83.38 4.62% and
The yeast cell populations and viability were determined by a 84.6 3.98% of theoretical value, respectively. The F-test analysis
direct microscopic count in a counting chamber after staining with of variance revealed that these were statistically insignicant
methylene blue. The viable lactic acid bacteria concentration in the (p > 0.05). It should be noted, that the cells number in the SR was
fermentation broth was determined in triplicate for each dilution shown to decrease steadily throughout the time course of SFF pro-
used, by plating an appropriate cell suspension onto a MRS agar cess while the cells number in WR remained almost constant.
medium and counting after incubation at 30 C in a CO2 incubator However, these differences were found to be insignicant at the
(Hereus, Germany). The results were expressed as Cfu per probability level of 95% and consequently were not further
milliliter. discussed.
Our results are, in contrast, opposite to those obtained by Wang
et al. (2005), who also studied ethanol production from corn starch
3. Results and discussion
using the same enzyme mix of A. kawachi a-amylase expressed in
T. reesei, and a glucoamylase from A. niger (STARGEN 001) sup-
Currently, the two main processes for producing ethanol from
plemented with acid fungal protease (GC 106). The nal ethanol
corn are practically utilized: wet milling, and dry grinding. The
concentration for the modied enzymatic dry grind corn process
dry grinding process traditionally generates only three products:
using STARGEN 001 was 122.3 g/l (15.5% v/v), whereas the starch
ethanol, a thin stillage and DDGS, an animal feed component. As
conversion efciency was 86.4%, respectively. The observed dis-
was mentioned previously, the thin stillage is usually concentrated
crepancy might be attributed to the fact that the starch during pro-
in a double effect evaporation unit. Evaporation condensates are
then directly recycled in the mashing process as a substitute for
the process water needed (Fig. 1a). The greatest drawback of this
technology is its very high energy consumption (Larsson et al.,
1997). In this study, a new ethanol production process was inves-
tigated which included the SSF of raw corn starch by using GSH en-
zymes, as well as the recycling of thin stillages, in order to
minimize the overall process cost (Fig. 1b). To verify the developed
technology, 20 experimental conrmation runs were performed
with recycled stillage in 5 l lab-scale fermentors. It is important
to note that in this process, the decanter centrifuge in the actual
plant was replaced with a laboratory centrifuge.

3.1. The course of the fermentation process

As shown in Fig. 2 minor differences were observed in the eth-

anol proles of conventional treatments without stillage recycling
(WR) and the repeated fermentation system (SR). Fig. 2. Time course of SFF processes with (SR) and without stillage recycling (WR).
For each WR process a higher nal ethanol concentration of s starch (SR), d starch (WR), D ethanol (SR), N ethanol (WR), h glucose
approximately 3.2 g/l was achieved. However, differences can be (SR), j glucose (WR), e cells number (SR),  cells number (WR).
 W. Biaas et al. / Bioresource Technology 101 (2010) 31263131
Fig. 3. Effect of recycling stillage on the yield of ethanol fermentation (a), the concentration of ethanol (b), residual starch (c) as well as residual glucose (d).
cessing was initially soaked with water at 55 C for 12 h and this
was followed by a one-step incubation, conducted with the en-
zyme mix mentioned above, at 48 C and pH 4.2 for 3 h with agita-
tion. According to Wang et al. (2005), this kind of pretreatment
before SSF (liquefaction) for GSH treatment is not required, but rec-
ommended by the enzyme manufacturer. Recently, Shariffa et al.
(2009) illustrated that heat treatment had a major inuence on
the raw starch granules susceptibility to enzymatic attack. There-
fore it is reasonable to assume that the degree of hydrolysis of raw
starch was probably enhanced by pre-treating the starch with heat
below its gelatinization temperature before being subjected to en-
zyme hydrolysis. Czarnecki and Grajek (1990) have also shown
that the ethanol yields increased with the increase of temperature
and a prolonged enzymatic conversion time.
