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Journal of Membrane Science 375 (2011) 212–219

Contents lists available at ScienceDirect

Journal of Membrane Science

journal homepage: www.elsevier.com/locate/memsci

Application of membrane distillation for ethanol recovery during fuel ethanol

production
Grażyna Lewandowicz ∗ , Wojciech Białas, Bartłomiej Marczewski, Daria Szymanowska
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Wojska Polskiego 48, 60-627 Poznań, Poland

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

Article history: The economic competitiveness of fuel ethanol in comparison with fossil fuels depends on the effective
Received 20 September 2010 lowering of costs on each step of the process. One of the most promising methods seems to be the appli-
Received in revised form 21 March 2011 cation of membrane separation technology. In this study, the focus is on improvement of brewer’s wort
Accepted 22 March 2011
fermentation. For this purpose, the membrane distillation process for ethanol recovery was implemented.
Available online 29 March 2011
The experimental system consisted of a bioreactor equipped with a capillary polypropylene microfiltra-
tion unit. Yeast cells’ count and viability, assimilation of sugars, production of ethanol and fermentation
Keywords:
of by-products (glycerol and lactic acid) were monitored during fermentations. The intercellular tre-
Membrane distillation
Bioreactor
halose as well as Hsp70 and Hsp104 heat shock proteins contents were determined. It was concluded
Ethanol fermentation that membrane distillation can be regarded as a straightforward method, which leads to an increase in
Yeasts stress ethanol production by facilitation of the continuous process, more complete fermentation of sugars, low-
ering the osmotic pressure in the fermentation broth, decreasing glycerol synthesis level and increasing
yeast cells’ number and viability.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
In the forthcoming years ethanol production will increase,
which is associated with its wide usage as a fuel additive. Ethanol
provides a renewable energy source, which is beneficial for a num-
ber of reasons, including limited greenhouse gas emissions and the
creation of new employment opportunities by enhancing economic
benefits in rural areas [1]. Depending on the feedstock (sugar-
cane, cereal grain, lignocellulosic biomass), ethanol production
involves several steps: pretreatment of the raw material, hydrol-
ysis of polysaccharides, fermentation of carbohydrates, recovery
and dehydration of ethanol, and therefore that technology is more
expensive in comparison with fossil energy. Moreover, the most
commonly used technique for fuel ethanol production is batch
fermentation, which has low volumetric productivities and is time-
consuming. Hence, to compete with the existing fossil fuel industry
and become commercially viable, the cost must be reduced. There
are several possible ways to approach this problem. The appli-
cation of membrane bioreactors is often suggested as a way of
increasing the effectiveness of different technological processes
[2]. The first attempts to accomplish ethanol fermentation in a
membrane bioreactor involved using a microfiltration unit. In this
type of fermentation, a fermentation medium from a bioreactor
is allowed to flow continuously through a microfiltration module
∗ Corresponding author. Tel.: +48 61 8466005; fax: +48 61 8466003.
E-mail address: gralew@up.poznan.pl (G. Lewandowicz).
where yeast cells are rejected by the membrane and recycled to the
reactor, resulting in an increase of cell densities with time. Further-
more, ethanol which affects the fermentation adversely as a process
inhibitor is removed from the fermentation system. This makes
the continuous process possible at high volumetric productivity.
However, in spite of these advantages, there are some drawbacks
of using a bioreactor coupled with a microfiltration module. Since
both substrates and products pass through the membrane, their
separation is not possible in this system [3]. Thus, an additional
step of ethanol recovery is still needed.
One of the ways to overcome this inconvenience and to increase
the productivity of alcoholic fermentation is to remove the product
by pervaporation or membrane distillation. While ethanol passes
through the selective membrane, yeast cells, substrates, and other
nutrients are retained on the feed side. Therefore, it can be both
purified and concentrated [3]. Moreover, coupling bioreactors with
the above mentioned separation systems reduces natural inhibi-
tions of cell growth caused by high concentrations of ethyl alcohol
[1,4–6].
Pervaporation and membrane distillation are both membrane
processes which involve energy consuming evaporation. This
shortcoming is on the other hand an advantage of the both pro-
cesses as it enables selective separation of volatile products from
the fermentation broth. Due to their similarity, pervaporation and
membrane distillation are often confused with each other. How-
ever, despite some similarities, there are fundamental differences,
mainly in the role that the membrane plays in separation. Mem-
brane distillation utilises a non-wetting, microporous membrane

0376-7388/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.memsci.2011.03.045
 Author's personal copy
G. Lewandowicz et al. / Journal of Membrane Science 375 (2011) 212–219
that prevents liquid solutions from entering its pores. Pervapo-
ration, on the other hand, makes use of dense membranes made
of swollen homogeneous polymers that render the membranes
permeable [7]. The driving force of membrane distillation is the
partial pressure difference between each side of the membrane
pores, whereas the selectivity varies according to the proper-
ties of the type of polymer used, the membrane characteristics,
and the operation conditions [8]. The driving force for perva-
poration is a low vapour pressure on the permeate side of the
membrane generated by cooling and condensing the permeate
vapour. The selectivity of separation depends on the affinity of
some specific components of a mixture to the membrane material
[9].Recently, a number of interesting papers concerning the pos-
sibility of using membrane distillation for ethanol recovery have
appeared [6,8–12]. However, despite the membrane distillation has
been studied extensively over the last few years, so far that tech-
nology has received limited acceptance in the industry. As opposed
to membrane distillation, pervaporation systems are available for
two commercial applications. The first application is the removal
of water from concentrated alcohol solutions, and the second is the
removal of small amounts of volatile organic compounds from con-
taminated water [13]. As can be seen, both membrane distillation
and pervaporation could be applied for the separation of ethanol
from the fermentation broth, however the research is needed in
order to improve their efficiency, mainly to increase the permeate
flux.
