b i o m a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 2 5 e3 2

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http://www.elsevier.com/locate/biombioe

Performances comparison between three technologies

for continuous ethanol production from molasses
Hassib Bouallagui*, Youssef Touhami, Nedia Hanafi, Amine Ghariani, Moktar Hamdi
Laboratory of Microbial Ecology and Technology, National Institute of Applied Sciences and Technology, BP 676, Tunis 1080, Tunisia

article info

abstract

Article history:

Molasses are a potential feedstock for ethanol production. The successful application of

Received 7 August 2009

anaerobic fermentation for ethanol production from molasses is critically dependent to the

Received in revised form

development and the use of high rate bioreactors. In this study the fermentation of sugar

16 October 2012

cane molasses by Saccharomyces cerevisiae for the ethanol production in a continuously

Accepted 20 October 2012

stirred tank reactor (CSTR), an immobilised cell reactor (ICR) and a membrane reactor

Available online 23 December 2012

(MBR) was investigated. Ethanol production and reactor productivities were compared
under different dilution rates (D). When using the CSTR, a decent ethanol productivity (Qp)

Keywords:

of 6.8 g L
1 h
1 was obtained at a dilution rate of 0.5 h
1. The Qp was improved by 48% and

Molasses

the residual sugar concentration was reduced by using the ICR. Intensifying the production

Ethanol

of ethanol was investigated in the MBR to achieve a maximum ethanol concentration and

Productivity

a Qp of 46.5 g L
1 and 19.2 g L
1 h
1, respectively. The achieved results in the MBR worked

CSTR

with high substrate concentration are promising for the scale up operation.
2012 Elsevier Ltd. All rights reserved.

ICR
MBR

1.

Introduction

In the past decades, natural energy resources such as petroleum have been consumed at high rates. Therefore, alternative resources as ethanol are becoming more important [1,2].
Microbial ethanol production has been considered as renewable fuel for future contributing to the reduction of environmental impacts [3e5]. Saccharomyces cerevisiae is the most
useful microorganism for the ethanol production by the
alcoholic fermentation of various raw materials rich in sugars
[6]. Molasses is an agro-industrial by-product often used in
alcohol distilleries due to the presence of fermentative sugars,
being an optimal carbon source for the microorganism
metabolism [7]. The use of molasses generated during the
process of sugar cane refining has attracted great interest

because they are rich in sucrose, which presents a substrate

not requiring pre-treatment prior to the fermentation.
During traditional batch fermentation for distilled ethanol
production using S. cerevisiae, inhibition of growth can be
caused either by product or substrate concentrations, and the
productivity is limited to only 1.8e2.3 g L
1 h
1 [8]. This leads
us to define new strategies for intensive production in order to
maximise Qp and yield. Use of continuous reactors for ethanol
production has been investigated to improve economics and
the performance of fermentation processes [9]. Although, high
Qp can be achieved if cell retention is also employed [10].
General strategies for cell retention include immobilisation of
microbial cells within the fermenter or membrane separation
from the product stream, followed by recycle to the fermenter
[11,12].

* Corresponding author. Tel.: 216 22524406; fax: 216 71704329.

E-mail address: hassibbouallagui@yahoo.fr (H. Bouallagui).
0961-9534/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biombioe.2012.10.018

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b i o m a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 2 5 e3 2

Immobilisation of microbial cells for fermentation has

been developed to eliminate inhibition caused by high
concentration of substrate and product, also to uncouple the
hydraulic retention time and the cellular retention time.
Immobilisation of cells to a solid matrix is an alternative
means of high biomass retention. Alginate is widely used in
food, pharmaceutical; textile and paper products. The use of
alginate is for thickening, stabilising, gel and film forming [6].
Recent works on ethanol production in an immobilised cell
reactor showed that production of ethanol using Zymomonas
mobilis and S. cerevisiae was increased significantly [13,14].
The Qp can be also improved by increasing the concentration of viable biomass in the reactor. In this approach many
researches studied cultures at high cell density. The best
performance was obtained using continuous systems with cell
recycling [15e17]. Membrane filtration is most frequently used
to increase the concentration of viable biomass in the reactor.
We must take into account the characteristics of the membrane
(material, porosity, size and number of pores, charge of
surface), the nature and composition of species present in the
solution and the operating conditions (applied pressure, the
solution concentration, temperature, pH and ionic strength).
The aim of this work was to study the alcoholic fermentation of sugar cane molasses by using CSTR, ICR and MBR
technologies. In order to find the better alternative with higher
ethanol productivity, the reactors performances were investigated at different loading rates.