Fig. 3 summarises the results on yield and ethanol concentra-
tions, as well as residual starch and glucose concentrations during
20 fermentation runs. It can be seen that the yield was not inu-
enced by the recycled stillage: the mean yield was 83.38% of the
theoretical value, with a standard deviation of 4.62% (Fig. 3a). In
fact, the attained yield was lower than the theoretical value, indi-
cating that the nal fermentation broth contained signicant
amounts of substrates which had not been utilized during fermen-
tation. No signicant trend (p > 0.05) of change in the ethanol con-
centration or residual starch at a particular cycle was observed
(Fig. 3b and c). The average glucose concentration for 20 runs
was 7.12 3.36 g/l. An analysis of linearity (Fig. 3d) indicated that
residual glucose declined in the succeeding runs, as evidenced by
the signicant negative slope of the equation for the regression
curve. It can be seen that the residual glucose content decreased
by about 0.49 g/l on average in each successive fermentation, to
reach concentrations of lower than 4 g/l. This indicates a low diver-
sion of glucose to other metabolites and cell production, as will be
discussed below. However, it should be noted that for SR process a
slightly higher standard deviation of experimental data was ob-
served comparing to WR process. This may be attributed mainly
to a large number of unit operations such as distillation and centri-
3129
fugation, repeatedly applied to the stillage suspension during SR
processes. These separation steps can be regarded as a major
source of error, due to possible loss of analytes (starch, ethanol,
glucose, etc.).
3.2. Accumulation of soluble substances during alcoholic fermentation
To determine whether other by-products of the fermentation
process might account for the reduced ethanol yield, analyses were
conducted for the presence of lactic acid in the recycled stillage, as
well as acetic acid and glycerol.
It is well known that minor quantities of acetic acid are pro-
duced by yeast cells during fermentation, whereas lactic acid could
be produced by contaminating lactic acid bacteria as a result of car-
bohydrate metabolism (Narendranath et al., 1997). Microbial
counts can be signicantly reduced by penicillin or virginiamycin,
the two antibiotics most frequently used commercially to control
bacterial contamination in the fuel ethanol industry. However, in
spite of this protection, bacterial contamination still persists in
many ethanol production plants (Narendranath et al., 1997; Chang
et al., 1997). Theoretically, technology that employs raw starch
digestion, as presented here, is particularly susceptible to micro-
bial contamination, because pasteurization of the mash does not
occur during the heating process. For this reason we used chloram-
phenicol in our study, to act as a contamination inhibitor. The re-
sults show (Fig. 4a) that a signicant quantity of lactic acid was
formed, whereas an insignicant amount of acetic acid was ob-
served in any particular cycle (Fig. 4b). It can be seen that the lactic
acid content increased by about 0.12 g/l on average in each succes-
sive fermentation, to reach a concentration of over 3 g/l. According
to Narendranath et al. (2001), lactic acid concentrations of 28 g/l
introduce stress to Saccharomyces yeast when growing on a chem-
ically dened minimal media. Stress, which could lead to a change
in yield, can also contribute to other by-product formation. In our
study however, the ethanol production remained fairly constant
throughout the 20 recycling operations on the stillage. In contrast
3130 W. Biaas et al. / Bioresource Technology 101 (2010) 31263131
Fig. 4. Changes in the organic acids (a and b), glycerol (c), and the osmotic pressure (d) during stillage recycling.
to the chemically dened minimal media required for this process,
corn mash as a complex media probably provides an additional
source of non-nutritional components which promote yeast
growth and survival.
Recently, Graves et al. (2006) illustrated that the presence of the
components mentioned above had a major inuence on the toxic-
ity of lactic, as well acetic acid, on the yeast cells. He reported that
the nal ethanol quantities were signicantly reduced when corn
mashes contained at least 40 g/l of lactic acid. Consequently, a
low lactic acid concentration in the recycled stillage indicates that
neither inhibition of ethanol production nor bacterial infections oc-
curred during the fermentation. This hypothesis was supported in
our analysis of the viable number of lactic acid bacteria. When the
same overall concentration of chloramphenicol was added to each
fermentation run, the bacterial viable cell number decreased grad-
ually from 1.0  105 Cfu/ml at the beginning of the SSF to 2.5  101
Cfu/ml at the end of the SFF process. These results show that the
chloramphenicol treatment is effective in controlling bacterial con-
tamination in a laboratory fermentor. It is worth noting that the re-
sults obtained in our investigation are in excellent agreement with
the earlier study of Wang et al. (2007), who also reported a maxi-
mum lactic acid concentration of about 3 g/l, and no bacterial
infections during the fermentation.
