Process Biochemistry 50 (2015) 15091515

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Accelerated bioethanol fermentation by using a novel yeast

immobilization technique: Microtube array membrane
Chien-Chung Chen a,1 , Chien-Hui Wu b , Jhih-Jhong Wu c , Chien-Chih Chiu a ,
Chien-Hsuan Wong c , Min-Lang Tsai c , Hong-Ting Victor Lin c,d,
a

Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, 250 Wu-Hsing St., Taipei 11031, Taiwan, ROC
Department of Seafood Science, National Kaohsiung Marine University, No. 142 Hai-Chuan Road, Nan-Tzu, Kaohsiung 81143, Taiwan, ROC
c
Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan, ROC
d
Center of Excellence for the Oceans, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan, ROC
b

a r t i c l e

i n f o

Article history:
Received 19 March 2015
Received in revised form 3 June 2015
Accepted 8 June 2015
Available online 14 June 2015
Keywords:
Repeated-batch
Fermentation
Bioethanol
Microtube array membrane (MTAM)
Kluyveromyces marxianus

a b s t r a c t
Cell immobilization is a way to isolate or localize intact cells in a certain space and maintain their catalytic
activity. Immobilized cells can effectively reduce the negative effects of inhibitors and the processing
cost of inoculum preparation for continuous or fed-batch fermentation of microorganisms. In this study,
a novel yeast immobilization technique using renewable poly-l-lactic acid (PLLA) microtube array membrane (MTAM) was thoroughly evaluated for bioethanol fermentation. PLLA-MTAM was shown to be
stable in 15% (v/v) ethanol solution during shaking cultivation. A yeast encapsulation efciency of 6770%
was obtained, and the yeasts in MTAMs with greater porosity showed greater bioethanol productivity.
The MTAM-immobilized Kluyveromyces marxianus, prepared using in situ and siphon methods, were evaluated using 5% (w/v) glucose fermentation. Improved glucose consumption and bioethanol production
were observed in batch bioethanol fermentation. In 7 cycles during repeated-batch fermentation, the
immobilized yeasts prepared using the in situ method showed a maximum CEtOH of 24.23 g/L, maximum
YP/S of 0.48 g/g, and r PEtOH of 2.69 g/L h. Our data indicated that the PLLA-MTAM immobilized yeasts
signicantly enhanced bioethanol productivity and was a novel, promising technology for bioethanol
fermentation.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
The rising prices of fossil fuels and environmental concerns have
urged people to search for renewable energy sources. Bioethanol
is a type of biofuel that can be produced through the fermentation of biomass [1]. First-generation bioethanol production utilizes
sugar and starch from crops as a feedstock and encounters multiple
challenges such as rising food prices and land shortage, resulting
in an urgent need for alternative solutions [2]. Third-generation
bioethanol production uses algae and requires the biomass to be
pretreated and hydrolyzed for saccharication as well as generation of fermentable sugars to be used by other microorganisms in
order to produce bioethanol through fermentation [2,3]. However,

Corresponding author at: Department of Food Science, National Taiwan Ocean

University, No. 2, Pei-Ning Road, Keelung 202, Taiwan, ROC.
Tel.: +886 2 24622192x5150; fax: +886 2 24624113.
E-mail addresses: polyjack@tmu.edu.tw (C.-C. Chen), hl358@ntou.edu.tw
(H.-T.V. Lin).
1
Tel: +886 2 2736 1661x5134.
http://dx.doi.org/10.1016/j.procbio.2015.06.006
1359-5113/ 2015 Elsevier Ltd. All rights reserved.

pretreatment and hydrolysis of biomass are often accompanied by

the generation of inhibitors, which seriously impede subsequent
microbial fermentation to produce bioethanol [4,5]. Thus, the key
challenges associated with this energy source are related to the
ability to produce bioethanol more efciently without competing
for food crop supply and cultivable lands.
Immobilization is a way to isolate or localize intact cells in
a certain space and maintain their catalytic activity. Immobilized cells can effectively reduce the negative effects of inhibitors
and the processing cost of inoculum preparation for continuous
or fed-batch fermentation of microorganisms [6]. Immobilization
techniques can be categorized as follows: (1) immobilization on
solid carrier surfaces, (2) entrapment within a porous matrix, (3)
mechanical containment behind barriers, and (4) cell occulation
(aggregation). Bioethanol fermentation using immobilized cells can
increase cell density, shorten fermentation times, increase ethanol
and inhibitor tolerances, and improve the feasibility of using continuous fermentation, resulting in higher bioethanol production
efciency [6]. Lee et al. [7] used sodium alginate beads immobilized
with Saccharomyces cerevisiae for bioethanol fermentation. They
found that immobilized yeast cells converted glucose into alcohol

