Optimization of Very High Gravity Fermentacion Process For Ethanol Production From Industrial Sugar Beet Syrup

b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1 e9
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Optimization of very high gravity fermentation

process for ethanol production from industrial
sugar beet syrup
Claire Joannis-Cassan a,b,*, Julien Riess a,b,c,d, Franck Jolibert c,

Patricia Taillandier a,b
a
Universite de Toulouse, INPT, Laboratoire de Genie Chimique, 4, Allee Emile Monso BP 44362, F-31432 Toulouse,
France
b
CNRS, Laboratoire de Genie Chimique, UMR 5503, F-31030 Toulouse, France
c
ADEME, 20 Avenue du Gresille, 49004 Angers Cedex 01, France
d
UNGDA, 174 boulevard Camelinat, 92247 Malakoff Cedex, France
article info abstract
Article history: In order to reduce production costs and environmental impact of bioethanol from sugar
Received 5 December 2012 beet low purity syrup 2, an intensification of the industrial alcoholic fermentation carried
Received in revised form out by Saccharomyces cerevisiae is necessary. Two fermentation processes were tested:
28 March 2014 multi-stage batch and fed-batch fermentations with different operating conditions. It was
Accepted 24 July 2014 established that the fed-batch process was the most efficient to reach the highest ethanol
Available online xxx concentration. This process allowed to minimize both growth and ethanol production in-
hibitions by high sugar concentrations or ethanol. Thus, a good management of the
Keywords: operating conditions (initial volume and feeding rate) could produce 15.2% (v/v) ethanol in
Ethanol 53 h without residual sucrose and with an ethanol productivity of 2.3 g L h1.
Sugar beet low purity syrup © 2014 Elsevier Ltd. All rights reserved.
Fed-batch
Very high gravity fermentation
Saccharomyces cerevisiae
used in France for the production of ethanol. However, fer-