The economic effectiveness of the whole process for the manu-
facturing of fuel ethanol depends not only on its effective recovery,
but also on the efficiency of its synthesis. In spite of the fact
that ethanol fermentation using Saccharomyces cerevisiae yeast is
a rather well-established technology, inhibition of this process by
the product is still a problem. The capability of producing ethanol
by these microorganisms is completely inhibited if the ethanol
concentration in the fermentation broth exceeds 110 g l−1 , below
30 g l−1 the inhibition is negligible [14]. In the course of ethanol fer-
mentation run on a commercial scale yeast cells are also exposed
to several other types of stress including: heat, osmotic, oxida-
tive, mechanical, pressure, and nutrient deficiency stress, stress
connected with low pH, the presence of toxins and inhibitors,
or microbial infection. The occurrence of stress conditions forces
living organisms to initiate defence mechanisms, making it possi-
ble for them to survive and function in such environments [15].
The metabolic response includes, inter alia, production of glycerol,
accumulation of trehalose, and synthesis of the so-called heat-
shock proteins (HSP), also referred to as chaperones [16–20].
A membrane distillation system that removes bioethanol from a
reactor can be a solution to overcome the disadvantages of conven-
tional fermentation systems. Theoretically, these systems enable
us to operate continuously and to reduce yeast stress commonly
occurring in industrial fermentation. In this study, the focus is on
improvement of brewer’s wort fermentation. For this purpose, the
membrane distillation process for ethanol recovery was imple-
mented. In addition, the role of trehalose in yeast stress tolerance,
together with heat shock proteins, was examined in an industrial
strain of S. cerevisiae.
2. Experimental
2.1. Membrane
The experiments were conducted using microporous hydropho-
bic capillary membrane of polypropylene (MD 020 CP 2N)
purchased from Microdyn-Nadir GmbH (Germany). The capillaries
have an inside diameter of 1.8 mm, an outside diameter of 2.6 mm,
and a 0.2 ␮m pore size. The total number of capillaries in the module
213
was 40, and the effective membrane area was 0.1 m2 . For each set
of experiments described in the manuscript, the same membrane
module was used.
2.2. Fermentation medium
Molasses wort of density of 18◦ Plato (18 g extract per 100 g liq-
uid) obtained from Kompania Piwowarska SA. (Poznań, Poland) was
used as a fermentation medium. The medium contained: 86.27 g l−1
maltose, 16.07 g l−1 glucose, 5.65 g l−1 fructose, and 21.33 g l−1
maltotriose. The pH and osmolality were 5 and 0.7 Osm kg−1 ,
respectively.
2.3. Yeast
Lyophilized S. cerevisiae yeast strains (strain Ethanol Red, Lesaf-
fre, France) were used in the study. According to the producer’s
information, the preparation contained ≥2.0 × 1010 cfu g−1 . Yeasts
were added to the medium in a concentration of 0.5 g l−1 , corre-
sponding to 2.5 × 106 cfu ml−1 after inoculation.
2.4. Chemicals
All chemicals used in this experiment were obtained from
Sigma–Aldrich (Germany) or POCh (Poland) and were of analytical
grade.
2.5. Experimental apparatus and procedure
The set-up used to conduct the direct contact membrane
distillation DCMD experiments is presented in Fig. 1. The cross-
flow filtering module was equipped with a plate heat exchanger
(Termtrans, Poland). The distillate side of the separation module
was filled initially with distilled water (2.2 l), which was pumped
via the heat exchanger in order to obtain the temperature difference
between the retentate and the permeate side of the module.
2.5.1. Evaluation of the membrane performance
Prior to fermentation experiment, performance of the mem-
brane module was evaluated by clean ethanol–water feed solutions
permeation. Several ethanol permeation measurements were
taken, and the effect of difference in the feed and distillate tem-
peratures (T) on flux and selectivity was examined by varying
T between 13 and 20 ◦ C. The membrane was run for 3 h using
clean 10 wt.% ethanol feed.
2.5.2. Fermentation
The ethanol fermentations were conducted in batch and in a
single stage continuous culture. Batch fermentation was carried
out in 5 l bioreactor (Bio Flo III, New Brunswick Scientific, USA)
with a working volume of 4 l, at a temperature of 35 ◦ C, with no
pH adjustment. Stirring speed was 120 rpm and the time course of
fermentation cycle was 72 h.
The continuous fermentation was carried out in an experimen-
tal set (Fig. 1) which was made of a 5 l bioreactor (Bio Flo III, New
Brunswick Scientific, USA), equipped with the membrane module
described above (Section 2.1). The distillate side of the separation
module was filled initially with distilled water (2.2 l), which was
pumped via the heat exchanger in order to obtain the temper-
ature difference between the retentate and the permeate side of
the module. The distillate was cooled to 15 ◦ C, and the fermenting
medium was heated in the bioreactor to 35 ◦ C, hence the temper-
ature difference mentioned above was 20 ◦ C. The total volume of
the fermentation medium was 4 l, and the process was carried out
at the stirring rate of 120 rpm, over 50 h, with no pH adjustment.
