Process Biochemistry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

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

Repeated-batch lactic acid fermentation using a novel bacterial

immobilization technique based on a microtube array membrane
Chien-Chung Chena,b,c,d, Chuan-Chi Lane, Chorng-Liang Pane, Mei-Ying Huangf, Chee-Ho Chewa,
⁎
Chin-Chieh Hunge, Po-Hsuan Chene, Hong-Ting Victor Line,g,
a
Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, 250 Wu-Hsing St., Taipei, 11031,
Taiwan, ROC
b
International PhD Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, 250 Wu-Hsing St., Taipei, 11031, Taiwan, ROC
c
International PhD Program for Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, 250 Wu-Hsing St., Taipei, 11031, Taiwan, ROC
d
PhD Program in Biotechnology Research and Development, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing St., Taipei, 11031, Taiwan, ROC
e
Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung, 202, Taiwan, ROC
f
Division of Marine Fisheries, Fisheries Research Institute, No. 199, Hou-Ih Road, Keelung, 202, COA, Taiwan, ROC
g
Center of Excellence for the Oceans, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung, 202, Taiwan, ROC

A R T I C LE I N FO A B S T R A C T

Keywords: Lactic acid has received considerable attention for its various applications. Although lactic acid is usually pro-
Repeated-batch duced by batch fermentation using free microbes, repeated-batch fermentation using immobilized cells could
Fermentation offer several advantages over this method. In the present study, a novel bacteria immobilization technique using
Lactic acid a renewable poly-L-lactic acid (PLLA) microtube array membrane (MTAM) was thoroughly evaluated in terms of
Microtube array membrane (MTAM)
its suitability for lactic acid fermentation. A bacteria encapsulation efficiency of 85–90% was obtained using a
Lactobacillus acidophilus
siphon approach, and bacteria in MTAMs with greater porosity showed greater lactic acid productivity. MTAM-
immobilized Lactobacillus acidophilus were evaluated in eight cycles of repeated-batch lactic acid fermentation of
commercial medium MRS (4% glucose) and red seaweed Gracilaria hydrolysate. The MTAM exhibited physical
stability during lactic acid fermentation and improved glucose consumption and lactic acid production were
achieved by immobilized cells. The immobilized bacteria showed a maximum CLA of 37.39 g/L, r PLA of 0.79 g/
L·h, and YL/S of 0.94 g/g in MRS fermentation, and a maximum CLA of 27.76 g/L, r PLA of 0.58 g/L·h, and YL/S of
0.92 g/g in seaweed hydrolysate fermentation. Our data indicate that PLLA-MTAM is a novel, promising im-
mobilization technology for lactic acid fermentation.

1. Introduction media have been thoroughly optimized for the production of L

Lactic acid, a colorless and odorless monocarboxylic acid [1], has
received considerable attention for its various possible applications in
food and non-food industries [2]. For example, there is increasing de-
mand for lactic acid in the manufacturing of biodegradable polylactic
acid materials used as alternatives to petroleum-derived plastics. It has
been estimated that worldwide demand for lactic acid will reach
1,000,000 metric tons by the year 2020 [3]. Lactic acid can be pro-
duced by either chemical synthesis or microbial fermentation. Two
optical isomers of lactic acid, L(+)- and D(-)-lactic acid, are produced
during its chemical synthesis; of these, D(-)-lactic acid is considered to
be the main entity involved in human acidosis [4]. Most lactic acid
production worldwide is based on the fermentation of lactic acid bac-
teria [5,6], and fermentation parameters along with the composition of
(+)-lactic acid [7,8].
Although lactic acid production is usually accomplished by batch
fermentation using free microbes, repeated-batch fermentation using
immobilized cells could offer several advantages [9], such as a higher
fermentation rate [10], the possibility to reuse cells, a protective effect
against possible inhibitors [11], decreased inoculum preparation pro-
cessing cost [12], and reduced product contamination. Cell im-
mobilization is performed to localize or isolate intact cells in a certain
space and maintain their catalytic activity. Effective entrapment of
whole bacterial cells onto /within solid materials can sometimes im-
prove cell growth, bioprocessing activities and inhibitors tolerance
[13,14]. Cell immobilization allows integration of biocatalysts in a
manner that maintains long-term cell viability and typically enhances
process output, and it has been used in many biotechnology purposes

