Vitalized yeast with high ethanol productivity

Article abstract of DOI:10.1039/c4ra08809c

Fuel ethanol is an attractive alternative to fossil-based fuels or fuel additives. Very-high-gravity (VHG) ethanol fermentation is a promising technology to reduce energy consumption in distillation. However, yeast cells subjected to a high concentration of ethanol and osmotic stress readily lose cell viability, resulting in reduced ethanol productivity. Here we report the beneficial effect of fully water-soluble polyethylene glycol (PEG) in chemically exo-protecting yeast cells during fermentation, resulting in largely boosted cell vitality and tolerance to high ethanol concentration. The final ethanol concentration and the yeast cell viability were substantially increased as compared to PEG-free fermentation. The recovered exo-protected yeast was further demonstrated to continue to deliver superior bio-catalytic performance in subsequent fermentations over that recovered from PEG-free broth. Furthermore, the water-soluble PEG was readily recycled for reuse after distillation of ethanol. This journal is

Full text of DOI:10.1039/c4ra08809c

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Vitalized yeast with high ethanol productivity†
Xiumei Liu, Wenjuan Xu, Chao Zhang, Peifang Yan, Songyan Jia, Zhanwei Xu
and Z. Conrad Zhang*
Cite this: RSC Adv., 2014, 4, 52299
Fuel ethanol is an attractive alternative to fossil-based fuels or fuel additives. Very-high-gravity (VHG)
ethanol fermentation is a promising technology to reduce energy consumption in distillation. However,
yeast cells subjected to a high concentration of ethanol and osmotic stress readily lose cell viability,
resulting in reduced ethanol productivity. Here we report the beneficial effect of fully water-soluble
polyethylene glycol (PEG) in chemically exo-protecting yeast cells during fermentation, resulting in
largely boosted cell vitality and tolerance to high ethanol concentration. The final ethanol concentration
and the yeast cell viability were substantially increased as compared to PEG-free fermentation. The
recovered exo-protected yeast was further demonstrated to continue to deliver superior bio-catalytic
performance in subsequent fermentations over that recovered from PEG-free broth. Furthermore, the
water-soluble PEG was readily recycled for reuse after distillation of ethanol.
Received 18th August 2014
Accepted 9th October 2014
DOI: 10.1039/c4ra08809c
www.rsc.org/advances
decreases process water requirements, but also reduces waste
Introduction
14,15
distillage discharged from the distillation system.
Thus VHG
Fuel ethanol represents the largest volume of renewable fuels fermentation has garnered great attention. However, VHG
world wide as an environmentally friendly alternative to fossil fermentation suffers from incomplete glucose utilization and
fuels. The production of bio-ethanol has been considerably decreased fermentation rate by subjecting yeast cells to high
1–3
16–21
increased over the last few years.
Feedstock and energy osmotic stress and severe ethanol inhibition in yeast cells.
consumption contribute to the major cost of bio-ethanol
Research has revealed that improved cell viability and yeast
production. The U.S. Department of Energy's research road- growth rate needed to achieve high ethanol concentration
4
map highlighted a number of challenging targets, including strongly depend on the supplementation with osmoprotectant.
high yield with complete sugar utilization, higher nal ethanol Thomas et al. reported that glycine can promote yeast cell
22
concentration (titer), higher overall volumetric productivity, growth and improve cell viability in VHG medium. Thomas
and high tolerance to inhibitors.