Membrane distillation unit was switched on in 20th hour of the
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214 G. Lewandowicz et al. / Journal of Membrane Science 375 (2011) 212–219
Fig. 1. Schematic diagram of the applied system. P-1, 2, 3 – pumps; L-1 – level sensor; CSTR – continuous stirred tank reactor with temperature control (Bio Flo III); V-1,
2, 3 – diaphragm valves; HE-1 – heat exchanger; P – pressure sensor; T – temperature sensor; MF-1 – microfiltration module; CT-1 – closed tank; OT-1 – open tank; F-1 –
flowmeter.
process. The system was equipped with a liquid-level sensor in the
bioreactor, which was connected to an electric relay switching on
a type-501U Watson-Marlow peristaltic pump (pump P-1, Fig. 1).
The liquid level in the bioreactor was controlled at a fixed level, so
that the inlet flow rate was exactly equal to the outlet flow rate. The
fresh fermentation medium was continually added throughout the
whole process, and ethanol was removed simultaneously.
After each fermentation experiment the membrane was chem-
ically cleaned according to the manufacturers recommendations.
The system was first rinsed with water. It was then cleaned with
a sodium hydroxide solution (10 g l−1 , 15 min), heated to 40 ◦ C and
rinsed to neutrality with high-purity water. Membrane selectivity
and ethanol flux were almost totally recovered by this procedure.
2.6. Calculations
The ethanol yield YEt/G (g ethanol g−1 glucose) was calculated
on the basis of the total amount of fermentable sugars added to
the fermentations, i.e. the sum of maltose, glucose, fructose and
maltotriose expressed as glucose.
The performance of the membrane bioreactor was evaluated
mainly by permeate flux J and the selectivity factor S. The permeate
flux was determined by the measurement of an increase of distillate
volume, whereas the volume flux was calculated from the following
equation:
V2 − V1
J= [l m−2 h−1 ]
At
where V1 is the volume of liquid on the distillate side at the begin-
ning of the process [l], V2 the volume of liquid on the distillate side
at the end of process [l], A the working area of the membrane (m2 )
and t the number of working hours [h].
The selectivity factor S of the aq. ethanol solution was calculated
from the following equation:
(1 − xp ) xf
S=
xp (1 − xf )
where xp and xf are the mass fractions of ethanol in the permeate
and feed, respectively.
2.7. Analysis
During each experiment samples were taken and analyzed for
yeast cell count and viability as well as for the maltose, glucose,
ethanol, lactic acid and glycerol concentration. The intercellular
trehalose content was determined as well as Hsp70 and Hsp104
heat shock proteins using Western blot method.
The yeast cell populations and viability were determined by a
direct microscopic count in a counting chamber after staining with
methylene blue.
All samples for chemical analysis were firstly centrifuged at
4000 × g for 10 min at 4 ◦ C (Multifuge 3SR, Germany), filtered
through a 0.22 ␮m membrane filter (Millex-GS, Millipore, USA),
and then analyzed using an HPLC system (Merck Hitachi, Germany).
Glucose, maltose ethanol, lactic acid, and glycerol were separated
using an Aminex HPX-87P (Bio-Rad, USA) at 30 ◦ C using a 5 mM
H2 SO4 solution as the mobile phase at the flow rate of 0.6 ml min−1 ,
and then detected with a refractive index detector (Model L- 7490,
Merck Hitachi, Germany).
Prior to the analysis, trehalose was extracted from yeast cells
for 1 h at 60 ◦ C, using a solution containing 60% acetonitrile, 15%
methanol and 25% water. Then, each of the samples was centrifuged
by a laboratory centrifuge and, after that, filtered through filter
paper and a 0.22 ␮m membrane filter. Incidentally, the same elu-
ent was used as a mobile phase during the trehalose determination
by means of HPLC method described below. Trehalose was eluted
isocratically on a carbohydrate analysis column (Waters, USA). A
50 ␮l sample was injected. All analyses were carried out at 25 ◦ C
and a flow rate of 2.0 ml min−1 .
Western blotting was carried out following electrophoresis to
(1) identify production of specific HSPs. Cells were harvested from
fermentation medium in log-phase and proteins extracts were pre-
pared by vortexing with glass beads in a solution containing 8 M
urea and protease inhibitor. Protein concentrations were deter-
mined by means of a Nanodrop spectrophotometer (Germany)
with bovine serum albumin as a standard. Protein samples were
separated in 12% SDS-PAGE electrophoresis gel against prestained
broad range molecular weight standards, transferred to nitrocel-
lulose membranes and probed with either monoclonal anti-Hsp70
(2) (Sigma–Aldrich) or polyclonal anti-Hsp104 (Abcam) mouse anti-
bodies raised against yeast heat shock proteins. Blots were then
probed with biotin-conjugated anti-mouse (or anti-rabbit) anti-
body and developed using Western DotTM625 Western Blot Kits
(Invitrogen). The mouse anti-␤-actin (Biomibo) antibodies raised
against yeast ␤-actin served as a reference for analysis of heat shock
proteins expression patterns during the fermentation process. The
digitalized image was obtained by scanning the gel with a den-
sitometer (Image Master, Amersham Pharmacia Biotech, Sweden)
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G. Lewandowicz et al. / Journal of Membrane Science 375 (2011) 212–219
Fig. 2. The effect of the difference of the temperatures between feed and distillate
on the total flux (a) and ethanol flux (b) (䊉) – T = 13 ◦ C; () – T = 15 ◦ C; () –
T = 20 ◦ C.