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C.-C. Chen et al. / Process Biochemistry 50 (2015) 15091515

more efciently than free yeast cells during batch fermentation, and
the beads immobilized with yeast cells could be reused for 5 consecutive batch runs. Eiadpum et al. [8] showed that using multi-hole
sterile loofah sponges or cicada cocoons for Kluyveromyces marxianus immobilization enhanced ethanol fermentation compared
with that using free cells. Furthermore, S. cerevisiae VS3 immobilized on rice straw boosted bioethanol fermentation and were
reusable for 8 consecutive batch runs [9].
The current techniques to immobilize cells have several drawbacks. The surface attachment of cells using chemical linking
agents, such as glutaraldehyde, may be unsuitable for the production of ethanol or beverages. Moreover, because there are no
barriers between solution and cells, cell detachment and relocation
may contaminate products. Another approach to cell immobilization is using a porous gel matrix, such as Ca-alginate, to entrap
cells and obtain high biomass loadings for fermentation; however,
the bead structure can destabilize in the presence of acid or diffusion limited gases, such as CO2 in ethanol fermentation, and result
in bead rupture [10]. Cell occulation is considered to be a relatively low-cost method; however, the occulation of some yeasts
is inhibited by the presence of sugars, such as glucose, and ethanol
[11]. Using the method of containment behind a barrier, such as that
used in microporous membrane lters, for cell immobilization is
most suitable when a cell-free product is required. However, there
are inherent problems such as possible membrane fouling, high
cost, and container recycling issues [12].
In this report, we utilized a highly porous microtube array
membrane (MTAM) [13] to immobilize K. marxianus for bioethanol
fermentation, a rst in bioethanol production. MTAMs have structures similar but superior to hollow bers and were developed
using coaxial electrospinning. Compared with a typical hollow
ber, MTAMs are 12 orders smaller in diameter and >2 orders thinner in the tube wall; most strikingly, they can self-assemble into a
single membrane sheet [14,15]. MTAMs are further functionalized
by creating nanopores on the surface using a pore-forming agent
and followed by a leaching out process [16]. With these structural
characteristics, MTAMs provide a large surface area per volume
and short diffusion distances with a self-supported barrier function. They are easily manipulated and have a low cost. MTAMs
may serve as an excellent substrate to immobilize yeast for fermentation processes in an industrial scale. In this study, we used
poly-l-lactic acid (PLLA), an environment friendly material [17], to
prepare MTAMs; further, we optimized the conditions to prepare
MTAM-immobilized cells and evaluated their potential for repeated
fed-batch bioethanol fermentation.
2. Materials and methods
2.1. Source of K. marxianus
K. marxianus (BCRC 21363) was purchased from Bioresources
Collection and Research Center (BCRC) in Hsinchu, Taiwan.