and Ingledew showed that the fermentation time was shortened
High ethanol concentration has always been pursued in the from 8 days to 3 days when 0.9% yeast extract was added in
23
fermentation industry, because signicant energy savings can wheat mash fermentation that produced 17.1% (v/v). Consid-
be achieved for downstream distillation and waste distillage ering that fresh yeast extract was too costly for the industrial
5,6
treatment. Bio-ethanol fermentation can be improved by use, Kawa-Rygielska and Pietrzak studied the spent brewer's
fermentation in media containing high concentrations of dis- yeast supplements and found that ethanol concentration can be
ꢀ1
ꢀ1 24
solved solids (above 300 g per liter), optimization of the ethanol enhanced from 120 g L to 140 g L
. In that study, it was also
fermentation process, and selection of robust microorgan- recognized that spent brewer's yeast cell lysis released various
7,8
isms. One of the process strategies currently applied in the hydrocolloids (b-glucan, polypeptides), which caused an
fermentation industry is very high gravity (VHG) technology. increase in mash viscosity. Overall, the use of the most of these
Compared to conventional fermentation process, industrial supplements is not economically feasible because of their
application of VHG ethanol fermentation technology has relatively high prices.
9
–12
exhibited obvious advantage,
as it proceeds in signicantly
Surfactants have lower vapor pressure and their physical
higher nal ethanol titer (usually above 15%, v/v) in the properties can be ne-tuned with appropriate choice of molec-
13
fermentation broth. This fermentation technology not only ular structure and weight. Surfactant Triton X-100 was found to
saves energy consumption for ethanol distillation and improve the cell viability during post-logarithmic cell growth
25
phase, but its effect in ethanol fermentation was not assessed.
PEG-6000/dextran aqueous two-phase system (ATPS) was used
as a means for extractive ethanol fermentation, which encour-
aged growth of microorganism and improved fermentation
State Key Laboratory of Catalysis, Dalian National Laboratory for Clean Energy,
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
E-mail: zczhang@yahoo.com; Fax: +86-0411-84379462; Tel: +86-0411-84379462
26,27
efficiency.
The ATPS fermentation took place in the bottom
O/
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4ra08809c
H
2
O/dextran phase with ethanol extracted to the upper H
2
1
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PEG-6000 phase. However, the ethanol concentration was very VHG fermentation
ꢀ
1
low (3.3 g L ) as compared to that in VHG fermentation. An
industrial application of the ATPS extractive fermentation may
also be limited due to the high cost of dextran.
The ethanol fermentation was performed in the air-locked
Erlenmeyer ask (50 or 100 mL). The initial yeast cell concen-
8
8
tration was approximately 3 ꢂ 10 –5 ꢂ 10 cells per mL. The
The objective of this work was to identify and to study
potential supplements that may provide excellent biocompati-
bility to microorganisms and may also enhance the vitality of
the microorganisms at higher ethanol concentrations. By using
such supplements, the yeasts may be reused and the distillation
cost in separating ethanol from the fermentation broth may be
reduced.
Up to now, there was no report in the literature on the effect
of fully water soluble PEGs directly in ethanol fermentations. In
this work, the addition of the PEGs to a fermentation broth was
found to induce a highly favourable effect in vitalizing yeast
ꢀ1
PEG concentration in water was approximately 0.25 g mL . The
2 4
pH of the media was adjusted to 3.9 with a H SO solution.
Glucose was used as a carbon source without nutrient supple-
ment. The fermentation was performed in batch mode and the
ꢁ
temperature was controlled at 30–36 C. During fermentation,
the asks were placed on a rotary shaker (ZWY-240) at 160 rpm.
Aer fermentation, the yeast cells and PEG-400 with H SO were
2 4
recycled for subsequent uses.
2 4
Recovery of yeast, PEGs and buffer H SO
cells, resulting in substantially enhanced cell tolerance to very A representative procedure for the recovery and reuse of yeast,
high ethanol concentrations. Complete glucose conversion and PEG-400 and H SO is shown in Fig. 1. Aer fermentation, the
2
4
substantially improved nal ethanol concentration were yeast cells were collected by centrifugation at 8000 rpm for 5
obtained in this work. It was further revealed that the vitality of minutes, and the yeast cells were further evaluated for their
the exo-protected yeast cells was maintained and the recycled activity in subsequent fermentations. And then the separated
cells displayed robust activity in subsequent fermentations.
liquid phase was subjected to vacuum distillation to separate
ethanol. PEG and H SO were reused by recycling the fermen-
2
4
tation broth aer distillation.