and analyzed quantitatively using Quantity One software (Bio-Rad,
USA). Volume was obtained for each band as a sum of the intensi-
ties of the pixels within the volume boundary × pixel area (volume
units = intensity units × mm2 ).All experiments were repeated at
least twice and the average values are given. The statistical t-tests
were also performed at the significance level ˛ = 0.05. The commer-
cial software STATISTICA, version 6.0 PL from StatSoft, Inc. (2004)
was used for the analyses of the data obtained.
3. Results and discussion
3.1. Membrane distillation module performance
The driving force of the membrane distillation process corre-
sponds to the differential equilibrium vapour pressure between the
two sides of the membrane. In the case of direct contact membrane
distillation (i.e. the process applied in this study), the difference in
the vapour pressure is caused by the difference in the feed and dis-
tillate temperatures as well as the feed and distillate composition
[12,21]. In the experiments on a model ethanol solution, increas-
ing the temperature difference proved to have a positive effect on
the distillate flux (Fig. 2a). The total flux of the distillation in all the
examined variants slightly increased at the beginning of the pro-
cess and then remained relatively stable. The presented results are
consistent to a large degree with the data reported earlier by Gryta
[6], who also studied the performance of the fermentation process
integrated with membrane distillation.
Furthermore, the time course of ethanol flux (Fig. 2b) followed
the changes of the total flux, which may suggest that membrane
selectivity remained unchanged during the experiments. Accord-
ing to Udriot et al. [22] the membrane selectivity S hardly varies as
215
Fig. 3. Selectivity of the applied membrane distillation unit ethanol flux. (䊉) –
T = 13 ◦ C; () – T = 15 ◦ C; () – T = 20 ◦ C.
a function of T. Generally, in most of the membrane processes,
as the feed temperature increases, the swollen polymeric mem-
brane brings the flux to increase, but the selectivity decreases [23].
As shown in Fig. 3, the highest value of selectivity was observed
during the experiment conducted in T equal to 13 ◦ C and increas-
ing the T led to a decrease in the process selectivity. However,
the differences between S coefficients of T equal to 15 and 20 ◦ C
were found to be insignificant at the probability level of 95%. It is
also interesting to note that the selectivity curve for the investi-
gated systems can be divided into two periods: the initial period,
characterized by a comparatively rapid decrease of S, and the final
period, after the first hour, where S reached almost a constant
level. In fact, at the beginning of the process a slightly higher
standard deviation of experimental data was observed compar-
ing to the end of the process. This may be attributed mainly to
the time required to attain the desired separation temperatures
on the feed as well as permeate side of the membrane. As these
data do not completely rule out an important influence of experi-
mental conditions on membrane performance, the module used in
the described system was assessed for operation stability in a few
experiments carried out at different time intervals within 2 years
(data not shown). The results of the each experiment were almost
the same and no membrane leakage was detected, which indicated
that the repeatability of the experiment was excellent as well as
membrane performance was quite stable over time. Therefore, it
may be stated that in our system, there was no evidence of a loss
of membrane hydrophobicity. The long-term stability of PP mem-
brane has also been recently assessed and confirmed by Gryta [6] by
testing the capillary modules made from polypropylene membrane
intermittently for a couple of months. Lastly, it was also stated that
polypropylene membranes have appropriate characteristics that
make them suitable for membrane distillation process [6,24].
In order to obtain efficient separation of the ethanol from the fer-
mentation medium, the temperature difference mentioned above
should be as high as possible. However, most of the microorganisms
utilized for the fermentation of sugar feedstock, e.g. S. cerevisiae
limit the temperature to below 35 ◦ C. Therefore, in the next step
of this experiment, the temperature difference T was maintained
at a constant level of 20 ◦ C, whereas the fermentation temperature
was controlled at 35 ◦ C.
3.2. Fermentation
The experiments were undertaken using the molasses wort of
18◦ Plato density. It should be noted that the traditional brewing
uses 10–12 ◦ P in the production of 4–5 vol.% ethanol content beer.
The high gravity fermentation has been implemented mainly to
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216 G. Lewandowicz et al. / Journal of Membrane Science 375 (2011) 212–219
Fig. 4. Time course of the concentration of sugars and ethanol in the fermentation
broths. () – total sugars in batch fermentation; () – total sugars in fermentation
with MD; () – ethanol in batch fermentation; () – ethanol in fermentation with
MD.
enhance the process performance. Increasing the concentration of
carbon source in the medium could lead to the enhanced ethanol
production without expanding the existing facilities. In general, fer-
mentation of wort of density exceeding 18◦ Plato is performed in
order to achieve a final ethanol concentration of at least 7.5 vol.%
[25]. However, when high-gravity wort is used as fermentation
medium, yeast cells are exposed to adverse conditions including
ethanol toxicity and osmotic stress [26].