1. Introduction mentations carried out at industrial scale usually provide final
ethanol concentration between 10 and 12% (v/v) [1,2].
France is currently the largest producer of sugar beet ethanol In order to reduce production costs and environmental im-
in the world. From an economic point of view, low purity pacts, it is essential to intensify these fermentations, that is to
syrup 2, is a good raw material for ethanol production due to increase the final ethanol concentration. For the same volume
its content of fermentable sugars. Indeed, low purity syrup 2 is of fermented juice, the increase of final ethanol concentration
obtained from the second thick juice crystallization and still could permit to reduce both water requirements for the prep-
contains 55e60% (w/w) of fermentable sugars and 10e15% (w/ aration of fermentation media and energy needs for distillation
w) of non-sugar. The low purity syrup 2 is now commonly while increasing the production capacity of plants without
* Corresponding author. CNRS, Laboratoire de Genie Chimique, UMR 5503, F-31030 Toulouse, France. Tel.: þ33 5 34 32 36 22; fax: þ33 5 34
32 36 97.
E-mail address: [email protected] (C. Joannis-Cassan).
http://dx.doi.org/10.1016/j.biombioe.2014.07.027
0961-9534/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Joannis-Cassan C, et al., Optimization of very high gravity fermentation process for ethanol
production from industrial sugar beet syrup, Biomass and Bioenergy (2014), http://dx.doi.org/10.1016/j.biombioe.2014.07.027
2 b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1 e9
investment [3]. Energy is the second largest cost after raw ma- sugars equivalent sucrose, 720 gDM kg1 of total solids (dried
terials and represents 30% of the ethanol production cost [4,5]. matter) and 494 mg kg1 of assimilable nitrogen (ammonia
To do this, very high gravity (VHG) fermentation which involves and amino acids).
use of medium containing high sugar concentration (>250 g L1)
must be implemented to achieve high ethanol concentration 2.2. Yeast
[6]. However, VHG fermentation leads to significant stress for
Saccharomyces cerevisiae due to osmotic pressure at the begin- The C10 strain of S. cerevisiae was provided by UNGDA (Union
ning of the fermentation and high ethanol content at the end Nationale des Groupements de Distillateurs d’Alcool) which is
[7,8]. This results in incomplete or stuck fermentation [9e11]. the French National Union of Alcohol-Distillers Groups. The
There are three main possibilities to limit drawbacks caused starter cultures were carried out in YEPD medium (40 g L1
by VHG fermentations [1]. The first one is the improvement of glucose, 15 g L1 yeast extract and 10 g L1 peptone). They
ethanol tolerance by genetic modification of strains but GMO were inoculated from YEPD agar used to store the C10 strain at
strains of S. cerevisiae are banned in France for ethanol pro- 4 C. Media were sterilized at 121 C for 20 min before use. The
duction. The second one is the optimization of the fermentation starter cultures were incubated at 30 C on a rotary shaker at
media composition. Many research works studied the role of 120 rpm (Infors AG CH-4103, Bottingen) for 15 h. The final
osmoprotectants or yeast growth factors by supplementing yeast cells concentration was about 2 108 cells mL1. A
media with various nutrients (available nitrogen sources, sufficient aliquot was centrifuged, then yeast cells were rinsed
protein-lipid complexes, calcium, magnesium, yeast extracts, with sterile water and centrifuged twice to allow the inocu-
…) [1,12e14]. However, supplementations should be as limited lation of fermentation media at 1 107 cells mL1.
as possible and low-cost to be realistic from an economic point
of view at the industrial scale. Although many studies were 2.3. Fermentation media
conducted on media optimization for VHG fermentation of
synthetic medium or starchy substrates, few concerned sac- In order to avoid sugar limitation during the fermentation,
charides substrates [1,15e18]. Therefore optimization of the media initially contained a total amount of 300 gsucrose L1
composition of low purity syrup 2 medium for high gravity (372 gdry matter L1) which has been determined has a large
fermentation was done in our laboratory [2]. This optimized excess of sugar for the fermentation of low purity syrup 2 [2].
medium, supplemented with nitrogen, phosphorus, yeast The media were made by diluting low purity syrup 2, in a