The sample of each fermentation broth was diluted in 2500
mL solution with deionized water, and ltered through a 0.22
mm lter. The glucose and ethanol concentrations of fermen-
tation samples were quantied using a high performance liquid
chromatography (HPLC). An Agilent 1260 Series HPLC system
equipped with a refractive index detector was used. Ion
exchange columns (HPX-87H, 300 ꢂ 7.7 mm) were used in
series. The column and detector temperatures were maintained
Experimental
Organisms and chemicals
A commercially available immobilized active dry yeast, Saccha-
romyces cerevisiae, was purchased from Angel Company (China),
which was named Angel Super-Alcohol Active Dry Yeast (starch
base). The yeast was kept at 4 C during storage, and was
weighted and directly added to specied fermentation media as
ꢁ
received right before each use. D-Glucose (C
erol (99 wt%), ethanol (99 wt%) were received from Sinopharm
China). Polyethylene glycol (PEG-400) was purchased from
Kermel (China). Sulphuric acid (98 wt%) and methylene blue
were provided by a local supplier. Deionized water (DI H O) was
6
H
12
O
6
$H
2
O), glyc-
ꢁ
ꢁ
2 4
at 65 C and 50 C, respectively, with 5 mM H SO as the mobile
ꢀ
1
phase at 0.6 mL min . The glucose conversion was calculated
based on initial glucose and consumed glucose. The ethanol
yield was calculated based on the theoretical ethanol yield from
consumed glucose; the concentrations of ethanol and glucose
were calculated based on the water volume in the fermentation
broth. Data analysis were performed using the Agilent Chem-
station soware and Microso Excel. The glucose conversion,
ethanol yield and ethanol concentration were calculated
according to the following equations:
(
2
produced by a Milli-Q Integral 5 system. All other chemicals
were of analytical quality.
Measurement of viable cell density
The yeast viability was measured according to the Methylene-
Violet Staining Procedure. A volume of 100 mL dilute sample
containing cells was mixed with 100 mL of a methylene blue
solution. Aer 20 min staining, the numbers of viable (living)
cells and of total cells were counted under a microscope (Nikon
Ci-L). The cell viability was calculated according to the following
equation:
viable cell numbers
Cell viability ¼ total cell numbers ꢂ 100%
ꢁ
Yeast culture: the yeast strain was grown at 33 C in YPD
ꢀ
1
ꢀ1
medium containing 10 g L yeast extract, 20 g L peptone and
2
ꢀ
1
0 g L glucose. The yeast cell concentration was determined
Fig. 1 Process scheme for recovery and reuse of yeast, PEGs and
by the optical density of the culture using a UV-vis spectro-
photometer at 600 nm (Shimadzu UV-2600).
2 4 2 4
H SO . Experiments on combined reuse of yeast, PEGs and H SO
followed similar recovery procedure.
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ðinitial glucoseðmolÞ ꢀ final glucoseðmolÞÞ
initial glucoseðmolÞ
ꢂ100%
Glucose conversion ¼
final ethanolðgÞ
Ethanol yield ¼ 0
ꢂ100%
:51 ꢂ initial glucoseðgÞ
final ethanolðgÞ
Ethanol concentrationðg=LÞ ¼
H
2
OðLÞ
Fermentation model and parameter estimation
The model for ethanol productivity from glucose was a modied
Logistic model. The parameters in the model were evaluated by Fig. 2 Effect of initial glucose concentration on glucose conversion
and ethanol concentration. (a) Effect of initial glucose concentration
on glucose conversion in pure water; (b) effect of initial glucose
concentration on ethanol concentration in pure water; (c) effect of
initial glucose concentration on glucose conversion in the presence of
using Matlab 7.5. Its parameter estimation feature seeks to
minimize the residual sum of squares between the model pre-
dicted values and the experimental values. These data were

tted to Logistic model as given below
PEG-400; (d) effect of initial glucose concentration on ethanol
ꢀ
ꢁ
concentration in the presence of PEG-400; fermentation conditions:
dp
p
8
ꢁ
v ¼
¼ vmax 1ꢀ p
approximately 5 ꢂ 10 cells per mL, 8 mL water, 33 C, 160 rpm, and
pdt
max
pH of 3.9. 2.0 g of PEG-400 in (c and d). All data and error bars
represent the averages and standard deviations of triplicate
measurements.