High concentration of ethyl alcohol causes: changes in the
metabolic pathways, the increase of the fermentation time as well
as changes in the structure and function of yeast cell walls and
membranes. As a result, ethanol can inhibit cell growth or cause
an increase in cell maintenance [27]. In order to overcome this
inhibition and increase the substrate conversion, mainly perva-
poration has been used to continuously remove ethanol from the
fermentation broth [28]. Besides pervaporation, the direct contact
membrane distillation has been suggested as an alternative way for
the ethanol recovery from alcoholic fermentations [10]. Hence, the
present work describes the development of an integrated process
of fermentation and ethanol recovery by MD.
As shown in Fig. 4, the yeast cultures were capable of con-
suming almost all fermentable sugars present in the medium, 4.54
and 1.73 g l−1 of total sugars remained in the reactor after the fer-
mentation activity had stopped in batch and continuous process,
respectively. The final ethanol amount in batch fermentation was
above 200 g and the product yield, YEt/Gl , on the total amount of
fermentable sugars was above 0.38 gg−1 , corresponding to 75% of
the theoretical maximum (Fig. 5). The application of membrane
Fig. 5. Total ethanol production in the continuous process with membrane distil-
lation in comparison to the batch fermentation. () – total ethanol production in
batch fermentation; () – total ethanol production in fermentation with MD.
Fig. 6. Productivity of the batch and continuous bioreactor. () – batch fermenta-
tion; () –continuous fermentation with MD.
distillation caused a decrease of the concentration of ethanol in
the fermentation broth (Fig. 4), however enabled a further linear
increase of the total amount of the ethanol produced (Fig. 5). Hence,
the total amount of the ethanol obtained during the continuous fer-
mentations was 426 g, corresponding to the conversion yield, YEt/Gl ,
of 0.45 gg−1 . Therefore, the conversion efficiency of carbohydrates
to ethanol was 88.2% of theoretical yield, that is 15.6% higher than
the one obtained during batch fermentation.
Furthermore, the presented results imply that the ethanol pro-
ductivity with MD after 30 h of process duration was almost 2 times
higher than without MD under the present fermentation conditions
(Fig. 6). The average ethanol productivity in continuous fermen-
tation was 2.15 g ethanol l−1 h−1 , while in the batch process was
1.4 g ethanol l−1 h−1 . This was nearly the same as the value obtained
by Kaseno et al. [29], who studied the performance of a fermenta-
tion system integrated with pervaporation. Moreover, according to
data presented in Fig. 6, ethanol productivity in batch fermentation
gradually decreased with the fermentation time. This significant
decrease in productivity can be attributed to a number of reasons.
The observed trend is probably a consequence of the inhibitory
effect of increasing concentration of ethanol as well as other by-
products accumulated in the broth. This hypothesis is supported
by Kaseno et al. [29] and Groot et al. [30], who also described
ethanol production from glucose in a system with cell retention
in the reactor coupled to a pervaporation module.
A common problem in the continuous fermentation is finding
a set of conditions for the input variables, especially dilution rate
D, which ensures the highest level of productivity and yield of the
final product. In the present work, the value of D was the same
as J, because the system was controlled by the level sensor. Since
the permeate flux was almost constant during the whole period of
fermentation, the dilution rate was also constant (Fig. 7). There-
fore, given the fact that the total concentration of carbohydrates at
steady state stage approached values below 2 g l−1 (Fig. 4), it can
be expected that the increase in the substrate concentration could
lead to the increased product yield in the actual process.
Additionally, the implementation of membrane distillation into
the fermentation system not only increased the throughput rate of
an ethanol plant but also caused a series of small but profitable
changes. As shown in Fig. 8, the osmolality of the fermentation
broth decreased and pH increased in the case of the fermentation
system coupled with the membrane distillation unit. These results
correspond well to the fact that there was no significant decline in
the number of cells and their viability (Fig. 9). In fact, the number
of cells and viability significantly decreased in the system without
MD. According to Nikolić et al. [31], the yeast viability significantly
decreased in batch fermentation most probably due to the great
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G. Lewandowicz et al. / Journal of Membrane Science 375 (2011) 212–219
Fig. 7. Time course of permeate flux for continuous fermentation with MD unit.
Fig. 8. Time course of the osmolality and pH. () – osmolality in batch fermentation;
() – osmolality in fermentation with MD; () – pH in batch fermentation; () – pH
in fermentation with MD.
Fig. 9. Changes in the cells number (a), and viability (b) during fermentation, ()
–fermentation with MD; () – batch fermentation.
217
Fig. 10. Glycerol/ethanol ration during course of the fermentation. () – fermenta-
tion with MD; () – batch fermentation.
accumulation of intracellular ethanol which altered the structure
of the membrane decreasing its functionality.
3.3. Stress
According to Lentini et al. [27] the most visible sign of yeast
stress induced by ethanol or high osmotic pressure is the induc-
tion of stress response genes. These genes were found to encode
for proteins involved in stress tolerance (i.e. heat shock proteins,
HSP) as well as the enzymes responsible for the formation of com-
patible solutes such as trehalose and glycerol. On the basis of the
present knowledge of the regulatory mechanisms that control the
stress tolerance in yeast, glycerol, beside trehalose, is the major
compatible solute in cells that can counterbalance the loss of water
due to osmotic stress [32]. According to data of Scanes et al. [33]
about 4–10% of all fermented carbon sources are converted into
glycerol, resulting in a decrease of 7–10% in ethanol production. It
is evident that glycerol overproduction is undesirable, as it lowers
the overall ethanol yield. The experiments in this study suggested
that osmolality is related not only to the concentration of sugars in
the medium but also to the ethanol produced. Thus, with the lower
osmolality, the glycerol concentration in the fermentation medium
would also decrease due to lowered environmental stress. From the
industrial point of view, membrane distillation can be regarded as
a straightforward method, which leads to decreasing glycerol syn-
thesis and increasing ethanol production. This working hypothesis
was born from the above mentioned results concerning osmolality.