2.

Materials and methods

2.1.

Yeasts and molasses sources

Sugarcane (Saccharum officinarum) cultivars employed in this

work were harvested in the central province of Santiago
(Cuba) on 2008. Molasses, from which fermentation medium
were prepared, were obtained from the STS Company of
Tunis. They have the following composition: dry matter
(72.2%), total sugars (48.5%), ash content (6.7%) and pH of 7.9.
The feedstock solution was boiled for 5 min, centrifuged and
filtered for pre-treatment and clarification. In the clarification
step, a part of the coloured material and unknown toxic
substances frequently included in the molasses were separated or inactivated and the molasses were diluted with
distilled water to give a total sugar concentration of 100 g L
1.
pH was adjusted to 5 with 10% sulphuric acid concentration.
Medium used for bioreactors feeding consisted of yeast
extract (10 g L
1), peptone (5 g L
1), NH4Cl (2 g L
1), KH2PO4,
MgSO4 H2O (0.5 g L
1), sugars (100 g L
1) and 200 mg L
1Nathioglyconate as the reducing agent at pH 5 [18]. The culture
medium was sterilized at 121  C for 15 min. The initial
oxidation reduction potential (ORP) of the medium was nearly

250 mV indicating the anaerobic conditions.
The different fermentations have been achieved using
fresh commercial bakers yeast S. cerevisiae (Tunisian Society
of Yeasts). The yeast strain viability was determined using
methylene blue staining technique. It was cultivated using an
incubator shaker under sterile conditions at pH 5, 30  C and
150 revolutions min
1. Pure cultures grown under anaerobic
conditions were used for inoculation of experimental reactors.

2.2.
Laboratory reactors configurations and operating
conditions
Three types of reactors were used to carry out the continuous
fermentation at 30  C (CSTR: Fig. 1, ICR: Fig. 2 and MBR: Fig. 3).
The fermentation in the CSTR was conducted in a 1 L Biolafit fermentor using a pre-culture of 200 mL seeded in
a medium with a sugar concentration of 50 g L
1. An aerated
batch phase for 48 h was provided to produce a sufficient
concentration of biomass of 18 g L
1. At the end of the batch
phase, the continuous culture was conducted in anaerobic
conditions to ensure the fermentation. The CSTR was fed with
a sugar concentration of 100 g L
1. Different dilution rates
were tested. A D of 0.12 h
1 (run 1) was applied during the first
246 h of monitoring, a D of 0.25 h
1 (run 2) was applied from
time 246 h to time 415 h and a D of 0.5 h
1 (run 3) was applied
during the last 85 h of work.
The fermentation set-up of the ICR was comprised of
a Biorad column packed with beads of immobilised cells. The
immobilisation of S. cerevisiae was performed by the enriched
cells cultured media harvested at the exponential growth
phase. The fixed cell loaded ICR was carried out at initial stage
of operation and the cell were entrapped by calcium alginate
(2%) [6,8]. The fermentation in the ICR has been done over
a period of 225 h. Ds were adapted to 0.12, 0.25 and 0.5 h
1,
respectively.
The MBR system (YUASA Membrane Systems: ED-03SPH) is
composed of two reactors: the fermentation tank and
a membrane tank (1 L each) maintained in anaerobic conditions. A recirculation of flux was maintained between the
fermentation tank and the membrane tank with a high flow
rate to maintain similar conditions in both tanks in terms of
mixed liquor total suspended solids. Then, the dilution rate
was calculated using the 2 L total volume of the fermentor and
the membrane unit. The filtration unit consisted of an external
filtration module with a nominal cut-off filter of 0.4 mm and
a filtration surface of 0.014 m2. The maximum allowable
pressure drop over the filter was 4 bars. The start-up of the MBR
culture was provided from the outlet of the CSTR with an initial
biomass concentration of 8 g L
1 and a pH of 4.8. The
fermentation tank was loaded with an initial sugar concentration of 100 g L
1. The system was operated 245 h without
cleaning resulting in a membrane flux of 21.4e35.7 L h
1 m
1.
There has been a decrease in the permeate flow over time

CO2

Feed Tank

Pump

Fermenter

Pump

Product Tank

Fig. 1 e Schematic of the laboratory scale continuously

stirred tank reactor.