ꢀ
1
ꢀ1
where n is specic rate of ethanol formation (g L
maximum specic rate of ethanol formation (g L
ethanol concentration (g L ), and Pmax, ethanol concentration
h
), nmax is
ꢀ1
ꢀ
1
h
), P is
ꢀ1
ꢀ
1
above which cells do not produce ethanol (g L ).
fermentations. The incomplete fermentation of glucose is
undesirable as it wastes the valuable carbohydrate resource as
well as brings difficulty for the subsequent treatment of waste
distillage. Therefore, it can be concluded that the highest
Results and discussion
Effect of PEG-400 on glucose conversion at high glucose
ꢀ
1
ethanol concentration of 160 g L which corresponds to the
initial glucose concentration is 37 wt% at full glucose conver-
sion in pure water for the yeast employed in this work. Further
increase in the initial glucose concentration has no positive
effect on ethanol production.
concentrations
Separation of bio-ethanol from the aqueous fermentation broth
is usually by distillation. Increasing ethanol concentration in
water can greatly decrease the ethanol separation cost, reduce
the amount of waste water, and improve the equipment utili-
zation efficiency.
In order to get high ethanol concentration, it's prerequisite
to use high glucose concentration. First, two high initial glucose
concentrations (37 wt% and 40 wt% solutions) were evaluated
for batch fermentation process in pure water as reference
experiments. The results are shown in Fig. 2a and b. The
glucose conversion of 100% and ethanol concentration of 160 g
were obtained in 72 h at the glucose concentration of 37
wt%.
To further increase the ethanol concentration, an initial
2
8
To break the ethanol concentration ceiling by the yeast in
pure water, we evaluated potentially low-cost and recoverable
supplements as osmoprotectant in the fermentation media. In
this study, we explored the efficiency of PEG-400 as an osmo-
protectant in VHG fermentation. The basis for focusing on PEG-
400 among several PEGs in this study is provided later in this
paper. To establish the role of PEG-400, the effect of the initial
glucose concentration in the presence of PEG-400 on glucose
conversion and ethanol concentration was studied. The results
are shown in Fig. 2c and d. Under glucose concentration of 37
wt%, it took 48 h to convert glucose completely, and the ethanol
ꢀ
1
L
glucose concentration of 40% was also evaluated. It was found
that the glucose conversion was decreased to 91% at this higher
glucose concentration, while the ethanol concentration (160 g
ꢀ1
concentration of 160 g L was obtained. The time to reach full
conversion for 37 wt% glucose was 48 h in the presence of PEG-
400, as compared to 72 h for that in pure water. Full conversion
ꢀ
1
L
) did not change. The results are consistent with the
of 40 wt% glucose was achieved in the presence of PEG-400 in 72
discussion of VHG process in the introduction; one disadvan-
tage of the VHG process is incomplete glucose conversion at
high glucose concentration. This phenomenon may be ascribed
to the particular yeast cell's tolerance to an ethanol concentra-
tion ceiling due to osmotic pressure and ethanol stress. This
may cause decreased yeast viability, and slow or stuck
ꢀ1
h. Moreover, ethanol concentration was increased to 175 g L
which was substantially higher than the ethanol concentration
ꢀ1
ceiling (160 g L ) in pure water for the same yeast. The results
indicate that the yeast cell's ethanol tolerance was improved
when the PEG-400 was supplemented to the reaction media.