extract, magnesium and antifoam, was used in this study. mixture of sterilized water and stillage (66/33% v/v). A sup-
Finally, the third possibility to limit yeast stress is to improve plementation of 31 mg L1 of nitrogen was provided by an
fermentation process [19e22]. Very few studies are reported in ammonia solution 35% (Analytical reagent grade, Fisher Sci-
the literature for sugar substrates [23]. Thus investigation on entific) and a supplementation of 151 mg L1 of phosphorus
the impact of the process on VHG fermentation of low purity was provided by a solution of 85% orthophosphoric acid (re-
syrup 2 medium is required. Usually, three fermentation pro- agent grade Laboratory, Fisher Scientific). Media were also
cesses are used for ethanol production: continuous, multi-stage supplemented with 1 g L1 of yeast extract (Prolabo),
batch and fed-batch [20]. Continuous processes are difficult to 120 mg L1 of magnesium [MgSO4,7H2O] (Prolabo) and
run for VHG fermentation because high ethanol content de- 50 mL L1 of an industrial antifoam Ouvrie 922k (PMC Ouvrie
creases viability and inhibits yeast growth which could lead to SAS).
biomass washout [24]. Moreover, oscillatory behaviors have The acidity of the media was adjusted to 2 gH2SO4 L1 with
been reported for VHG continuous process [25]. For these rea- sulfuric acid at 98e100% (Normapur Analytical reagent, Pro-
sons, only multi-stage batch and fed-batch processes were labo). The acidity is more relevant in the industry than pH
investigated in this study. Fed-batch process seems adapted for because this parameter does not depend on the dry matter
VHG fermentations because it could minimize inhibition ef- content which varies according to the substrate and its con-
fects of high concentrations of substrate and ethanol, which centration [26]. This acidity corresponded to a pH range from
occur at the beginning and the end of the process. 4.7 to 5.2 according to the dry matter content in the medium.
This paper compares different processes (two-stage batch,
three-stage batch and fed-batch with 4 feeding rate strategies) 2.4. Culture conditions
for VHG fermentation of sugar beet low purity syrup. The
objective was to reach the highest final ethanol concentration Multi-stage batch fermentations were performed in 1 L Setric
together with the highest ethanol productivity and the lower reactors at 30 C with 1 vvh aeration and 120 rpm stirring
residual sugars concentration. speed. For the two-stage batch fermentation, first step used
for biomass propagation was made in a low gravity medium
containing 160 g L1 of sucrose. Then, one third of the vessel
2. Materials and methods content (333 mL) was transferred to another vessel of the
same capacity and filled with two-thirds (667 mL) of fresh
2.1. Substrates medium containing 370 g L1 of sugars (Fig. 1a). In this second
stage, the medium obtained after mixing contained
Industrial sugar beet low purity syrup 2 and stillage (residue of 240 gsucrose L1 plus the residual sugar from phase 1.
the distillation step) were provided by a French ethanol pro- The three-stage batch fermentation (Fig. 1b) was conduct-
ducer group. These two industrial products were not sterilized ed similarly as previously except that the stage 2 consisted in a
before use. The low purity syrup 2 contained 580 g kg1 of total fermentation in a medium containing 190 gSucrose L1 plus
b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1 e9 3
Fig. 1 e Experimental device and culture conditions for (a) two-stage batch fermentation and (b) three-stage batch
fermentation.
residual sugars from stage 1 during 24 h. This stage was until two successive samples provided the same ethanol
transferred for two-thirds (667 mL) in another vessel and filled concentration.
with one-third (333 mL) of fresh medium containing 420 g L1
of sugars. The stage 3 contained after mixing 140 gSucrose L1 2.5. Analytical methods
plus residual sugars from phase 2.
Fed-batch fermentations were performed in 4 L (LH Inceltech Viable yeast cells concentration was determined by the methy-
Series 210) at 30 C with 1 vvh aeration and 150 rpm stirring lene blue technique. A solution of methylene blue (1 g L1 in
speed (Fig. 2). Reactor feeding was made by a peristaltic pump 20 g L1 Na3 citrate) was mixed volume to volume with the