In order to verify whether the MD influenced the alcoholic perfor-
mance of S. cerevisiae, additional analyses were conducted for the
presence of glycerol, trehalose as well as the selected heat shock
proteins.
Since the membrane distillation operates on the principle of
vapour–liquid equilibrium, non-volatile constituents of the fer-
mentation broth such as glycerol are completely rejected by the
membrane. Consequently, the observed level of glycerol in contin-
uous fermentation coupled with MD cannot be readily compared
to the result obtained during classical batch fermentation. Keeping
these caveats in mind, the relative production rate of glycerol was
computed by combining the measured concentrations of ethanol
and glycerol. As shown in Fig. 10, the implementation of the
membrane distillation unit caused significant changes in glycerol
production. After 20th hour of the process, the regular decrease in
the production of glycerol per 1 g of ethanol was observed.
Moreover, these results were also supported by the analysis of
the concentration of the trehalose, one of the main yeast’s metabo-
lites accumulated in the cells under stress conditions. This sugar is
accumulated in cells exposed to heat, osmotic and ethanol stress.
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218 G. Lewandowicz et al. / Journal of Membrane Science 375 (2011) 212–219
Fig. 11. Trehalose concentration in yeast cells. () – batch fermentation without
MD; () – fermentation with MD. 1 – 0 h; 2 – 24 h; 3 – 48 h; 4 – 72 h.
Its amount in cells may exceed 100 m gg−1 d.m. It is positively
correlated with thermotolerance and tolerance of a given strain
to ethanol [34]. As can be seen in Fig. 11, when cells were intro-
duced into fresh fermentation medium, trehalose concentration
decreased considerably. According to Verstrepen et al. [35] the
observed changes in the trehalose level at the beginning of the
fermentation can be attributed to the activation of the cyclic AMP-
protein kinase A pathway. However, the trehalose concentration
started to increase again after 24 h, especially in the case of fer-
mentation conducted without MD. The reason for this discrepancy
seems to be clear, the explanation can be found in the conditions
that differ between the two types of fermentations.
Besides the well-known defence mechanisms such as accumu-
lation of glycerol or trehalose, the tolerance to stress can also be
acquired by means of the synthesis of heat shock proteins (HSPs).
All studies published so far have been focused on an expression of
particular HSPs in yeast strains cultivated in a standard bioreactor,
and no data have been presented about the effect of the other mode
of fermentation e.g. continuous fermentation integrated with MD.
In this study, for the first time a short report is given on the heat
shock proteins present in yeast growing in the above mentioned
system with the emphasis on the distribution of Hsp70 and Hsp104.
The investigated proteins have been found previously to be respon-
sible for protecting yeast cells against heat as well as ethanol stress
[36].
Although this work is preliminary, our results suggest that mem-
brane distillation may considerably influence the level of gene
expression responsible for the synthesis of HSPs in the yeast cells.
As can be seen in Fig. 12, the major changes in protein expression
were observed after 20th hour of process duration. It is inter-
esting that the level of Hsp104 during batch fermentation was
two times higher than during continuous fermentation with MD
(Table 1). These results definitely confirm that Hsp104 is respon-
sible for protecting yeast cells against ethanol stress. Furthermore,
Table 1
Quantitative analysis of Western blots for batch and continuous fermentation.
Time [h] Band volume [intensity units × mm2 ]
Batch fermentation Continuous
fermentation with MD
Hsp70 Hsp104 Hsp70
0 4601.7 4764.8 4352.5
20 9160.8 4615.7 12210.7
25 5778.8 10935.1 8112.8
28 4526.2 10143.1 7037.3
48 5233.5 10001.5 6062.1
Fig. 12. Detection of the hsp104 and hsp70 protein by western blot analysis for the
batch (a), and continuous with MD (b) fermentations, A – Hsp104, B – Hsp70, C –
actin which was used as a loading control. 1 – 0 h, 2 – 20 h, 3 – 25 h, 4 – 28 h, 5 –
48 h.
the membrane distillation exerts a serious impact on the fermen-
tation performance. Conversely, the reason for the variation at the
level of Hsp70 is not clear but may indicate that under these con-
ditions other stress factors that can induce the synthesis of this
protein occurred. The effect of applying the MD on the synthesis of
Hsp70 will be investigated in detail in our laboratory in the nearest
future.
4. Conclusions
Continuous fermentation processes based on a bioreactor cou-
pled with a membrane distillation unit have the potential of
maximising the volumetric productivity and thus minimising the
production costs in the biofuel industry. According to the present
results, MD can be regarded as a straightforward method, which
leads to an increase in ethanol production by decreasing glyc-
erol synthesis level as well as an increase in yeast cells’ number
and viability. However, the main problem of membrane distilla-
tion is associated with too low membrane selectivity as well as
progressive wettability of the membranes observed during increas-
ing concentration of volatile compounds [37,38]. Compared to
other membrane processes, such as ultrafiltration (UF), or reverse
osmosis (RO), membrane distillation is more difficult to apply
on industrial scale because of some serious engineering prob-
lems. These include module design as well as heat loss during
the MD process, which may lead to uncertain economic costs.