⁎
Corresponding author at: Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung, 202, Taiwan, ROC.
E-mail address: HL358@ntou.edu.tw (H.-T.V. Lin).

https://doi.org/10.1016/j.procbio.2019.09.016
Received 16 April 2019; Received in revised form 18 August 2019; Accepted 13 September 2019
1359-5113/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Chien-Chung Chen, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.09.016
C.-C. Chen, et al.
[14], such as biocontrol, bioremediation, biodegradation and produc-
tion of various compounds, such as amino acids [15], organic acids
[16], biofuels [17]. Cell immobilization can be achieved in several
ways: (1) encapsulation within a porous matrix, (2) immobilization on
solid carrier surfaces, (3) mechanical containment behind barriers, and
(4) cell flocculation (aggregation). The techniques currently used to
immobilize lactic acid bacteria have several drawbacks. The surface
attachment of cells onto solid carriers by chemical linking agents or
physical absorption might contaminate products due to cell detachment
and relocation because there are no barriers between solution and cells.
Already, approximately 40–70% of operating and capital costs are as-
sociated with the separation of lactic acid from the culture medium
[18], and cell contamination could make separation and purification
steps even more difficult. Calcium alginate is the most common im-
mobilization matrix used at present and has already been investigated
for application in lactic acid production [19]; however, lactic acid may
interact with calcium alginate, destabilize the bead structure of the
matrix, and result in bead rupture [20]. A method of cell immobiliza-
tion incorporating containment behind a barrier, such as that used in
microporous membrane filters, is most suitable when a cell-free product
is required. However, there are inherent problems in such methods,
such as possible membrane fouling, high cost, and lack of possibilities
for recycling containers [21].
In the present study, we used a highly porous microtube array
membrane (MTAM) [22] to immobilize Lb. acidophilus for lactic acid
fermentation, a first in lactic acid production. MTAMs were developed
using coaxial electrospinning and have structures similar but superior
to hollow fibers. MTAMs are 1–2 orders smaller in diameter with tube
walls > 2 orders thinner than typical hollow fibers; most strikingly,
they can self-assemble into a single membrane sheet [23,24]. As a re-
sult, MTAMs provide a large surface area to volume ratio and short
diffusion distances with a self-supported barrier function. In the present
work, MTAMs were prepared using poly-L-lactic acid (PLLA), a low-cost
and environmentally friendly material [25]. We optimized conditions
to prepare MTAM-immobilized lactic acid bacteria and evaluated their
potential for repeated fed-batch lactic acid fermentation with com-
mercial medium. Recently, seaweeds have drawn considerable atten-
tion in the production of bio-based chemicals [26,27] and biofuels
[28,29], which is an indication of their great potential as carbon
sources for lactic acid fermentation. Rich in carbohydrates, seaweeds
have a short generation cycle and can easily be cultivated in various
aquatic environments. They are not considered to be a primary food
crop and do not contain lignin, which makes saccharification more
feasible. Thus, we further established the application of this novel
bacterial immobilization technology in repeated fed-batch fermentation
of seaweeds for lactic acid production.
2. Materials and methods
2.1. Source of Lb. acidophilus
Lb. acidophilus (BCRC 10695) was purchased from Bioresources
Collection and Research Center (BCRC) in Hsinchu, Taiwan.