27

b i o m a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 2 5 e3 2

using a standard curve of absorbance against dry cell mass

(TSS: total suspended solids). The TRS concentration was
measured by using the phenol acid method [19]. The samples
were analysed in triplicates and results were reproducible. pH
and ORP were measured using a pH metre (WTW).
Ethanol production was analysed by high performance
liquid chromatography on an Agilent Model 1200 series
liquid chromatograph equipped with four solvent pumps,
a programmable multi-wavelength detector and a data
module. The mobile phase was 1 mol m
3 H2SO4. Aliquots of
20 mL were injected into a silica column (ZORBAX C18)
(150 mm  3.9 mm) at ambient temperature. The flow rate of
the mobile phase was 0.3 mL min
1 and the analysis was done
under isocratic mode (no composition change of the solvent).
Quantification of ethanol was done by using standard ethanol.
Samples were diluted with ultra pure water and filtered with
millipore membranes (0.22 mm pore size).

Immobilised Cell Reactor

Valve

Pump

Valve

Feed Tank

2.4.

Product Tank

Pump

Fig. 2 e Schematic of the laboratory scale immobilised cell

reactor.

which corresponded to the membrane fouling. Consequently

the dilution rate initially set at 0.5 h
1 fall during the first 90 h of
working to stabilise at 0.31 h
1. All the experiments occurred in
triplicate and the relative standard deviations among the
results did not exceed 5%.

2.3.

Technical analysis

Kinetic of fermentations was monitored by measuring the

concentration of biomass, total reduced sugars (TRS) and
ethanol during the time. The samples were centrifuged
(10,000  g) and the biomass concentration was determined by
measuring the optic density (OD) of diluted sample at 600 nm

Statistical analysis

In order to determine whether the observed differences

between reactors performances were significantly different,
data were subjected to the analysis of variance by ANOVA
tests (p < 0.05) [20].

3.

Results and discussion

3.1.

Performances of the CSTR

The alcoholic fermentation provided in the CSTR has been

achieved by varying the D from 0.12 h
1 to 0.5 h
1.
Fig. 4 shows the variation of total sugar and ethanol
concentrations. During the first run (D 0.12 h
1) the
concentration of residual sugar was stabilised at an average
value of 18.3 g L
1 equal to a substrate conversion yield (Ys) of
81.7%. This condition permitted to succeed to an average
ethanol concentration of 27.9 g L
1 and a yield ethanol for
substrate (YP/S) of 0.34 g g
1. Average Qp of 3.34 g L
1 h
1 has
been determined.

(Permeate)
(Concentration)

Permeate Tank

CO2
P
(V)

Feed Tank

Feed Pump

Fermenter

Circulation
Pump

(V)

(V)

(V)

Circulation
Tank

Circulation
Pump

Membrane

Fig. 3 e Schematic of the laboratory scale membrane bioreactor (P [ pressure measurement and V [ valve).