Thus, considerable effort should be directed towards the design
and construction of a novel membrane module to permit a suc-
Hsp104 cessful industrial application of this separation technique. It is
4572.3
worth mentioning that a MD module design for a particular sep-
4044.5 aration is a trade-off of a number of factors, such as cost, as well
3139.4 as membrane performance including thermal stability, selectivity
5749.3 and low thermal conductivity. However, there are many different
5450.2
hydrophobic membrane modules available in the market which
 Author's personal copy
G. Lewandowicz et al. / Journal of Membrane Science 375 (2011) 212–219
has been extensively investigated during the past years. In addi-
tion, since the number of research papers published in MD field
continues to increase, it is reasonable to suppose that in the near
future membranes with higher selectivity and improved ethanol
resistance may become available.
Summing up the conclusions arrived at, it may be stated that for
the economic use of membrane distillation in ethanol fermenta-
tion process, future investigations should mainly concentrate on
the systematic modeling of the proposed system as well as the
development of high-flux and selective membrane. It is worth to
point-out that the numerical results of mathematical models may
be beneficial at the earlier stages of process scale-up.
Acknowledgements
The authors gratefully acknowledge the support by the Ministry
of Science and Higher Education (grant No. N N312 311437).
References
[1] C.E. Wyman, Applications of corn stover and fiber, in: P.J. White, L.A. Johnson
(Eds.), Corn Chemistry and Technology, Second ed., American Association of
Cereal Chemists, St. Paul MN, 2003, pp. 723–750.
[2] H.-J. Huang, S. Ramaswamy, U.W. Tschirner, B.V. Ramarao, A review of sepa-
ration technologies in current and future biorefineries, Sep. Purif. Technol. 62
(2008) 1–21.
[3] T. Matsuura, Synthetic Membranes and Membrane Separation Processes, CRC
Press, 1993.
[4] K. Kargupta, S. Datta, S.K. Sanyal, Analysis of the performance of a contin-
uous membrane bioreactor with cell recycling during ethanol fermentation,
Biochem. Eng. J. 1 (1998) 31–37.
[5] M. Gryta, A.W. Morawski, M. Tomaszewska, Ethanol production in membrane
distillation bioreactor, Catal. Today 56 (2000) 159–165.
[6] M. Gryta, The fermentation process integrated with membrane distillation, Sep.
Purif. Technol. 24 (2001) 283–296.
[7] R. Hatti-Kaul, Downstream processing in industrial biotechnology, in: W.
Soetaert, E.J. Vandamme (Eds.), Industrial Biotechnology, Sustainable Growth
and Economic Success, WILEY-VCH Verlag, 2010, pp. 279–323.
[8] M. Khayet, Membrane distillation, in: N.N. Li, A.G. Fane, W.S.W. Ho, T. Matsuura
(Eds.), Advanced Membrane Technology and Applications, John Wiley & Sons,
2008, pp. 297–371.
[9] R.W. Baker, Membrane Science and Technology, John Wiley & Sons, 2004.
[10] F.A. Banat, F. Abu Al-Rub, M. Shannag, Modeling of dilute ethanol–water mix-
ture separation by membrane distillation, Sep. Purif. Technol. 16 (1999) 119.
[11] A.C.M. Franken, J.A.M. Nolten, M.H.V. Mulder, C.A. Smolders, Ethanol–water
separation by membrane distillation: effect of temperature polarization, in: B.
Sedlacek, J. Kahovec (Eds.), Synthetic Polymeric Membranes, Walter de Gruyter,
Berlin, 1987, p. 531.
[12] M.A. Izquierdo-Gil, G. Jonsson, Factors affecting flux and ethanol separation
performance in vacuum membrane distillation (VMD), J. Membr. Sci. 214
(2003) 113.
[13] R.L. Calibo, M. Matsumura, J. Takahashi, H. Kataoka, Ethanol stripping by per-
vaporation using porous PTFE membrane, J. Ferment. Technol. 65 (1987) 665.
[14] K. Kargupta, S. Datta, S.K. Sanyal, Analysis of the performance of a contin-
uous membrane bioreactor with cell recycling during ethanol fermentation,
Biochem. Eng. J. 1 (1998) 31–37.
[15] W. Grajek, D. Szymanowska, The influence of environmental stresses on Sac-
charomyces cerevisiae yeast in ethanol fermentation (Stresy środowiskowe
˛
działajace na drożdże Saccharomyces cerevisiae w procesie fermentacji
etanolowej), Biotechnologia 82 (3) (2008) 46–63 (in Polish).
[16] B. Shen, S. Hohmann, R.G. Jensen, H.J. Bohnert, Roles of sugar alcohols in osmotic
stress adaptation. Replacement of glycerol by mannitol and sorbitol in yeast,
Plant Physiol. 121 (1999) 45–52.
219
[17] V. Ansanay-Galeote, B. Blondin, S. Dequin, J.-M. Sablayrolles, Stress effect of
ethanol on fermentation kinetics by stationary-phase cells of Saccharomyces
cerevisiae, Biotechnol. Lett. 23 (2001) 677–681.