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ꢁ
Effect of yeast cell loading and temperature
glucose conversion at 36 C can be ascribed to high cell death
rate at this high fermentation temperature, as the cell viability
The loading of yeast cell was determined for the batch
fermentation process in the presence of PEG-400 at 33 C and at
ꢁ
has dropped to 3%. The cell viability of 24% for fermentation at
3 C in 72 h represents a moderate cell death rate at the highest
ethanol concentration, a most severe condition for the yeast
cells.
ꢁ
3
40 wt% of glucose concentration. The results are shown in
Fig. 3a and b. It is clear that when the cell concentration was
8
below approximately 5 ꢂ 10 cells per mL, glucose was not
completely converted in 72 h. The highest ethanol concentra-
ꢀ
1
Effect of PEG concentration in water
tion of 175 g L was obtained. Therefore, the cell concentration
8
of approximately 5 ꢂ 10 cells per mL was chosen for VHG PEG-400 concentration was optimized for the batch fermenta-
fermentation process in this study. It should be pointed out that tion process. It is shown in Fig. 4 that when the concentration of
this yeast loading ensures full glucose conversion in 72 h at the PEG-400 added was increased, the glucose conversion, ethanol
initial 40% glucose concentration in the presence of PEG-400. yield and concentration also increased. As the concentration of
Further increase in the yeast loading may only shorten the PEG-400 achieved 0.25 g mL , the glucose conversion, ethanol
time to reach full glucose conversion, but is not expected to yield and concentration reached maximum. When the concen-
ꢀ
1
increase the ethanol concentration ceiling.
tration of PEG-400 was further increased, the glucose conver-
The effect of fermentation temperature was investigated in sion, ethanol yield and concentration start to decrease. Thus,
ꢀ1
the presence of PEG-400. The results are shown in Fig. 3c and d. 0.25 g mL was chosen as the appropriate PEG-400 concen-
The glucose conversion was 90% with the ethanol concentration tration applied in this paper.
ꢀ
1
ꢁ
ꢁ
of 152 g L at 36 C in 72 h. When the temperature was 33 C,
ꢀ
1
the ethanol concentration of 175 g L and glucose conversion
of 100% were obtained in 72 h. However, at 30 C, glucose
conversion of 91% and the ethanol concentration of 151 g L
Selection of PEGs
ꢁ
Effects of different PEGs on batch ethanol fermentation were
investigated. Four types of PEG were used in this paper. The
results are shown in Fig. 5. It can be seen that when PEG-200
was added, the glucose conversion, ethanol yield and concen-
tration were decreased. However, addition of PEG-400, PEG-600
and PEG-1000 resulted in higher glucose conversion, ethanol
yield and concentration than that of the control (without adding
PEGs). The three PEGs increased fermentation productivity.
In this study, PEG-400 was chosen and studied in greater detail
as the appropriate additive in improving fermentation
productivity.
ꢀ
1
were obtained, indicating that the end of fermentation was not
reached in 72 h.
We measured the cell viabilities, dened as the percent of
survived living cells, from tests at the three temperatures to
account for the different glucose conversions. The lower glucose
ꢁ
conversion at 30 C is largely due to the lower activity of the cells
because the cell viability (52%, Fig. S1†) at this temperature is
the highest among the three. On the other hand, the lower
Effect of exo-protection by PEG-400 on yeast performance and
viability
In order to understand the effect of PEG-400 on the increased
ethanol productivity in VHG fermentation, fermentation
performances of the yeast with and without PEG-400 were
further investigated as shown in Fig. 6, even though the glucose
conversion rate was faster in the rst 36 h in pure water than
Fig. 3 Effect of yeast cell loading and temperature on glucose
conversion and ethanol concentration. (a) Effect of yeast cell
concentration on glucose conversion. (b) Effect of yeast cell
concentration on ethanol concentration. (c) Effect of temperature on
glucose conversion. (d) Effect of temperature on ethanol concentra- Fig. 4 Effect of PEG-400 concentration in water. Fermentation
8
tion. Fermentation conditions: 3.5 g D-glucose, 8 mL water, 2.0 g of conditions: 7.0 g D-glucose, approximately 5 ꢂ 10 cells per mL, 16 mL
ꢁ
PEG-400, 160 rpm, and pH of 3.9. All data and error bars represent the water, 33 C, 160 rpm, and pH of 3.9. All data and error bars represent
averages and standard deviations of triplicate measurements.