2.2. The preparation of PLLA MTAM-immobilized Lb. acidophilus
The Lb. acidophilus was cultured in a Man Rogosa Sharpe (MRS)
medium at 37 °C to reach a cell density of 108, 109 and 1010 cfu/mL for
immobilization. Materials used were PLLA (BioTechOne, Taiwan),
polyethylene glycol/polyethylene oxide (PEG/PEO; Sigma–Aldrich),
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 [22]. An elec-
trostatics charger (ChargeMaster, Simco-Ion, Alameda, CA, USA) or a
2
Process Biochemistry xxx (xxxx) xxx–xxx
high voltage power supply unit (You-Shang Co., Fongshan city, Taiwan)
was used as the source of electrostatics [22]. 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 Scientific, Holliston, MA, USA) at the rate of 4–9 mL/h, with
5–7 kV of applied voltage and 3–5 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
bacteria-containing PLLA MTAM was accomplished by using typical
MTAM electrospinning process as described above, with inner solution
of mixed solution of bacteria cell culture (cell density of 108/mL and
109/mL) and PEG solution (1:9 in volume). The obtained MTAM sam-
ples 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 lactic acid bacteria
cell-containing PLLA MTAM was prepared by simply placed one open
end of dried PLLA-MTAM (3 cm × 0.5 cm × 0.0004 cm) into lactic acid
bacteria culture (cell density of 109 and 1010 cfu•mL–1). After the ca-
pillary force siphoned the solution up through-out MTAM, both ends
were folded about 2 mm and pressed to seal.
2.3. Analysis of encapsulation efficiency
The PLLA MTAM-immobilized Lb. acidophilus was torn by using a
scissors and soaked in 0.1% (w/v) peptone. The mixture was thor-
oughly vortexed to release the immobilized cells into the peptone. The
mixture was then diluted and plated out onto MRS plates and the plates
were incubated at 37 °C for 48 h. The colonies were calculated to obtain
the actual cell counts of the immobilized lactic acid bacteria. The actual
cells counts were divided by the initial cell numbers of live bacteria of
the immobilized bacteria to obtain the encapsulation efficiency. The
initial cell numbers of live bacteria mean the total numbers of the live
bacteria to be added for cell immobilization, and the actual cell counts
mean the numbers of live bacteria that were actually immobilized in
MTAM.
2.4. Optimal surface porosity of PLLA-MTAM
PLLA-MTAM has a nanopore diameter of approximately 30 nm on
the surface of each hollow tube. The addition of 10% (v/v) pore-
forming agent PEG in PLLA-MTAM preparation could produce greater
porosity with nanopore diameters of 0.31 ± 0.08 μm [30]. Use two
types of PLLA-MTAM above to prepare immobilized Lb. acidophilus.
Both original and greater porosity PLLA-MTAM-immobilized Lb. acid-
ophilus were placed into 100 mL of MRS medium containing 4% glucose
at 30 °C for 48 h. The final glucose usage and lactic acid production
were monitored by using HPLC analysis.
2.5. Optimal buffering agent for Lb. acidophilus fermentation
Fermentations were performed with 100 mL MRS medium con-
taining 4% glucose and inoculated with 1% (v/v) Lb. acidophilus at
30 °C. To determine the optimal buffering agent, 0.2 M disodium
phosphate, 0.1 M trisodium citrate and 0.2 M sodium acetate were
added into MRS medium to control the pH value during fermentation,
respectively. The MRS medium was added with the buffering agent and
the pH was adjusted to the optimum pH for Lb. acidophilus (pH 6 to pH
6.5) before fermentation. The glucose usage and lactic acid production
were monitored at 0, 6, 12, 18, 24, 36, 48 and 72 h. by using HPLC
analysis.
2.6. Repeated-batch fermentation of MRS medium using immobilized Lb.
acidophilus
For lactic acid fermentation, the PLLA MTAM-immobilized Lb.
C.-C. Chen, et al. Process Biochemistry xxx (xxxx) xxx–xxx