28

b i o m a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 2 5 e3 2

In the second run, the increase of the D to 0.25 h
1 caused

a disruption of the system balance which resulted in a significant drop in the residual sugar concentration, accompanied
by an increase in the alcohol concentration. The important
input of substrate excited the fermentation metabolism of
cells to produce high quantity of ethanol which reached
a maximum concentration of 40.9 g L
1. This result showed
the coupling between biomass and ethanol productions
because the biomass concentration has risen in this phase to
reach a maximum value of 9 g L
1. At the end of this run
ethanol and residual sugar concentrations were stabilised at
an average values of 21.6 g L
1 and 31.1 g L
1, respectively,
equal to a Ys yield of 68.9%. The YP/S yield decreased to
0.31 g g
1 and the Qp increased to 5.4 g L
1 h
1 by the application of a D of 0.25 h
1. In fact, the Qp depends on the ethanol
concentration and the dilution rate that has been increased.
The highest Qp of 6.8 g L
1 h
1 for the CSTR was observed at
a D of 0.5 h
1 (run 3). Applying a high D caused a progressive
decrease in the concentration of ethanol to achieve an average
value of 11.6 g L
1, accompanied by an increase in the
concentration of TRSs (51.5 g L
1). Then, the Ys fell to 48.5%
corresponding to an YP/S of 0.24 g g
1. At high D, the assimilation declined and a significant part of sugars was washed
out. This result showed that cells fail to produce high quantity

0,6

50

0,5

40

0,4

30

0,3

20

0,2

10

0,1

0
0

Biomass concentration
(g L-1)

50

100

150

200

250

300

350

400

450

D (h-1)

60

0
500

Time (h)
20

18

5,5
5

16

4,5

14

12

3,5

10

3
2,5

pH and D (h-1)

TRS and ethanol concentrations

(g L-1)

of ethanol at high D due to the wash out of cells at high flow

rate. The same Qp in order of 5.98 g L
1 h
1 with a high YP/S
yield of 0.47 g g
1 were obtained by Purwadi and Taherzadeh
[9] in a continuous fermentation of glucose by applying
a dilution ratio of 0.86 h
1. However, using hydrolysed lignocelluloses wood after an acid pre-treatment has led to a high
Qp in the order of 20 g L
1 h
1 by conducting a continuous
fermentation reactor in a volume equal to 0.45 L, with a D of
0.3 h
1 [21].
At run 1 (D 0.12 h
1), the biomass concentration dropped
considerably from 18 g L
1 to 4.8 g L
1 after passing through
fluctuations (Fig. 4). This decrease corresponds to a transitional state of cells adaptation characterised by a very
important wash out of cells out side the reactor. The stabilisation of biomass concentration was reached after 75 h of
operation of the system. The increase in the D from 0.12 to
0.25 h
1 caused an acceleration of cell multiplication to achieve a biomass concentration of 9 g L
1, followed by a fall, then
stabilised. The fall of the biomass concentration was caused
by the important flow rate that created a significant wash out
of cells. After 108 h of operation under this condition the
biomass stabilised and reached steady state at a concentration of 4.9 g L
1. The second change of the D (run3) led to
a further reduction in the biomass concentration to be

1,5

0,5

0
0

50

100

150

200

250

300

350

400

450

0
500

Time (h)

Fig. 4 e Evolution of (a): total reducing sugar (TRS) (>), ethanol (B), (b): biomass (>) and pH (B) during CSTR fermentation of
sugar cane molasses under different Ds (D).

29

b i o m a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 2 5 e3 2

2.4 g L
1. The application of a high D of 0.5 h
1 caused

a significant wash out of cells. The continuous increase of the
D to a value exceeding the maximum specific growth rate
(mmax) induced the phenomenon of leaching. In these conditions growth does not compensate the biomass washed out
due to the increase of the flow rate. However, if the out flow of
biomass exceeds the growth yield of yeasts, the continuous
cultivation fails and the biomass is washed out. Taherzedeh
et al. [22] showed that the continuous fermentation of
a fermentable hydrolysate by yeast can be successful at a D of
0.1 h
1, but fails at a D of 0.2 h
1 or higher.
The initial pH of feed was regulated to 5.5 by adding
a diluted sulphuric acid. Fig. 4 showed that the pH decreased
slightly during the fermentation and stabilised around values
of 4.6e5. This slight decrease can be explained by the CO2
production during the fermentation and the accumulation of
ions HCO

3 which represent the soluble form of CO2. It can be
also explained by the production of metabolites other than
ethanol (acetate, succinic acid, etc.) by the yeast during the
fermentation. S. cerevisiae have been reported to increase
ethanol production at pH 5.0 and 5.5 as opposed to pH 4.0 and
4.5 and its optimum pH is from 5.0 to 5.2 [23].