the averages and standard deviations of triplicate measurements.
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ꢀ1
the maximum ethanol concentration reached 175 g L at the
end of fermentation (Fig. 6a), with little residual sugar le when
2.0 g of PEG-400 was used. We also performed verication tests
of the same experiments in 40 mL. The results obtained at
smaller volumes were reproduced. Such remarkably benecial
effect of PEG-400 on the fermentation suggests that PEG-400
may have played a role of osmoprotectant for improved cell
tolerance to higher ethanol concentration. This hypothesis was
tested by measuring the yeast cell viability from fermentations
29
with and without PEG-400 (Fig. 6b). It was found that although
the yeast cell viability was the same with and without PEG-400
within 36 h, it was much decreased in pure water than that in
the presence of PEG-400 in 48 h. The cell viabilities in systems
with and without PEG-400 started to progressively diverge aer
3
6 h. The viability results clearly indicate that exo-protection of
the yeast cells by PEG-400 signicantly increased ethanol
mately 5 ꢂ 10 cells per mL, 16 mL water, 33 C, 160 rpm, and pH of concentration under VHG conditions. The mechanism of PEG
Fig.
5 Effect of different PEGs on batch ethanol fermentation.
Fermentation conditions: 7.0 g D-glucose, 4.0 g of PEGs, approxi-
8
ꢁ
3
.9. All data and error bars represent the averages and standard additives on the improvement of cell viabilities under high
deviations of triplicate measurements.
ethanol concentrations is an important subject of future study.
Reduced osmotic pressure to the yeast cells may be caused by
employing PEG-400 as an osmoregulator. Chemical interac-
tion of PEG-400 with ethanol could play an important role in the
fermentation broth.
30
Effect of PEG-400 on recovered yeast fermentation
performance
We further studied the performance of recycled yeast cells from
both fermentation systems with and without PEG-400. According
to the results in Fig. 6a, with fresh yeast, the ethanol concentra-
tion was near the same at 48 h in both fermentation systems with
and without PEG-400. It should be noted that the ethanol
concentration in 48 h is already close to the ethanol concentra-
tion ceiling in the pure water system. Therefore, the yeast cells
were recycled from both systems aer 48 h for the subsequent
fermentation. As shown in Fig. 7a and b, although in general the
activity of the recycled yeast was lower than that of fresh yeast and
fermentation productivity decreased with each recycle, there was
a very pronounced difference between yeast recycled from prior
fermentation in the presence of PEG-400 and that recycled from
fermentation in the absence of PEG-400. The rst recycled batch
ꢀ
1
ꢀ1
of yeast produced 62 g L ethanol as compared to 150 g L
produced by fresh yeast in pure water system (Fig. 7b). In
comparison, in the system with PEG-400, the rst recycled batch
of yeast produced 126 g L ethanol, which is more than doubled
Fig. 6 Comparison of glucose fermentation at different time. (a)
Ethanol concentration and glucose consumption (expressed without
ꢀ1
monohydrate) with and without PEG-400; (b) yeast cell viability with that produced from recycled yeast in pure water. The results of
and without PEG-400 from the VHG fermentation processes.
additional recycled runs further conrm the benecial effect of
PEG-400 exo-protection for the yeast cells. It was noted that the
yeast cells recycled from the PEG-400-free batches ceased to
produce ethanol in the 4th reuse. Comparing the total amount of
ethanol produced in the ve runs, the fermentation operated in
the presence of PEG-400 produced 7.16 g of ethanol, equivalent to
Fermentation conditions: 3.5 g D-glucose as monohydrate, approxi-
8
ꢁ
mately 5 ꢂ 10 cells per mL, 8 mL water, 2.0 g of PEG-400, 33 C, 160
rpm, and pH of 3.9. All data and error bars represent the averages and
standard deviations of triplicate measurements.