acidophilus were placed into 100 mL of MRS medium containing 4%

glucose and 0.2 M sodium acetate at 30 °C. The optimum batch fer-
mentation time enabling maximum lactic acid production was observed
and applied in repeated batch fermentation. Subsequently repeated
batch lactic acid fermentation was accomplished with the same growth
conditions as batch fermentation. At the end of each fermentation cycle
(48 h each batch), the PLLA MTAM-immobilized Lb. acidophilus cells
were washed with sterile peptone water and then transferred to the
similar volume of fresh medium for the next cycle of fermentation. The
cycles were repeated for eight cycles. The glucose and lactic acid fer-
mentation was measured at 0, 12, 24, 36 and 48 h during each cycle of
fermentation. The parameters for lactic acid fermentation were shown
in CLA (Lactic acid concentration), r PLA (Lactic acid production rate)
and YL/S (Lactic acid yield) [10]. CLA (g/L) is the maximum lactic acid
concentration to be achieved during fermentation; r PLA (g/L∙h) is that
CLA divided by the time (h) consumed to reach CLA; YL/S (g/g) is ob-
tained by dividing total produced lactic acid during fermentation (g) by
total sugar consumed during fermentation (g).

2.7. Preparation of Gracilaria sp. hydrolysate

Gracilaria sp. was purchased from a local market in Chao-Ching Park

in Keelung city. The red seaweed was washed by water and air-dried
(50 °C) in an oven. The dried seaweeds were ground by a grinding
machine, sieved through 0.25 mm pores and stored at −20 °C until use. Fig. 1. Representative scheme of the co-axial electrospinning with a drum
A 16% (w/v) of dried Gracilaria sp. samples were pretreated by 5% (v/ collecting unit.
v) sulfuric acid. The mixture was treated at 121 °C for 30 min in an
autoclave, and the obtained slurry was centrifuged at 5,500 × g for
2.10. Statistical analysis
10 min followed by filtration. The liquid was neutralized with po-
tassium hydroxide until the pH value reached 6.6–6.9, and then filtered
Data were analyzed statistically using SPSS Version 12.0 (SPSS Inc.,
to remove the precipitate. After acid hydrolysis, the residual salts and
Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to
organic acids in Gracilaria sp. hydrolysate was removed by electro-
determine statistical differences between sample means, with the level
dialysis (Micro Acilyzer S3, Astom, Tokyo, Japan) until the salinity
of significance set at p < 0.05. Multiple comparisons of means were
dropped to 7 g/100 g.
done by Duncan test. All data are expressed as mean ± SD.

2.8. Repeated-batch fermentation of Gracilaria sp. hydrolysate using
immobilized Lb. acidophilus
For lactic acid fermentation, the PLLA MTAM-immobilized Lb.
acidophilus were placed into 100 mL of Gracilaria sp. hydrolysate con-
taining 1% (w/v) yeast extract and 0.2 M sodium acetate at 30 °C. At
the end of each fermentation cycle (48 h for each batch), the PLLA
MTAM-immobilized Lb. acidophilus cells were washed with sterile
peptone water and then transferred to the similar volume of fresh hy-
drolysate for the next cycle of fermentation. The cycles were repeated
over and over until the production of lactic acid was not efficient. The
glucose and lactic acid fermentation was measured at 0, 12, 24, 36 and
48 h during each cycle of fermentation. The parameters for lactic acid
fermentation were shown in CLA (Lactic acid concentration), r PLA
(Lactic acid production rate) and YL/S (Lactic acid yield) [10]. CLA (g/L)
is the maximum lactic acid concentration to be achieved during fer-
mentation; r PLA (g/L∙h) is that CLA divided by the time (h) consumed to
reach CLA; YL/S (g/g) is obtained by dividing total produced lactic acid
during fermentation (g) by total sugar consumed during fermentation
(g).
2.9. Analyses of glucose and lactic acid using HPLC
3. Results and discussion
3.1. Encapsulation of Lb. acidophilus in PLLA-MTAM
MTAM consists of a single layer of hollow tubes, each with a dia-
meter of approximately 40 μm and a hollow tube wall pore diameter of
approximately 30 nm [30]. Lactic acid bacterium Lb. acidophilus is ob-
ligately homofermentative, and producing mainly lactic acid from a big
variety of sugars, such as cellobiose, sucrose, lactose, glucose, galactose,
maltose, and mannose [31]. The representative scheme for PLLA-
MTAM preparation using a co-axial electrospinning step with a drum
collecting unit was shown in Fig. 1. The detailed procedures for in situ
and siphon preparation of bacteria-containing PLLA-MTAM was de-
scribed in Section 2.2. Free Lb. acidophilus cells and MTAM-immobilized
Lb. acidophilus cells are shown in Fig. 2a and b. PLLA-MTAM entrapped
and immobilized the bacterial cells 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 [32], suggesting that PLLA-
MTAM is thermostable at all known lactic acid fermentation tempera-
tures. Furthermore, PLLA is insoluble in water and its solubility in
solvents is highly dependent on the molar mass and degree of crystal-
linity [32].