An electrostatics charger (ChargeMaster, Simco-Ion, Alameda, CA,

USA) or a high voltage power supply unit (You-Shang Co., Fongshan
city, Taiwan) was used as the source of electrostatics. Typically,
electrospinning was performed by delivering the PLLA (shell) and
PEG (core) solutions through a house-made co-axial spinneret with
a syringe pump (KDS-100, KD Scientic, Holliston, MA, USA) at
the rate of 49 mL/h, with 57 kV of applied voltage and 35 cm
of distance to a rotating drum collector. All electrospinning procedures were performed in a chamber at a relative humidity of
50 5% and a temperature of 25 1 C. Nano-porous PLLA MTAMs
were obtained by washing to remove the core component of PEG,
followed by drying. In situ preparation of yeast-containing PLLA
MTAM was accomplished by using typical MTAM electrospinning
process with similar parameters described above, with inner solution of mixed solution of yeast cell (cell density of 108 mL1 and
109 mL1 ) medium solution and PEG solution (1:9 in volume). The
obtained MTAM samples were then cut into the dimensions of
3 cm 0.5 cm 0.0004 cm and both ends sealed by a heated metal
plated. The siphoned yeast cell-containing PLLA MTAM was prepared by simply placed one open end of dried PLLA-MTAM into a
xed volume (10 L) of yeast cellmedium solution (cell density
of 108 mL1 and 109 mL1 ). After the capillary force siphoned the
solution up through-out MTAM, both ends were sealed by a heated
metal plate.
2.3. The stability test of PLLA MTAM at various rpm and ethanol
concentration
The PLLA MTAMs were made as described previously and stored
in 0.1% (w/v) peptone water at 4 C. For the stability test of PLLA
MTAM at various ethanol concentrations, the prepared MTAMs
were placed into ethanol solution at a concentration of 3%, 6%, 9%,
12%, and 15% (v/v), individually, at room temperature. The PLLA
MTAM structure at each ethanol concentration was observed by
naked eyes and microscopes for any breakage. For the stability test
of PLLA MTAM, the prepared PLLA MTAM were incubated in 0.1%
(w/v) peptone water at various 100, 200, 300, 400 and 500 rpm,
individually, at room temperature. The PLLA MTAM structure at
each ethanol concentration was observed by naked eyes and microscopes for any breakage.
2.4. Analysis of encapsulation efciency
The PLLA MTAM-immobilized K. marxiansu was torn by using a
scissors and soaked in 0.1% (w/v) peptone. The mixture was thoroughly vortexed to release the immobilized cells into the peptone.
The mixture was then diluted and plated out onto YPD plates and
the plates were incubated at 42 C for 24 h. The colonies were calculated to obtain the actual cell counts of the immobilized yeasts.
The actual cells counts were divided by the theoretical cell counts
of the immobilized yeast to obtain the encapsulation efciency.
2.5. Batch and repeated-batch of bioethanol fermentation using
immobilized K. marxinaus

2.2. The preparation of PLLA MTAMs-immobilized K. marxianus

The K. marxianus was cultured in a YPD medium (1%
yeast extract, 2% bactopeptone, and 2% dextrose) at 42 C to
reach a cell density of 108 and 109 cfu/mL for immobilization.
Materials used were PLLA (BioTechOne, Taiwan), polyethylene glycol/polyethylene oxide (PEG/PEO; SigmaAldrich), N,N-dimethyl
formamide (DMF; Tedia, USA), and dichloromethane (DCM;
Mallinckrodt, USA). To prepare the electrospinning dopes, PLLA
was dissolved in mixed DCM/DMF (8/2) solvents at room temperature to obtain a 10% solution. PEG was added to the PLLA solution
to obtain PEG/PLLA (30/70) solutions, as described previously.

For bioethanol fermentation, the PLLA MTAM-immobilized K.

marxianus were placed into 100 mL of YPD medium containing 5%
glucose (1% yeast extract, 2% bactopeptone, and 5% glucose) at
42 C with a shaking of 200 rpm. The glucose usage and ethanol
production in batch fermentation were monitored at 0, 6, 9, 12,
15 and 24 h. by using HPLC analysis. The optimum batch fermentation time enabling maximum ethanol production was observed
and applied in repeated bat fermentation. Subsequently repeated
batch bioethanol fermentation was accomplished with the same
growth conditions as batch fermentation. At the end of each fermentation cycle, the PLLA MTAM-immobilized K. marxianus cells

C.-C. Chen et al. / Process Biochemistry 50 (2015) 15091515

Table 1
The effects of agitation and shaking on MTAM.

Table 2
The effects of encapsulation methods and cell numbers on encapsulation efciency.

Shaking incubator (rpm)

Breakage time (h)

Cell numbers (CFU/mL)

50

100

150

200

>600

>600

144168

7296

Agitation in fermentor (rpm)

Breakage time (h)

1511

108
109

Encapsulation efciency (%)

In situ preparation

Siphone

70 2.0%
27 8.5%

67 15.0%
57 28.0%

Data are expressed as mean SD (n = 3).