sugar in the culture medium. Therefore the YP/S yield reduced

and the concentration of residual sugars become more
important (9.6 g L
1). The decrease in the concentration of
ethanol could be caused by the combined effect of increasing
the medium flow rate and the loss of cell viability over time.
The maximum Qp of 10.1 g L
1 h
1 was obtained with a D of
0.5 h
1. Goksungur and Zorlu [24] achieved a Qp very close
(10.16 g L
1 h
1) to that of our work by applying a D of 0.22 h
1.
While, Baptista et al. [8] achieved a higher Qp of 16 g L
1 h
1
with a D of 0.4 h
1, with the same type of bioreactor but using
cells of S. cerevisiae immobilized on polyurethane cubes.
Monitoring of pH during this fermentation showed
a decrease of values from 5.5 to 4.3e5.3. This decrease was due
to the increase of dissolved CO2 in the reactor as mentioned
previously. The dissolved CO2 in the solution reacts with
water to form carbonate ions and proton H. According to the
literature, the intracellular pH varies slightly for an extracellular pH ranging between 4 and 7. In this range of pH, cell
viability can not be affected by the pH because its value
remained constant and greater than 4 [23].

3.2.

Fig. 6 shows a very important increase in the concentration of

biomass in the MBR from an initial value of 8 g L
1 to a final
value of 41.1 g L
1 after 245 h of work. This increase in the
biomass concentration was accompanied with a gradual
decrease in the permeate flux due to the membrane fouling.
The concentration of biomass at the outlet of the MBR was
very low; it is equal to 0.05 g L
1. This result confirms the
proper functioning of the membrane system that can retain
all the cells inside the bioreactor. pH within and in the outlet
of the bioreactor were the same which showed that the
membrane system does not affect the pH.
Fig. 6 shows an almost consumption of total sugars. The Ys
yield was close to 97.7e99.3% with a residual concentration
lower than 2.5 g L
1. The consumed sugar was headed towards
the growth of yeasts resulting in a very important activity
and ethanol production with average concentrations of

Performances of the ICR

The ICR fermentation monitoring has been done over a period

of 225 h. The applied Ds were adapted to 0.12, 0.25 and 0.5 h
1.
The results of the different runs are presented in Fig. 5 which
showed that the production of ethanol was steady after 18 h of
operation and maximum concentration of ethanol was
reached during the first 115 h of the fermentation. The average
concentration of ethanol during the stationary phase was
44.1 g L
1. In fact, Qp reached during this D was 5.28 g L
1 h
1.
The concentration of ethanol was affected by the media
flow rates and the residence time distribution. Following
a first increase in the D from 0.12 h
1 to 0.25 h
1 after 115 h of
fermentation, a reduction in the ethanol concentration was
observed and the Qp increased significantly (P < 0.05). Indeed,
with the increase of the D, yeasts fail to completely consume

3.3.

Performances of the MBR

60

50

5
4,5

40

4
3,5

30

3
2,5

20

D (h-1) and pH

TRS and ethanol c onc entrations

(g L-1)

5,5

2
1,5

10

1
0,5

0
0

25

50

75

100

125

150

175

200

0
225

Time (h)

Fig. 5 e Evolution of total reducing sugar (TRS) (>), ethanol (,) concentrations and pH (D) during ICR fermentation of sugar
cane molasses under different Ds (B).

30

45
40
35
30
25
20
15
10
5
0
0

Ethanol concentration (g L-1)

30

60

90

120

150

180

210

240

Time (h)

60

8
7

50

6
40

5
4

30

20

pH and TRS (g L-1)

and permeate flux (L h-1 m-2)

Biomass concentration (g L -1)

b i o m a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 2 5 e3 2

2
10

(p < 0.05). The decrease of the Qp can be avoided by studying

the membrane behaviour during the operation to learn the
moment when it must be regenerated by flow reversal or
chemical cleaning.
The intensification of ethanol production from hydrolysed
wood in a membrane bioreactor was studied by Lee et al. [15].
They obtained a similar Qp of 16.9 g L
1 h
1 with ethanol
concentration of 76.9 g L
1 and a YP/S of 0.43 g g
1. However,
higher Qp has been also obtained by Ben Chaabene et al. [17] in
a two stage MBR. They mentioned an important Qp in the
order of 41 g L
1 h
1 and an ethanol concentration of 80 g L
1.
Escobar et al. [25] have implemented a membrane bioreactor
of 7000 L. They obtained a Qp of 3.4 g L
1 h
1 with ethanol
concentrations ranged between 80 and 90 g L
1. However, the
authors mentioned some problems of membrane fouling with
biomass concentrations above 100 g L
1. They also observed
a drop in the cell viability due to the stress caused by the
pump.