(Gilson). As it is done in the industry, the fed-batch fermen- diluted yeast suspension, shaken and after 5 min incubation,
tations were divided in three phases: the first one was made in placed in a Thoma's counting chamber. The number of un-
batch mode in a low gravity media for yeast propagation, the stained (active cells) was counted in five different fields. Under
second one consisted in reactor feeding and corresponded to these conditions viability was estimated with an accuracy of
ethanol production and the third one was made in batch mode 12.2% (experimentally measured on five repetitions).
to exhaust as much sugar as possible. These phases are called: Ethanol, fructose, glucose and sucrose concentrations were
propagation phase, feeding phase and exhausting phase determined in the supernatant after centrifugation and
respectively (Fig. 2). Concentrations and durations of these filtration at 0.2 mm, by high performance liquid chromatog-
different phases are shown in Table 1. raphy equipped with a Rezex ROA-Hþ column 300 7.8 mm
Fermentation kinetics were monitored by measuring viable (Phenomenex) maintained at 30 C and a refractive index de-
yeast cells, sugar and ethanol concentrations at 2 h intervals, tector maintained at 40 C. The mobile phase was constituted
Fig. 2 e Experimental device for fed-batch experiments.
of ultrapure water with 1.9 mmol L1 of sulfuric acid at a flow viable yeast cells concentration was 1.5$108 cells mL1 at 30 h
rate of 170 mL min1. Sugar concentrations were expressed in against 2.5$108 cells ml1 for the three-stage batch fermenta-
sucrose equivalent where 1 g of monosaccharide corresponds tion. A decrease in ethanol productivity was also observed
to 0.95 g of sucrose. Ethanol and sucrose concentrations were between 12 h and 30 h (3.9 g L1 h1 for the three-stage against
estimated with an experimental error of 1% and 8% 3.1 g L1 h1 for the two-stage fermentation). For the two-stage
respectively. batch fermentation, a drop of viable yeasts and a decrease in
ethanol production rate were observed after 30 h. This could
be explained by the additional stress caused by high concen-
3. Results and discussion tration of ethanol. Even if the mechanisms of inhibition of
ethanol are not completely elucidated, multiple effects have
3.1. Multi-stage batch fermentations been reported in the literature as inhibition of cell growth and
viability or a decrease in specific rate of fermentation [23]. The
The kinetics of ethanol production, sugars consumption and inhibition effect of ethanol could occur at relative low con-
yeast growth are presented on Fig. 3 for both two-stage and centration (between 8 and 10% v/v) and depends on other
three-stage fermentations. The final ethanol concentration, parameters of the fermentation (temperature, substrate con-
residual sugar and fermentation duration are given in Table 2. centration …). It seemed that a synergetic effect of about 8%
One major difference between these two fermentations was (v/v) of ethanol and about 150 g L1 of sugar was inhibitory for
the cell growth. During the second stage, the specific growth yeast viability in the two-stage batch fermentation. These
rate was higher for the three-stage fermentation. It was conditions were not met in the three-stage batch fermenta-
explained by a lower initial sugar concentration (200 g L1 tion. In the third stage (Fig. 3b), no growth occurred but con-
instead of 270 g L1) in this fermentation. It is well known that centration of viable yeast cells remained constant during 40 h
high sugar concentrations cause osmotic pressure stress that and allowed the transformation of sugar to ethanol. There-
leads to growth inhibition and loss of yeast viability, and in- fore, it seemed that adding sugar progressively could improve
creases formation of by-products, including glycerol. Then a yeast viability and activity as often reported in the literature
decrease in ethanol production is usually observed [27,28]. [19,30,31]. An other major difference between the two fer-
These effects could occur at concentration of 150 g L1 of mentations was sugar consumption. The total amount of
glucose [29]. In our experiments, substrate inhibition has led consumed sugar was higher for the three-stage process. This
to a growth limitation in the two-stage batch fermentation as could be explained by the growth observed during the second
Table 1 e Summary of operating conditions applied in fed-batch fermentations.