100

200

300

400

500

60

36

12

12

12

were washed with sterile peptone water and then transferred to

the similar volume of fresh YPD medium containing 5% glucose for
the next cycle of fermentation. The cycles were repeated over and
over until the production of bioethanol was not efcient. The glucose and bioethanol fermentation was measured at 0, 3, 6, 9 12 h
during each cycle of fermentation. The parameters for bioethanol
fermentation were shown in CEtOH (Ethanol concentration), r PEtOH
(Initial ethanol production rate) and Max. YP/S (Maximum ethanol
yields) [5]. CEtOH (g/L) is the maximum ethanol concentration to be
achieved during fermentation; r PEtOH (g/L h) is that CEtOH divided
by the time (h) consumed to reach CEtOH ; Max. YP/S (g/g) is obtained
by dividing total produced ethanol during fermentation (g) by total
sugar consumed during fermentation (g).
2.6. Analyses of glucose and ethanol using HPLC
The glucose (g) and ethanol (g) were all analyzed using an
Aminex HPX-87H column (Bio-Rad, Sunnyvale, CA, USA), along
with a refractive index detector with a 5 mM H2 SO4 eluent at a
ow rate of 0.6 mL/min at 60 C. All the tested samples were ltered
through a 0.22 m membrane lter prior to the HPLC analysis.
2.7. Statistical analysis
Data were analyzed statistically using SPSS Version 12.0 (SPSS
Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was
used to determine statistical differences between sample means,
with the level of signicance set at P < 0.05. Multiple comparisons of means were done by Tukey test. All data are expressed
as mean SD.
3. Results and discussion
3.1. Encapsulation of K. marxianus in PLLA MTAM
The scanning electron microscope (SEM) section image of MTAM
is shown in Fig. 1(a). MTAM consists of a single layer of hollow tubes
with a diameter of approximately 40 m and a hollow tube wall
pore diameter of approximately 30 nm [16]. Free K. marxianus cells
and MTAM-immobilized K. marxianus cells are shown in Fig. 1(b)
and (c). PLLA-MTAM entrapped the yeast cells for immobilization
to obtain high biomass loadings in fermentation. Enantiomerically, pure PLLA is a semicrystalline polymer with a glass transition
temperature of approximately 55 C and a melting point of approximately 180 C [18], suggesting that PLLA-MTAM is thermostable
at all known bioethanol fermentation temperatures. Furthermore,
PLLA is insoluble in water and its solubility in solvents is highly
dependent on the molar mass and degree of crystallinity [18].
3.2. Effects of ethanol and agitation on the stability of MTAM
To determine the effect of agitation and ethanol, MTAMimmobilized K. marxianus was incubated at different rpm settings
and ethanol concentrations. As shown in Table 1, MTAM retained a

consistent solidity at 3%, 6%, 9%, 12%, and 15% (v/v) ethanol for more
than 5 weeks (840 h) (data not shown). A previous report indicated
that 8 repeated-batch fermentations using corncob-immobilized S.
cerevisiae needed approximate 520 h [19], and our data indicated
that MTAM was stable at 15% (v/v) ethanol for more than 840 h
and that cell immobilization may be applicable for repeated-batch
and continuous fermentations. The effect of agitation (100, 200,
300, 400, and 500 rpm) on MTAM was determined using 2 different methods, a rotary shaker and a fermenter with an impeller.
As shown in Table 1, MTAM started to tear at an incubation time
of 7296 h on a rotary shaker at an rpm of 200. As the rpm was
decreased, the time required for any tear to appear on MTAM was
increased. At the rpm settings of 50 and 100, MTAM showed no
tearing within 600 h (25 days). In the fermenter, we observed that
the stability of MTAM decreased as the agitation increased. MTAM
showed no obvious tearing at 100 rpm for up to 60 h and at 200 rpm
for up to 36 h. However, agitation greater than 200 rpm degraded
MTAM within 12 h. The tearing of MTAM could be because of the
mechanical shear force from the agitation in solution [20] and the
physical conicts between the impellers and MTAM. Our data suggested that the current PLLA-MTAM was applicable for anaerobic
and aerobic fermentation with shaking, but not for aerobic fermentation with an impeller. However, it is possible to reinforce
PLLA-MTAM with other composite brous structures to improve
its mechanical strength.