The glucose (g) and lactic acid (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 H2SO4 eluent at a flow rate of
0.6 mL/min at 60 °C. All the tested samples were filtered through a
0.22 μm membrane filter prior to the HPLC analysis.
3.2. Effects of cell numbers on encapsulation efficiency
Two approaches, in situ and siphon preparation, were tested for
MTAM-immoblized lactic acid bacteria encapsulation efficiencies. As
shown in Table 1, encapsulation of lactic acid bacteria at cell densities

3
C.-C. Chen, et al.
Fig. 2. The light microscopy images of (a) free Lb. acidophilus and (b) MTAM-immobilized Lb. acidophilus prepared by siphon method.
Table 1
The effects of encapsulation methods and cell numbers on encapsulation effi-
ciency.
Cell numbers (CFU/mL) Encapsulation efficiency (%)
In situ preparation Siphone
108 N.D. 90.0 ± 20.7%a
109 N.D. 85.2 ± 8.8%a
N.D.: Not detected.
Data are expressed as mean ± SD (n = 3). Different letters in the same row
show significant differences (p < 0.05).
of 108 and 109 cfu/mL by siphon methods showed encapsulation effi-
ciencies of 90.0 ± 20.7% and 85.2 ± 8.8%, respectively. The data
showed no statistical differences in encapsulation efficiency between
the two cell densities, although a cell density of 109 CFU/mL produced
a smaller standard deviation compared with a cell density of 108 CFU/
mL. Intriguingly, lactic acid bacteria immobilized by in situ preparation
were not observed on MRS plates, indicating that the cells were either
in a viable but non-culturable state or were inactivated by the 5–7 kV
voltage applied during in situ preparation. Rodrigo, et al. [33] indicated
that such a high-voltage environment could cause cell inactivation, cell
membrane damage, or even apoptosis in lactic acid bacteria. Potha-
kamury, et al. [34] reported that an electric field intensity of 16 kV/cm
or greater is usually sufficient to reduce the viability of Gram-positive
bacteria by 3–4 log cycles. Our data indicate that the siphon method
results in a better encapsulation efficiency than in situ preparation
methods.
3.3. Effects of pore-forming agent PEG in MTAM preparation on lactic acid
fermentation
Mass transfer limitation can sometimes occur in immobilized cell
systems and may reduce the supply of nutrients and oxygen and cause
the accumulation of inhibitory metabolites, which results in a decrease
in cell growth or low production efficiencies. PLLA-MTAM has a na-
nopore diameter of approximately 30 nm on the surface of each hollow
tube. The addition of 10% (v/v) pore-forming agent PEG in PLLA-
MTAM preparation could produce nanopores with greater diameters of
0.31 ± 0.08 μm [30]. Hung, et al. [35] indicated that the permeation
of glucose and protein bovine serum albumin across the lumen wall (in
and out) is significantly higher in MTAMs that were formed with higher
percentage of porogen PEG, suggesting that the increase amount of
porogenic material used would directly result in a greater diffusion of
material across the lumen wall, most likely due to a greater porosity.
We thus determined whether the addition of pore-forming agent PEG in
the MTAM preparation would accelerate sugar consumption and lactic
4
Process Biochemistry xxx (xxxx) xxx–xxx
acid production, possibly because of greater mass diffusion across the
barrier. We used 10% (v/v) polyethylene glycol as a pore-forming agent
in the preparation of PLLA-MTAM to generate greater porosity with
nanopore diameters of 0.31 ± 0.08 μm on the surface of the MTAM
hollow tube wall [30]. As shown in Table 2, Lb. acidophilus immobilized
with more porous MTAM exhibited a CLA of 23.87 ± 0.04 g/L, r PLA of
0.50 ± 0.00 g/L∙h, and YL/S of 0.93 ± 0.03 (g/g) during fermentation,
and the CLA and the r PLA of the Lb. acidophilus immobilized with more
porous MTAM were statistically higher than the control group. In im-
mobilized systems, mass transfer limitation could sometimes reduce the
cell growth and therefore reduce fermentation efficiency. Our data in-
dicated that the bacteria encapsulated in more porous MTAM exhibited
better fermentation efficiency, making it a better candidate for bacterial
immobilization. Hence, we used 10% (v/v) polyethylene glycol to
create MTAM-immobilized Lb. acidophilus for the rest of the study.
3.4. Effect of buffering agent on Lb. acidophilus growth and batch lactic
acid fermentation
Lactic acid bacteria produce acid (mainly lactic acid) during growth,
which lowers the pH value of the media and thus slows down or inhibits
growth [36]. Therefore, in lactic acid fermentation, buffering agents
used to stabilize the pH of the fermentation substrate are very im-
portant. Sodium acetate, trisodium citrate, and disodium phosphate are
essential buffering agents that are commonly used in commercial cul-
ture media for lactic acid bacteria, such as MRS and M17. To determine
the effects of different buffering agents, Lb. acidophilus was incubated in
MRS medium containing 4% (w/v) glucose with, separately, 0.2 M