0
0

30

60

90

120

150

180

210

3.4.

Comparison of reactors performances

240

Time (h)

Fig. 6 e Evolution of (a): biomass concentration (>),

permeate flux rate (,), (b): total reducing sugar (TRS) (>),
ethanol concentrations (D) and pH (,) during MBR
fermentation of sugar cane molasses.

41.4e46.5 g L
1. The high consumption of sugars and

production of ethanol are independent to the loading flow
rate. They are the result of the high concentration of biomass
in the bioreactor, which provided an important YP/S with
a constant rate, even during the change of flow. The stabilisation of biomass concentration in the MBR can be explained
by the fact that the high concentration of ethanol becomes
a limiting factor for the multiplication of cells and induced the
yeast metabolism for the alcoholic fermentation.
During the MBR system monitoring results showed two
phases characterised by two levels of Qp. The first Qp corresponds to a D of 0.31e0.5 h
1 during the first 90 h which was
about 12.83e19.2 g/L h. The second phase was characterised
by a Qp of 14.4 g L
1 h
1 which corresponds to a D of 0.31 h
1.
After the membrane fouling, the Qp decreased significantly

A comparison between performances of bioreactors used for

the ethanol production has been made.
Table 1 summarizes the results obtained with different
configurations of reactors. The average ethanol concentrations decreased by increasing the D however, productivities
increased. The Qp depends not only to the ethanol concentration but also to the D hence the interest in working with
high D exceeding the mmax. This was achievable only by
working with the MBR which provided high cell densities
within the reactor.
Data showed that ethanol concentration and Qp during the
fermentation of sugar cane molasses in the ICR are higher
than those obtained with the CSTR. In addition, YP/S yields are
more important in the ICR. This was mainly caused by the
immobilisation of cells in the reactor which slowed cell
proliferation and promotes the metabolism to the alcoholic
fermentation. Subsequently, yeasts consumed the substrate
in advantage to produce ethanol.
The fermentation of molasses through the MBR bioreactor
improved the Qp, the concentration of ethanol and the YP/S
yield. In fact, Qp achieved in this reactor was three times
higher than that in the CSTR and it was two times higher than
that in the ICR. The Ys yield showed very high levels

Table 1 e Performances of the CSTR, ICR and MBR bioreactors used for the ethanol production at different dilution rates.
Reactors

CSTR

1

Dilution rate D (h )
Ethanol concentration (g L
1)
Productivity: Qp (g L
1 h
1)
Substrate inlet (g L
1)
Substrate outlet (g L
1)
Sugar consumption: Ys (%)
Biomass in the reactor (g L
1)
YP/S yield (g ethanol
g substrate
1)