ꢀ
9
that in the presence of PEG-400, it slowed down aerward. Even
0
.90 ꢂ 10 g of ethanol per yeast cell, while that in the absence
ꢀ
1
ꢀ9
aer extending the fermentation to 96 h, 42.0 g L of glucose
of PEG-400 produced 3.88 g of ethanol, equivalent to 0.48 ꢂ 10
was unconverted in the pure water system. The maximum
g of ethanol per yeast cell. We also measured the yeast cell
viability aer each recycled use (Fig. 7c). Overall, the yeast cell
viability decreased in the rst two recoveries, and then became
ꢀ
1
ethanol concentration was 159 g L , which is near the ethanol
concentration ceiling for the yeast in pure water. In comparison,
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The recovery of PEG-400 and H
2
SO
4
is integrated as a natural
step of ethanol separation by distillation. The PEG-400 and
H SO were readily recovered from the residual mother liquor of
2
4
the distillation. By following the process as shown in Fig. 1, we
have veried that PEG-400 and H SO were recoverable and
remained effective without additional makeup. The results of
recycled PEG-400 and H SO in subsequent fermentation cycles
2
4
2
4
are shown in Fig. 8. There was little change in glucose conver-
sion, ethanol yield and concentration from the four repeated
uses of the initially loaded PEG-400 and H SO .
2
4
Integrated recycle process for yeast, PEG and H
We further studied an integrated recycle process for yeast, PEG-
00 and H SO . In the experiments, fresh yeast cells were sup-
2 4
SO
4
2
4
plemented in late cycles to complete glucose-to-ethanol
conversion. The yeast cells were recovered by centrifugation,
and then PEG and H SO were recycled by reusing the
2
4
fermentation broth aer distillation. The living cells and PEG-
00 were reused in subsequent cycles of the fermentation
4
process. Fresh yeast cells were supplemented in cycles 2–4 with
the amount pre-determined based on the death rate of cells
(40%, 60%, 65%, respectively) from the results in Fig. 9. The
results obtained in recycled use of spent yeast with supplement
yeast, and of PEG-400 and H SO are shown in Fig. 9.
2
4
Comparing the total amount of ethanol produced in the four
runs, the fermentation operated in an integrated recycle process
for yeast and PEG produced 10.37 g of ethanol, equivalent to
ꢀ
9
0
.86 ꢂ 10 g of ethanol per yeast cell while that in process for
Fig. 7 The performance of recycled yeast from fermentation systems
with and without PEG-400. (a) The ethanol concentration over
recovered yeast with PEG-400. (b) The ethanol concentration over
reuse of recovered yeast produced 7.16 g of ethanol, equivalent
ꢀ
9
to 0.90 ꢂ 10 g of ethanol per yeast cell. The results not only
recovered yeast without PEG. (c) Comparison of yeast cell viability with verify the superior productivity of PEG-400 exo-protected yeast
and without PEG-400. (d) Comparison of yeast cell vitality with cycle 2,
cells in cycles 2–4, but also demonstrate that the spent yeast can
be reused with the supplementation of a much reduced
amount of fresh yeast cells. In combination with the recycled
cycle 3 and cycle 4. Fermentation conditions: 6.5 g D-glucose,
8
approximately 5 ꢂ 10 cells per mL, 16 mL H
2
O, 4.0 g of PEG-400,
ꢁ
3
3 C, 48 h, 160 rpm, and pH of 3.9. All data and error bars represent
the averages and standard deviations of triplicate measurements.