[18] J.C. González-Hernández, M. Jiménez-Estrada, A. Peña, Comparative analysis of
trehalose production by Debaryomyces Hansenie and Saccharomyces cerevisiae
under saline stress, Extremophiles 9 (2005) 7–16.
[19] M.S. Tracey, K. Watson, Stress tolerance in a yeast sterol auxotroph: role of
ergosterol, heat shock proteins and trehalose, FEMS Microbiol. Lett. 169 (1998)
191–197.
[20] R.J. Karreman, W.F. Brandt, G.G. Lindsey, The yeast Saccharomyces cerevisiae
stress response protein Hsp12p decreases the gel strength of agarose used as
a model system for the ␤-glucan layer of the cell wall, Carbohydr. Polym. 60
(2005) 193–198.
[21] M.C. Garcia-Payo, M.A. Izquierdo-Gil, C. Fernández-Pineda, Air gap membrane
distillation of aqueous alcohol solutions, J. Membr. Sci. 169 (2000) 61–80.
[22] H. Udriot, S. Ampuero, I.W. Marison, U. von Stockar, Extractive fermenta-
tion of ethanol using membrane distillation, Biotechnol. Lett. 11 (7) (1989)
509–551.
[23] Ch.H. Lee, W.H. Hong, Effect of operating variables on the flux and selectivity
in sweep gas membrane distillation for dilute aqueous isopropanol, J. Membr.
Sci. 188 (1) (2001) 79–86.
[24] M. Gryta, J. Grzechulska-Damszel, A. Markowska, K. Karakulski, The influence
of polypropylene degradation on the membrane wettability during membrane
distillation, J. Membr. Sci. 326 (2009) 493–502.
[25] L. Blieck, G. Toye, F. Dumortier, K.J. Verstrepen, F.R. Delvaux, J.M. Thevelein,
P. Van Dijck, Isolation and characterization of brewer’s yeast variants with
improved fermentation performance under high-gravity conditions, Appl. Env-
iron. Microbiol. 73 (3) (2007) 815–824.
[26] P.A. White, A.I. Kennedy, K.A. Smart, The osmotic stress response of ale and
lager brewing yeast strains, in: K.A. Smart (Ed.), Brewing Yeast Fermentation
Performance, Second ed., Blackwell Science, 2003, pp. 46–59.
[27] A. Lentini, P. Rogers, V. Higgins, I. Dawes, M. Chandler, G. Stanley, P. Chambers,
The impact of ethanol stress on yeast physiology, in: K.A. Smart (Ed.), Brew-
ing Yeast Fermentation Performance, Second ed., Blackwell Science, 2003, pp.
25–38.
[28] D.J. O’Brien, J.C. Craig, Ethanol production in a continuous fermenta-
tion/membrane pervaporation system, Appl. Microbiol. Biotechnol. 44 (1996)
699–704.
[29] Kaseno, I. Miyazawa, T. Kokugan, Effect of product removal by a pervaporation
on ethanol fermentation, J. Ferment. Bioeng. 86 (5) (1998) 488–493.
[30] W.J. Groot, M.R. Kraayenbrink, R.G.J.M. van der Lans, K.Ch.A.M. Luyben, Ethanol
production in an integrated fermentation/membrane system. Process simula-
tions and economics, Bioprocess Eng. 8 (1993) 189–201.
[31] S. Nikolić, L. Mojović, M. Rakin, D. Pejin, Bioethanol production from corn
meal by simultaneous enzymatic saccharification and fermentation with
immobilized cells of Saccharomyces cerevisiae var. ellipsoideus, Fuel 88 (2009)
1602–1607.
[32] M.G. Walker, P. van Dijck, Physiological and molecular responses of yeasts to
the environment, in: A. Querol, G.H. Fleet (Eds.), Yeasts in Food and Beverages,
Springer-Verlag, Berlin Heidelberg, 2006, pp. 112–152.
[33] K.T. Scanes, S. Hohmann, B.A. Prior, Glycerol production by the yeast Saccha-
romyces cerevisiae and its relevance to wine: a review, S. Afr. J. Enol. Vitic. 19
(1998) 17–22.
[34] J.J. Mansure, R.C. Souza, A.D. Panek, Trehalose metabolism in Saccha-
romyces cerevisiae during alcoholic fermentation, Biotechnol. Lett. 19 (1997)
1201–1203.
[35] K.J. Verstrepen, D. Iserentant, P. Malcorps, G. Derdelinckx, P. Van Dijck, J.
Winderickx, I.S. Pretorius, J.M. Thevelein, F.R. Delvaux, Glucose and sucrose:
hazardous fast-food for industrial yeast? Trends Biotechnol. 22 (2004)
531–537.
[36] P.W. Piper, The heat shock and ethanol stress responses for yeast exhibit
extensive similarity and extensional overlap, FEMS Microbiol. Lett. 134 (1995)
121–127.
[37] C. Adiche, K. Sundmacher, Experimental investigation on a membrane distilla-
tion based micro-separator, Chem. Eng. Proc. 49 (2010) 425–434.
[38] M.C. Garcia-Payo, M.A. Izquierdo-Gil, C. Fernández-Pineda, Wetting study of
hydrophobic membranes via liquid entry pressure measurements with aque-
ous alcohol solutions, J. Colloid Interface Sci. 230 (2000) 420–431.