ICR

Run 1

Run 2

Run 3

Run 1

Run 2

0.12
27.9  1.9
3.34  0.2
100
18.3  1.7
81.7  2.1
4.8  0.4
0.34  0.01

0.25
21.6  1.9
5.4  0.3
100
31.1  2.9
68.9  2.4
4.9  0.16
0.31  0.01

0.5
11.6  1.6
6.8  0.3
100
51.5  1.2
48.5  1.8
2.4  0.13
0.24  0.01

0.12
44.06  1.9
5.28  0.2
100
2.9  0.4
97.1  3.2
e
0.45  0.02

0.25
32  1.1
8.0  0.4
100
9.6  0.6
90.4  4.1
e
0.35  0.01

MBR
Run 3
0.5
20.2
10.1
100
26.8
73.2
e
0.27

 0.6
 0.6
1
 3.8
 0.01

Run 1

Run2

0.31e0.5
41.4  1.8
12.83e19.2
100
2.3  0.2
97.7  2.8
8.4e26.7
0.42  0.02

0.31
46.5  1.5
14.41  0.6
100
0.7  0.1
99.3  2.9
26.13e41.1
0.46  0.01

e
0.0032
0.0061
0.004
0.0012
e
0.0056

b i o m a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 2 5 e3 2

exceeding 98%, especially for MBR and ICR configurations

which indicated that sugars were completely consumed by
yeast.
The experimental YP/S yield compared to the theoretical
performance is more important in both ICR and MBR reactors
than in the CSTR because the microbial activity in both cases
is more important than for the CSTR. The best YP/S of 0.46 g g
1
and the Ys of 99.3% were obtained with the MBR. However, in
the case of CSTR YP/S decreased as the cell concentration
within the reactor was diluted by increasing the D.
The mass balance in the systems was calculated. The mass
substrate was converted in to ethanol, carbon dioxide,
biomass, glycerol and organic acids. The total feed mass was
calculated from the mass of sugars feeding per day. The
biomass generated may be accumulated in the reactors or
washed out in the effluent daily. It was measured by TSS in the
CSTR and the MBR and by volume variation in the ICR. Results
showed that mass recovery from substrates ranged between
78% for the CSTR to 99% for the MBR. It is very likely that the
low mass balance in the CSTR was due to an ethanol lost in the
carbon dioxide event and the secondary metabolites production like glycerol and organic acids.

4.

Conclusion

Results showed that the performance of the MBR was more

advantageous than the CSTR and the ICR for anaerobic sugar
cane molasses fermentation since a higher concentration of
ethanol (41.4 g L
1) and Qp (12.83e19.2 g L
1 h
1), with low
residual substrate content were achieved. These results
showed the interest of using the MBR to reach a high ethanol
concentration which confirmed the effectiveness of this
technology.

Acknowledgements
The authors wish to acknowledge the Ministry of Superior
Education and Scientific Research and Technology, which has
facilitated the carried work.

Abbreviations

CSTR
continuously stirred tank reactor
D
dilution rate (h
1)
ICR
immobilised cell reactor
MBR
membrane bioreactor
OD
optic density
m max (mu-max) maximum specific growth rate (h
1)
ORP
oxidation reduction potential (mV)
Qp
ethanol productivity (g L
1 h
1)
TRS
total reduced sugars (g L
1)
mass ratio between ethanol and sugar (g g
1)
YP/S
WTW
WTW GmbH, Weilhem, Germany
Ys
substrate conversion yield (%)