Glycerol is the third most important product of yeast alcohol
fermentation after ethanol and CO2, and its synthesis serves a
number of important physiological functions. Glycerol helps to
maintain the cells redox balance and might also protect cells
against high temperature as well as oxidative stress. Glycerol also
plays an essential role as a compatible solute in yeast osmoadapta-
tion (Tams and Hohmann, 2003). Russel (2003) has observed that
the typical glycerol concentrations are 1215 g/l in conventional
dry grind corn fermentation without efuent recycling. From an
economic viewpoint however, glycerol production is undesirable
because it lowers the ethanol yield. As shown in Fig. 4c, the glyc-
erol content of successive fermentation broths increased from an
initial 0.5 41 g/l in run 14 and then declined, to reach a nal con-
centration of c. 35 g/l. Accordingly, from the linear regression
equation presented in Fig. 4c, it can be seen respectively that the
glycerol content increased an average of about 1.4 g/l in each suc-
cessive fermentation. Julian et al. (1990) completed a comprehen-
sive study on recycling thin stillage in the traditional fermentation
of corn to ethanol and also found that the glycerol content of suc-
cessive fermentations at rst increased, and then declined. How-
ever, he reported that the glycerol content increased only by
about 0.4% on average in each successive fermentation. A possible
cause of the subsequent reduction of glycerol observed during
recycling could be the inhibitory effect of the glycerol that accumu-
lated in the stillage. This phenomenon might also be attributed to
the utilization of glycerol by the yeast at higher glycerol concentra-
tions, and the decreased formation of glycerol due to the availabil-
ity of amino acids to perform cell synthesis from the recycled
stillage (Nordstrom, 1966). As it was mentioned above, excessive
glycerol production is an indicator of yeast osmotic stress. This the-
ory was also conrmed in our study, where it was found that the
glycerol concentration was highly correlated with the osmotic
pressure of the recycled stillage (Fig. 4d), as shown by the value
of Pearsons correlation coefcient r, where 0.91 is equal with
p < 0.001. Another factor that must be considered therefore, is
the increase of the salt concentration in the mash and its effect
on the osmotic pressure of the medium.
It is well established that at high concentrations of a solute, the
effect of the osmotic pressure on the yeast cells decreases the met-
abolic activity, which could be the cause of a lower rate of ethanol
fermentation (Reddy and Reddy, 2006). On the other hand, some
metal ions such as magnesium and zinc exert signicant effects
on the cell viability and ethanol production (Dombek and Ingram,
1986; Zhao et al., 2009). As shown in Fig. 5, the recycling of the thin
stillage from SSF results in an increase in various ions such as K+,
Mg2+ and Fe2+. The statistical signicance of the linear model equa-
tions was evaluated by an F-test analysis of variance, which
revealed that these regressions are statistically signicant, show-
ing p < 0.0001. It can be seen that K+, Mg2+ and Fe2+ content in-
creased an average of about 176.7, 42.5 and 1.68 mg/kg in each
successive fermentation, respectively. However, in the present
 W. Biaas et al. / Bioresource Technology 101 (2010) 31263131
Fig. 5. Effect of stillage recycling on concentrations of inorganic ions. j Mg2+ =
523.1 + 42.5x (p < 0.0001); s Fe2+ = 31.70 + 1.68x (p < 0.0001); h K+ = 1598.4 +
176.7x (p < 0.0001); d Zn2+ = 7.75 + 0.0029x (p > 0.05); D Cu2+ = 0.30 0.0038x
(p > 0.05); x number of the cycle.
study there were no signs in the fermentation performance (etha-
nol concentrations and yield) of a negative impact of the ions men-
tioned, even where there were high concentrations of ions. Similar
observations on the relationship between the concentrations of the
ions and the ethanol yield have been reported by Lu et al. (2003).
They show that all the ions approached their equilibrium level
but continued to increase very slowly after about 56 operation cy-
cles. However, as shown in Fig. 2 only the smaller amount of bio-
mass was generated during the process, due to a high osmotic
pressure resulting from the accumulation of the metal ions.
Other investigated ions such as Zn2+ and Cu2+ remained fairly
constant throughout the 20 recycling operations on the stillage
(p > 0.05). This is probably due to the fact that zinc as well as cop-
per ions may bind to proteins and amino acids released from the
yeast cells during distillation. Since the majority of the amino acids
and proteins are precipitated at temperatures above 90 C during
the fermented mash distillation, a fraction of the bound metal ions
may also be lost.
4. Conclusions
It was shown that the yield of ethanol production was not inu-
enced by the recycling of the stillage, and that a mean yield was
equal to 83.38% of the theoretical value. No signicant trend for
change was observed in the ethanol concentration or in the resid-
ual starch at any particular cycle. In addition, our results clearly
indicate that the recycling of the stillage into the fermentation
phase would enable both water consumption and wastewater
rejection to be reduced. In summary, the results reported in this
work show that a repeated batch SSF process conducted on native
starch, using novel GSH enzymes, stands as an excellent alternative
for traditional technology in the production of fuel ethanol.
Acknowledgements
This work was supported by the Polish Ministry of Science and
Higher Education under R&D Grant No. 0619/P01/2007/02.
3131
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