3.3. Effects of encapsulation methods and cell numbers on the

encapsulation efciency
We evaluated the encapsulation efciency of yeast K. marxianus for 2 encapsulation methods, in situ and siphon preparations.
As shown in Table 2, encapsulation of 108 yeast cells using the
in situ and siphon methods showed encapsulation efciencies of
70.0 2.0% and 67.0 15.0%, respectively. Encapsulation of 109
yeast cells using the in situ and siphon methods showed encapsulation efciencies of 27.0 8.5% and 57.0 28.0%, respectively. These
data indicated that the encapsulation efciencies were greater for
both encapsulation methods at a cell density of 108 compared with
that at a density of 109 . The siphon method showed better encapsulation efciency than the in situ preparation method at a cell density
of 109 , whereas the 2 methods showed similar encapsulation efciencies at a cell density of 108 . In addition, the encapsulation
efciency using the in situ method produced a smaller standard
deviation compared with that using the siphon method. Accordingly, we used the cell density of 108 CFU/mL for cell encapsulation
in MTAM for better encapsulation efciency. Although the in situ
preparation of yeast-containing PLLA-MTAM was accomplished
using a typical MTAM electrospinning process, in which the electrical eld may affect cell viability, our encapsulation efciency
data suggested that the K. marxianus cells survived the process of
electrospinning. This nding is consistent with that of a previous
report, in which the mammalian cell line PC12 remained viable
after in situ MTAM encapsulation [21]. In addition, the yeast S. cerevisiae EBY100 maintained viability as well as the ability to express
surface-display enhanced green uorescent protein (eGFP) after

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C.-C. Chen et al. / Process Biochemistry 50 (2015) 15091515

Fig. 1. The SEM section image of (a) MTAM and the light microscopy images of (b) free K. marxianus and (c) MTAM-immobilized K. marxianus prepared by in situ method.

the cells were immobilized and cross-linked to polyvinyl alcohol

nanobers by electrospinning [22].

3.4. Effects of encapsulation methods and porosity of MTAM on

yeast growth and batch bioethanol fermentation
Fig. 1(a) showed that MTAM was a single layer with a hollow
tube diameter of approximately 40 m, and a nanopore diameter of approximately 30 nm on the surface of each hollow tube,
allowing simple sugars, alcohol, and other small molecules to diffuse in and out. In order to determine whether greater porosity on
the surface of the tube would accelerate sugar consumption and
ethanol production, possibly because of greater diffusion of sugars and ethanol across the barrier, we used 10% (v/v) pore-forming
agent polyethylene glycol in PLLA-MTAM preparation to produce
greater porosity with nanopore diameters of 0.31 0.08 m on the
surface of the MTAM hollow tube wall [16]. As shown in Table 3, the
in situ preparation of MTAM-immobilized K. marxianus with greater
porosity showed a CEtOH of 8.76 0.07 g/L, r PEtOH of 0.73 0.01 g/L

Table 3
The effect of porosity of MTAM on bioethanol fermentation in 12 h.
CEtOH (g/L)

r PEtOH (g/L h)

Max. YP/S (g/g)

In situ preparation
MTAM with greater porosity
MTAM

8.76 0.07a
7.86 0.08b

0.73 0.01a
0.66 0.01b

0.43 0.01a
0.39 0.01b

Siphon
MTAM with greater porosity
MTAM

8.13 0.39b
7.89 0.10b

0.67 0.03b
0.66 0.01b

0.39 0.02b
0.39 0.01b

CEtOH (Ethanol concentration); r PEtOH (Initial ethanol production rate); Max. YP/S
(Maximum ethanol yields).
Data are expressed as mean SD (n = 3). Different letters in column show signicant
differences (p < 0.05).

h, and maximum YP/S of 0.43 0.01a (g/g) during bioethanol fermentation, which were better than the corresponding values for
the original MTAM. With regard to MTAM-immobilized K. marxianus with greater porosity prepared by the siphon method, there
were no obvious differences compared with the original MTAM

C.-C. Chen et al. / Process Biochemistry 50 (2015) 15091515

(A)

Glucose concentration (g/L)

60
Free cells
In situ preparation
Siphone

50
40
30
20
10
0
0

12

15

18

21

24

Time (h)

(B)