unchanged. It is remarkable that the yeast cell viability in the
presence of PEG-400 was much higher than that in the absence of
PEG-400, supporting the hypothesis that PEG-400 improved cell
viability. However, the yeast cell viability in cycle 3 and cycle 4
(Fig. 7c) did not appear to correlate with the corresponding
fermentation performances as shown in Fig. 7a. This observation
could be explained by decreased cell vitality with the increasing
cycle times. To verify this hypothesis, the recovered cells were
cultured in YPD broth, and the results are shown in Fig. 7d. It
became evident that cell growth in cycle 4 was slower than that in
cycle 2 and cycle 3, and that cell in the cycle 3 and cycle 4 started
to die rapidly aer 24 h. The results imply that the cell
vitality decreased gradually with cell aging during the multiple
cycles.
Fig. 8 Results of four successive fermentation runs by recovering
PEG-400 together with H SO . Green bar, glucose conversion; red
2 4
bar, ethanol yield; (-), ethanol concentration based on water;
8
fermentation conditions: 7.0 g D-glucose, approximately 5 ꢂ 10 cells
Processes for PEG and H
Even though PEG-400 is a low cost surfactant, it is still
economically meaningful to recycle and reuse this surfactant. deviations of triplicate measurements.
2 4
SO recycle and reuse
ꢁ
per mL, 16 mL water, 4.0 g of PEG-400, 72 h, 33 C, 160 rpm, and pH of
.9. All data and error bars represent the averages and standard
3
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Conclusions
In summary, our results demonstrate that fully water-soluble
PEGs are effective for the vitalization of yeast cells in VHG
fermentation for the production of ethanol in very high
concentration. The nal ethanol concentration was increased
by over 10% with fresh yeast and no residue sugar remained in
the fermentation broth in the presence of PEG-400 supplement.
Very importantly, the yeast cells exhibited substantially
extended viability for reuse, and the PEG and H SO are readily
2
4
recoverable for multiple subsequent recycled use. It is also
demonstrated that the spent yeast with highly maintained
viability due to the presence of PEG-400 can be reused in
multiple cycles only by supplementing a much reduced amount
Fig. 9 Results of four successive fermentation runs by recovering of fresh yeast cells. The combination of recycled use of spent
yeast, PEG-400 together with H SO . Fermentation conditions: 6.5 g yeast, PEG-400 and H SO , allows the subsequent fermentations
2
4
2
4
ꢁ
D-glucose, 16 mL water, 4.0 g of PEG-400, 48 h, 33 C, 160 rpm, and
pH of 3.9. All data and error bars represent the averages and standard
deviations of triplicate measurements.
at the full efficiency due to the presence of PEG-400. The results
of the present study also imply that reduced water consumption
and energy consumption can be achieved by using an optimized
amount of the PEG. Largely enhanced ethanol productivity can
be achieved by recycling the yeast, which can be expected to
help reduce the overall cost. The results of this work further
imply that the method of yeast cell exo-protection may be
applicable beyond ethanol production; it may be used in
fermentation processes to produce other types of renewable
chemicals and bio-fuels.
use of PEG-400 and H SO , only supplemental amount of yeast
is needed to carry out the subsequent fermentations at the full
efficiency due to the presence of PEG-400.
2
4
The kinetics of ethanol production in the absence and
presence of PEG-400
Acknowledgements
Fig. 10 shows the product ethanol proles during VHG batch
fermentation in pure water and in the presence of PEG-400. The This work was funded by the Chinese Government “Thousand
experimental data of variation in specic ethanol productivity Talent” program funding.
on glucose in the absence and presence of PEG-400 were
obtained. The experimental data were also tted to estimate the
3
1
ꢀ1
kinetics parameters nmax and Pmax
.
Only 162 g L of Pmax was Notes and references
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ꢀ1
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