Step Propagation Feeding Exhausting sugar Total sucrose Total duration
(g L1) (h)
Sucrose Volume Duration Sucrose Volume Flow rate Duration Duration
1 1 1
(g L ) (L) (h) (g L ) (L) (mL h ) (h) (h)
FB 1 130 1.33 6 385 2.67 55.5 48 20 300 74
FB 2 130 1.33 6 385 2.67 variable 52 17 300 75
FB 3 160 2.67 9 580 1.33 55.5 24 24 300 57
FB 4 160 3.08 9 580 0.92 55.5 16.6 27.4 255 53
Fig. 3 e Kinetics of sugar ( ), ethanol ( ) and viable yeast cells ( ) concentrations during: (a) the two-stage batch fermentation
and (b): the three-stage batch fermentation. Dashed lines indicate the transition of one stage to the following.
stage that caused a sucrose use to produce biomass. Indeed, (Table 2). Therefore equilibrium has to be found between
the yield observed was 0.458 gethanol g1 sugar and ethanol production and yield.
0.492 gethanol g1
sugar in the second and the third stage respec- These first results are already very interesting because they
tively. Yield is an important parameter for the industry, and demonstrate the feasibility of VHG fermentations from sugar
should be between 85 and 90% of the theoretical yield calcu- beet substrates with high ethanol concentrations reached
lated from sucrose to make the process profitable and avoid (14.9% (v/v)). However, the process could be improved by
residual sugars [1]. In the three-stage batch fermentation, the adding sugar progressively. From the previous experiments,
second growth caused a decrease of the yield from 84 to 76% of sugar concentration should not exceed 150 g L1. Therefore
the sucrose theoretical yield (Table 2). Nevertheless, there was fed-batch fermentations were tested, since this process cor-
a slight improvement of 0.2% (v/v) in ethanol production responds to fermentation with an infinite number of stages.
Table 2 e Ethanol production for the different operating conditions tested.