Ethanol concentration (g/L)

25
20
15

1513

A maximum bioethanol concentration of 18.46 g/L was obtained

from fermentation using free cells after 15 h. The immobilized yeast
cells in MTAM from the in situ and siphon preparations showed
maximum bioethanol concentrations of 20.7 g/L (9 h) and 19.9 g/L
(12 h), which were 10% and 8% higher than that of free cells,
respectively. In batch fermentation, the relatively rapid sugar consumption by immobilized yeasts compared with that by free cells
has been commonly observed in previous reports [7,9,23]. However, the results related to enhanced ethanol productivity using
immobilized cells have been diverse. Mariam et al. [24] indicated
that the maximum bioethanol concentration obtained using free S.
cerevisiae was 7.3% higher than that obtained using immobilized
cells. Laopaiboon and Laopaiboon [19] reported that free and corncob immobilized yeast cells showed similar ethanol production in
the batch fermentation of sweet sorghum juice. Singh et al. [25] also
concluded that the concentrations of bioethanol produced by free
and calcium alginate-immobilized yeast cells were similar. Yu et al.
[23] reported that the ethanol production using immobilized cells
fermenting sorghum bagasse was 2.24 times higher than that using
free cells. In addition, S. cerevisiae VS3 cells showed 10% greater
ethanol production than that using free cells in batch fermentation [9]. In this study we used MTAM-immobilized K. marxianus for
batch fermentation and improved ethanol productivity by 810%
compared with free cells depending on the preparation method
used.
3.6. Repeated-batch bioethanol fermentation of YPD using
MTAM-immobilized K. marxianus

10
5

Free cells
In situ preparation
Siphone

0
0

12

15

18

21

24

Time (h)
Fig. 2. The (a) sugar usage and (b) bioethanol production in batch fermentation of
YPD using free and immobilized K. marxianus. Batch fermentation of YPD medium
containing 5% (w/v) glucose was incubated at 42 C with a shaking of 200 rpm. Data
are expressed as mean SD (n = 3).

in the parameters of bioethanol fermentation. The encapsulation

efciency using this method showed a relatively higher standard
deviation, suggesting a large discrepancy between the number of
cells involved in each encapsulation of the original MTAM and
MTAM with a greater porosity. We used 10% (v/v) polyethylene
glycol to create MTAM-immobilized K. marxianus for the rest of the
study.

3.5. Sugar usage and bioethanol fermentation of glucose using

free and MTAM-immobilized K. marxianus
The bioethanol fermentation of YPD by free and MTAMimmobilized K. marxianus is shown in Fig. 2. The starting cell density
of free and MTAM-immobilized K. marxianus in fermentation were
kept constant at 105 CFU/mL. The results showed that compared
with free cells, batch fermentation with yeast cells immobilized
in MTAM required less fermentation time to convert glucose to
bioethanol. Glucose at a concentration of 50 g/L was consumed
by the free cells in 24 h, whereas MTAM-immobilized cells prepared by the in situ and siphon methods depleted glucose in 9 h and
12 h, respectively, indicating that MTAM-immobilized K. marxianus
improved efciency.