disodium phosphate, 0.1 M trisodium citrate, or 0.2 M sodium acetate.
As shown in Fig. 3, lactic acid bacteria grown in MRS medium with
0.1 M trisodium citrate or 0.2 M sodium acetate exhibited superior
sugar usage and lactic acid production as compared with the control.
The control, consisting of lactic acid bacteria grown in MRS containing
4% (w/v) glucose with no extra buffering agents, used 82.88% glucose
and produced 28.00 g/L lactic acid with a final pH of 3.47 in 72 h
Table 2
The effect of porosity of MTAM on lactic acid fermentation in 48 h.
CLA(g/L) r PLA (g/L h) YL/S (g/g)
Siphon
MTAM with greater porosity 23.87 ± 0.04a 0.50 ± 0.00a 0.93 ± 0.03a
MTAM 22.93 ± 0.34b 0.48 ± 0.01b 0.92 ± 0.02a
CLA (Lactic acid concentration); r PLA (Lactic acid production rate); YL/S (Lactic
acid yields).
Data are expressed as mean ± SD (n = 3). Different letters in column show
significant differences (p < 0.05).
C.-C. Chen, et al.
Fig. 3. Effects of buffering agents (a) none, (b) 0.2 M disodium phosphate, (c) 0.1 M trisodium citrate and (d) 0.2 M sodium acetate on the batch fermentation of MRS
using free Lb. acidophilus. Batch fermentation of MRS medium containing 4% (w/v) glucose was incubated at 30 °C. Data are expressed as mean ± SD (n = 3).
(Fig. 3a). Lactic acid bacteria grown in MRS with 0.2 M disodium
phosphate consumed up to 63.52% of available glucose and produced
lactic acid at a concentration of 23.68 with a final pH of 4.33 at 72 h
(Fig. 3b). Lactic acid bacteria grown in MRS with 0.1 M trisodium ci-
trate consumed 88.98% of available glucose and produced lactic acid at
a concentration of 30.28 g/L with a final pH of 4.09 at 72 h (Fig. 3c).
Lactic acid bacteria grown in MRS with 0.2 M sodium acetate consumed
all sugars (100% sugar usage) and produced lactic acid within 36 h, and
gave the best lactic acid concentration at 32.42 g/L with a pH of 4.05 at
36 h (Fig. 3d). Other groups failed to consume all of the available
glucose even after 72 h fermentation (Fig. 3a–c). Similar results were
obtained in a previous report in which lactobacilli grew more ex-
tensively when acetate was present because an internal pH > 4.4 was
maintained for a longer period when no sodium acetate was added
[37]. Iino, et al. [38] investigated the lactic acid fermentation by 49
strains of lactic acid bacteria under various buffer conditions, including
sodium acetate, disodium phosphate and sodium citrate buffers; it was
concluded that the effects of buffer agents for lactic acid fermentation
could be various between different species, and Lb. sakei could produce
twice or more lactic acid in the presence of sodium acetate, suggesting
sodium acetate might possibly play not only the role of a buffer but also
other roles in the production of lactic acid. Intriguingly, it was shown
that sodium acetate could change the activities of some metabolic en-
zymes, therefore changing the production yield and of lactic acid [39].
Addition of higher concentration disodium phosphate may chelate
metal ions essential for bacterial growth and/or cell integrity [40]. The
Gram-positive bacteria Geobacillus stearothermophilus was reported to be
less resistant to heat as the concentration of phosphate buffer increased
5
Process Biochemistry xxx (xxxx) xxx–xxx
[41]. This might be the reason that the lactic acid production by Lb.
acidophilus was not improved in the presence of 0.2 M disodium phos-
phate. In the present study, sodium acetate was chosen as a buffering
agent to control pH changes during lactic acid fermentation using
MTAM-immobilized Lb. acidophilus, and the optimal sodium acetate
concentration (0.2 M) was also determined (data not shown).
3.5. Repeated-batch lactic acid fermentation of MRS using MTAM-
immobilized Lb. acidophilus
Next, glucose consumption and lactic acid production in repeated-
batch fermentation using MTAM-immobilized Lb. acidophilus were stu-
died (Fig. 4). Only the first batch of MTAM-immobilized cells had a lag
phase of about 12 h at the beginning of fermentation. In batches II to
VIII, the same MTAM-immobilized lactic acid bacteria were reused, and
it was shown that the glucose consumption, final lactic acid con-
centration, and fermentation rate of each batch were higher than those
of the first batch. Our attempt to encapsulate Lb. acidophilus in MTAM
for repeated-batch lactic acid fermentation successfully reduced the
processing cost of inoculum preparation and enhanced glucose con-
sumption and lactic acid production. PLLA is highly hydrophobic ma-
terial, with a slow degradation rate, which sometimes limits its use in
certain applications [42]. The degradation of PLLA is a hydrolysis
process, and the hydrolysis rate depends on a number of physical
characteristic, such as molecular weight, morphology, crystallinity, and
water diffusion rate into the polymer [43]. The PLLA-MTAM used in
this study made of the PLLA with over 140 kDa weight and had a
morphology of single layer sheet, which would have an even slower
C.-C. Chen, et al. Process Biochemistry xxx (xxxx) xxx–xxx