Ethanol Productivity Residual sucrose Yielda Total
(% v/v) (g L1 h1) (g L1) (% of theoretical yield) duration (h)
Two-stage batch 14.7 ± 0.1 1.5 ± 0.2 45 ±4 84 75
Three-stage batch 14.9 ± 0.1 1.6 ± 0.2 13 ±1 76 75
FB 1 13.4 ± 0.1 1.5 ± 0.2 70 ±6 85 74
FB 2 14.0 ± 0.1 1.5 ± 0.2 45 ±4 80 75
FB 3 15.4 ± 0.2 2.1 ± 0.3 46 ±4 89 57
FB 4 15.2 ± 0.2 2.3 ± 0.3 1 ±1 88 53
a
Theoretical yield with sucrose is 0.539 gethanol g1
sucrose.
Moreover, fed-batch fermentations could allow to decrease increased and the feeding media was more concentrated in
yeast stress during the fermentation by medium feeding sugar.
management [20]. Fed batch 3 provided higher ethanol concentration and
yield than previous fed-batches (Table 2). During the feeding
3.2. Fed-batch fermentations phase, sucrose concentration was quite constant at 100 g L1
(Fig. 4c). In this phase, ethanol yield was 0.490 gethanol g1
sugar.
Table 1 shows the operating conditions applied for the That indicates that even if the conditions were stressful
different fed-batch processes. For fed-batch 1, 3 and 4, the (synergy of ethanol and sugar inhibition), the duration was
feeding flow was constant to 55.5 mL h1. A variable feeding not sufficient to decrease cell activity. Moreover yeast cells
rate was applied for fed-batch 2 according to sugar con- concentration was already high at the beginning of the
sumption rate calculated from fed-batch 1 in order to avoid feeding phase (1.5$108 cells mL1) due to a longer propagation
sugar accumulation during the feeding step. phase. Furthermore, the fermentation duration was the
Two strategies were tested. In the first one, the operating shortest observed in all the experiments so the productivity
conditions were adapted from what is currently done in in- reached 2.13 g L1. These results confirmed that the feeding
dustry (fed-batches 1 and 2) with a short propagation phase in time must be short, since the yeast activity decreased
a small volume and then a long feeding step. The second throughout the fermentation due to high ethanol concentra-
strategy consisted in a propagation medium in a higher vol- tion but also to the duration of exposure to ethanol. However,
ume for a period of 9 h to obtain at the end of the propagation these experimental conditions did not allow the exhaustion of
phase the maximal cell concentration (Fed-batches 3 and 4). sugars. In the sugar industry, residual reducing sugars and
For these two experiments, sucrose concentration of the total sugars must be controlled and should not exceed 2 g L1
feeding medium was 580 gsucrose L1, which corresponded to and 5 g L1 respectively [23]. Therefore, fed-batch 4 was run
the average low purity syrup 2 sucrose concentration. under similar conditions than fed-batch 3 except a lower total
For fed-batch 1, the operating conditions caused a signifi- sugar provided (255 instead of 300 g L1) in order to obtain less
cant variation in sugar concentration profile during fermen- than 2 g L1 of residual sugar at the end of fermentation. The
tation (Fig. 4a). After 25 h, sugars accumulated and a slight total sugar provided has been decreased by reducing the
decrease of ethanol productivity was observed. As previously, feeding volume and then the feeding duration (Table 1).
inhibition by ethanol explained these results [32]. In this As presented in Table 2, fed-batch 4 was the most efficient
experiment, the inhibition occurred when ethanol concen- process for ethanol production due to its highest ethanol final
tration was superior to 9% (v/v). After 30 h, the viable biomass concentration, its shortest fermentation duration, its high
concentration decreased. It could be due to a delayed response ethanol yield and its lowest residual sucrose content. During
of yeast cells to ethanol inhibition [23] or a synergetic effect of this fermentation, ethanol productivity stayed almost con-
ethanol concentration and sugar concentration that increased stant from the beginning of the feeding phase to the end of the
at this time. In consequence, a second fed-batch (fed-batch 2) fermentation (Fig. 4d). During the feeding phase, yeast
was carried out with the objective to maintain the sugar viability was maintained. In the exhausting phase, the viable
concentration during the feeding phase at about 80 g L1 by cell concentration decreased but the productivity of ethanol
varying the feeding rate. It could be seen that until 45 h, the was maintained. It has been reported in the literature that
viable yeast cells concentration was almost constant even if ethanol inhibition could not affect the ethanol yield even if a
the ethanol concentration reached 12% (v/v) (Fig. 4b). After growth inhibition occurred [32].
45 h, the cell viability decreased and ethanol production quite Thereby, sugar and ethanol inhibition could be overcome
stopped. At this moment, the dilution due to the fermentor with a good fermentation management of fed-batch process.
feeding was too important in comparison with the ethanol The propagation phase should provide sufficient quantity of
production rate. This could be explained by a too long expo- viable yeasts and the feeding phase should be as short as
sure to high ethanol concentration (above 10% (v/v)) and possible to limit synergic inhibition by high concentration of
comforted the possibility of a latency time in the response of both sugar and ethanol.
yeasts to ethanol stress [23]. This latency time was shorter The comparison of the fed-batch 4 performances with
when yeasts were submitted to additional stress as high sugar literature was difficult as few works have been done on VHG
concentration (fed-batch 1). Thus, controlling the feeding rate fermentation of industrial saccharide substrates and oper-
to maintain a concentration of sugar below 100 g L1 (fed- ating conditions, such as temperature could vary [1]. Some
batch 2) allowed to increase the final ethanol concentration in studies were carried out at 30 C in flasks with synthetic me-
comparison to fed-batch 1 (Table 2) even if the ethanol yield dium containing glucose (between 300 and 330 g L1) supple-
decreased from 85 to 80% of the theoretical yield. The yield mented with various compounds (malted cowpea flour, soya
decrease could be explained by the duration of the stress flour, yeast extract, corn steep liquor, urea, magnesium sul-
caused by higher ethanol content in fed-batch 2. However, for fate, …) [28,29,33,34]. In these works, final ethanol concen-
these two fed-batch fermentations, ethanol concentration tration reached 15e18.6% (v/v) with productivity ranging from
and productivity were lower than for the multi-stage batch 2 to 3.3 g L1 h1 and fermentation durations comprised be-
fermentations. To improve fed-batch process, it is therefore tween 28 and 96 h. Some experiments were carried out in fed-
necessary to limit high ethanol exposure duration while batch process. Alfenore et al. [19] reported a final ethanol
having a sugar concentration not too high. Consequently the concentration of 19% (v/v) and a productivity of 3.3 g L1 h1
feeding phase has been shortened for fed-batches 3 and 4 using micro-aeration (0.2 vvm) and vitamin supply. Seo et al.
(Table 1). For that, the volume of the propagation phase was [35] reached up to 20% (v/v) of ethanol in 24 h and a
Fig. 4 e Kinetics of sugar ( ), ethanol ( ) and viable yeast cells ( ) concentrations during: (a) the fed-batch 1, (b): the fed-batch
2, (c): the fed-batch 3 and (d): the fed-batch 4. Dashed lines indicate the transition of one phase to the following.
productivity of 2 g L1 h1 with micro-aeration (0.13 vvm) and research update. J Ind Microbiol Biotechnol
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Optimization of Very High Gravity Fermentacion Process For Ethanol Production From Industrial Sugar Beet Syrup

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Optimization of very high gravity fermentation

Claire Joannis-Cassan a,b,*, Julien Riess a,b,c,d, Franck Jolibert c,

article info abstract

used in France for the production of ethanol. However, fer-

Fig. 2 e Experimental device for fed-batch experiments.

Table 1 e Summary of operating conditions applied in fed-batch fermentations.

Table 2 e Ethanol production for the different operating conditions tested.

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