Glucose consumption and ethanol production in repeated-batch

fermentation using MTAM-immobilized K. marxianus were studied
as a function of time (Fig. 3). The rst batch of MTAM-immobilized
cells consumed 17 g/L of the starting 50 g/L glucose (34%) at 6 h.
From batch II to VII, the same MTAM was reused and each batch
consumed more than 95% glucose at 6 h (Fig. 3(a)). Furthermore, all
the batches, including the rst batch, consumed all the glucose in
9 h. As a result, the highest ethanol concentration in all batches
was obtained at 9 h of fermentation, which started to decrease
at 12 h, possibly because of ethanol oxidation for metabolism
when glucose was unavailable [26]. The standard deviations of
ethanol concentration at 6 h for all the batches were greater than
those at any other time point, suggesting that some immobilized
MTAMs could achieve maximum ethanol production before 9 h.
Our attempt to encapsulate K. marxianus in MTAM for repeatedbatch bioethanol fermentation successfully reduced the processing
cost of inoculum preparation, and accelerated glucose consumption and ethanol production. Evaluation of the kinetic parameters
for ethanol production using free cells in fermentation and MTAMimmobilized K. marxianus in repeated-batch fermentation is shown
in Table 4. As expected, MTAM-immobilized cells in every batch
showed improved performance in terms of sugar consumption and
ethanol production compared with free cells. Bioethanol fermentation using free K. marxianus cells showed a glucose consumption
of 93%, CEtOH of 18.46 0.38 g/L, maximum YP/S of 0.40 0.002 g/g.
and r PEtOH of 1.23 0.02 g/L h at 15 h. For the immobilized cells,
the rst batch showed the lowest ethanol productivity in CEtOH
(20.78 0.16 g/L), maximum YP/S (0.42 0.003 g/g), and r PEtOH
(2.31 0.02 g/L h) at 9 h. In subsequent batches, the ethanol productivity increased gradually until batch VI, and the CEtOH , maximum
YP/S , and r PEtOH obtained in batches III, IV, V, and VI were higher
than those of the rst 2 batches. The immobilized cells prepared by
the in situ preparation gave a maximum CEtOH of 24.23 0.63 g/L,
maximum YP/S of 0.48 0.012 g/g, and r PEtOH of 2.69 0.07 g/L h for
the 6 cycles of repeated-batch fermentation with a slight reduction
in the last cycle of fermentation. Ethanol productivity started to
decrease in batch VII, possibly because of aging yeast cells; these

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C.-C. Chen et al. / Process Biochemistry 50 (2015) 15091515

Table 4
Evaluation of kinetic parameters for ethanol production by free cells in batch fermentation and MTAM-encapsulated K. marxianus in repeated-batch fermentation.
Batch

Substrate utilization (%)

CEtOH (g/L)

Free cells
I
II
III
IV
V
VI
VII

93
100
100
100
100
100
100
100

18.46
20.78
21.36
23.39
22.79
22.94
24.23
22.57

0.38f
0.16e
0.04d
0.36b
0.30c
0.21bc
0.63a
0.15c

Max. YP/S (g/g)

0.40
0.42
0.43
0.47
0.46
0.46
0.48
0.45

0.002e
0.003d
0.001d
0.007ab
0.006bc
0.004bc
0.012a
0.003c

r PEtOH (g/L h)
1.23
2.31
2.37
2.60
2.53
2.55
2.69
2.51

0.02d
0.02c
0.04c
0.04ab
0.03b
0.02b
0.07a
0.02b

Time (h)
15
9
9
9
9
9
9
9

CEtOH (Ethanol concentration); r PEtOH (Initial ethanol production rate); Max. YP/S (Maximum ethanol yields).
Data are expressed as mean SD (n = 3). Different letters in column show signicant differences (p < 0.05).

(A)
I Batch
II Batch
III Batch
IV Batch
V Batch
VI Batch
VII Batch

Glucose concentration (g/L)

50
40
30

Acknowledgements
This work was supported by a grant from the Center of
Excellence for the Oceans, National Taiwan Ocean University
(NTOU-RD-AA-2014-1-02011 and 103J29001F).

20
10

References

0
0

12

12

Time (h)
(B)
I Batch
II Batch
III Batch
IV Batch
V Batch
VI Batch
VII Batch

25

Ethanol concentration (g/L)

in physical properties, cell encapsulation efciency and immobilization technology was performed, and our data indicated that
MTAM-immobilized yeasts showed its great potential in repeatedbatch and continuous bioethanol fermentation. To my knowledge,
we are the rst to introduce this cell immobilization technique to
bioethanol fermentation.

20
15
10
5
0
0

Time (h)
Fig. 3. The (a) sugar usage and (b) bioethanol production in repeated batch fermentation of YPD using immobilized K. marxianus. Repeated-batch fermentation of
YPD medium containing 5% (w/v) glucose was incubated at 42 C with a shaking of
200 rpm. Data are expressed as mean SD (n = 3).

observations in the later batches have also been reported in many

previous reports regarding repeated-batch bioethanol fermentation [7,9,19,24].
4. Conclusion
Here we provide a novel cell immobilization technique for
bioethanol production by taking advantage of the properties
of PLLA-MTAM, a low-cost, easily manipulated, renewable, and
microporous membrane. A thorough evaluation of PLLA-MTAM

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