31

references

[1] Stewart L, Lambert DM. Spatial heterogeneity of factors

determining ethanol production site selection in the U.S.,
2000e2007. Biomass Bioenerg 2011;35(3):1273e85.
[2] Letti LAJ, Karp SG, Woiciechowski AL, Soccol CR. Ethanol
production from soybean molasses by Zymomonas mobilis.
Biomass Bioenerg 2012;44(1):80e6.
[3] Shenoy D, Pai A, Vikas RK, Neeraja HS, Deeksha JS, Nayak C,
et al. Biomass Bioenerg 2011;35(3). 4107e4011.
[4] Wilkins MR, Widmer WW, Grohmann K. Simultaneous
saccharification and fermentation of citrus peel waste by
Saccharomyces cerevisiae to produce ethanol. Process Biochem
2007;42(12):1614e9.
[5] Siqueria PF, Krap SG, Carvalho JC, Sturm W, RodriguezLeon JA, Tholozan JL, et al. Production of bio-ethanol from
soybean molasses by Saccharomyces cerevisiae at laboratory,
pilot and industrial scales. Bioresour Technol 2008;99(17):
8156e63.
[6] Najafpour G, Younesi H, Ismail KSK. Ethanol fermentation in
an immobilized cell reactor using Saccharomyces cerevisiae.
Bioresour Technol 2004;92(3):251e60.
[7] Cazetta ML, Celligoi MAPC, Buzato JB, Scarmino IS.
Fermentation of molasses by Zymomonas mobilis: effects of
temperature and sugar concentration on ethanol production.
Bioresour Technol 2007;98(15):2824e8.
[8] Baptista CMSG, Coias JMA, Oliveira ACM, Oliveira NMC,
Rocha JMS, Dempsey MJ, et al. Natural immobilization of
microorganisms for continuous ethanol production. Enzyme
Microb Technol 2006;40(1):127e31.
[9] Purwadi R, Taherzadeh MJ. The performance of serial
bioreactors in rapid continuous production of ethanol from
dilute-acid hydrolysates using immobilized cells. Bioresour
Technol 2008;99(7):2226e33.
[10] Loukatos P, Kanellaki M, Komaitis M, Alhanasiadis I,
Koutinas AA. A new technological approach proposed for
distillate production using immobilized cells. J Biosci
Bioengen 2003;95(1):35e9.
[11] Palmquist E, Galbe M, Hhn-Hagerdel B. Evaluation of cell
recycling in continuous fermentation of enzymatic
hydrolysates of spruce with Saccharomyces cerevisiae and online monitoring of glucose and ethanol. Appl Microbiol
Biotechnol 1998;50(5):545e51.
[12] Sreenath HK, Jeffries TW. Production of ethanol from wood
hydrolyzate by yeasts. Bioresour Technol 2000;72(3):253e60.
[13] Yamada T, Fatigati MA, Zhang M. Performance of
immobilised Zymomonas mobilis 31821 (pZB5) on actual
hydrolysates produced by Arkenol technology. Appl Biochem
Biotechnol 2002;98(100):899e907.
[14] Liu R, Shen F. Impacts of main factors on bioethanol
fermentation from stalk juice of sweet sorghum by
immobilized Saccharomyces cerevisiae (CICC 1308). Bioresour
Technol 2008;99(4):847e54.
[15] Lee JM, Pollard JF, Coulman GA. Ethanol fermentation with
cell recycling: computer simulation. Biotechnol Bioeng 1983;
25(2):497e511.
[16] Hojo O, Hokka CO, Major AM. Ethanol production by
a flocculant yeast strain in a CSTR type fermentor with cell
recycling. Appl Biochem Biotechnol 1999;77e79:535e45.
[17] Ben Chaabene F, Aldiquier AS, Alfenore S, Camelevre X,
Blanc P, Bidereux C, et al. Very high ethanol productivity in
an innovative continuous two-stage bioreactor with cell
recycles. Bioprocess Biosyst Eng 2006;29(1):49e57.
[18] Ozmihci S, Kargi F. Kinetics of batch ethanol fermentation of
cheese-whey powder (CWP) solution as function of substrate
and yeast concentrations. Bioresour Technol 2007;98(16):
2978e84.

32

b i o m a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 2 5 e3 2

[19] Menezes JC, Alves SS, Lemos JM, Azevedo SF. Sugar
analysis of industrial penicillin fermentation broths
containing complex nutrient sources. Biotechnol Tech
1992;6(1):1e4.
[20] StatSoft Inc. Statistica for windows, computer program
manual; 1997. Tulsa.
[21] Tang Y, An M, Liu K, Nagai S, Shigematsu T, Morimura S,
et al. Ethanol production from acid hydrolysate of wood
biomass using the flocculating yeast Saccharomyces cerevisiae
strain KF-7. Process Biochem 2006;41(4):909e14.
[22] Taherzedeh MJ, Millati R, Niklasson C. Continuous
cultivation of dilute acid hydrolysates to ethanol by

immobilised Saccharomyces cerevisiae. Appl Biochem

Biotechnol 2001;95(1):45e57.
[23] Nielsen MK, Arneborg N. The effect of citric acid and pH on
growth and metabolism of anaerobic Saccharomyces cerevisiae
and Zygosaccharomyces bailii cultures. Food Microbiol 2007;
24(1):101e5.
[24] Goksungur Y, Zorlu N. Production of ethanol from beet
molasses by Ca-alginate immobilized yeast cells in a packedbed bioreactor. Turk J Biol 2001;25(3):265e75.
[25] Escobar JM, Rane KD, Cheryan M. Ethanol production in
a membrane bioreactor: pilot-scale trials in a corn wet mill.
Appl Biochem Biotechnol 2001;91-93:283e96.