Fig. 4. The sugar consumption and lactic acid production in repeated batch Fig. 5. The sugar consumption and lactic acid production in repeated batch
fermentation of MRS broth using immobilized Lb. acidophilus. Repeated-batch fermentation of Gracilaria sp. hydrolysate using immobilized Lb. acidophilus.
fermentation of MRS medium containing 4% (w/v) glucose was incubated at Repeated-batch fermentation of Gracilaria sp. acid hydrolysate was incubated at
30 °C. Data are expressed as mean ± SD (n = 3). 30 °C. Data are expressed as mean ± SD (n = 3).

degradation rate [43]. Our observation indicated that the PLLA-MTAM repeated-batch fermentation, with a slight reduction in the last cycle.
showed it physical stability during the repeated-batch lactic acid fer- Lactic acid productivity began to decrease in batch VIII, possibly be-
mentation for eight consecutive cycles. cause of lactic acid bacteria cell aging [47].
An evaluation of the kinetic parameters of lactic acid production
using free cells in fermentation and MTAM-immobilized Lb. acidophilus
3.6. Repeated-batch lactic acid fermentation of Gracilaria sp. hydrolysate
in repeated-batch fermentation is shown in Table 3. MTAM-im-
using MTAM-immobilized Lb. acidophilus
mobilized cells in batches II to VIII showed improved performance in
terms of sugar consumption and lactic acid production compared with
Seaweeds, which contain high amounts of polysaccharides and do
free cells. Lactic acid fermentation using free Lb. acidophilus cells
not affect food supply, forests, or arable lands, offer a suitable carbon
showed an overall glucose consumption of 90.8%, CLA of
source for lactic acid fermentation [48]. Seaweeds, which contain high
35.47 ± 0.07 g/L, r PLA of 0.74 ± 0.00 g/L∙h, and YL/S of
amounts of polysaccharides and do not affect food supply, forests, or
0.95 ± 0.01 g/g during 48 h of fermentation. Immobilized cells from
arable lands, offer a suitable carbon source for lactic acid fermentation
batches II to VII exhibited better lactic acid productivity than free cells
[48]. Chemical proximate analyses indicated that dried red seaweeds
and batch I with a glucose consumption of more than 94%, CLA of more
Gracilaria species could have 5 − 15% moisture, 57 − 80% carbohy-
than 37 g/L, and r PLA between 0.77 and 0.79 g/L∙h in each batch. An
drate, 1 − 23% proteins, 0.3 − 2% Lipid, and 5 − 29% ash [49–53].
increase in the glucose consumption of immobilized cells prepared by
The dried Gracilaria used in this study had a carbohydrate content of
the siphon method indicates a more efficient utilization of nutrients in
79% (data not shown), making it a great candidate for lactic acid fer-
the fermentation substrate relative to free cells. Zhao, et al. [9] im-
mentation. At present, the red alga Gracilaria sp. is the only alga to be
mobilized Lactobacillus rhamnosus in novel mesoporous silica-based
cultivated on a large scale in Taiwan. In the present work, Gracilaria sp.
materials with a immobilization efficiency of 78.77%, and the im-
hydrolysate was used under optimum conditions for repeated-batch
mobilized cells exhibited an elevated glucose conversion yield of 92.4%
fermentation to produce lactic acid. As shown in Fig. 5, MTAM-im-
as compared to the free cells. The greater lactic acid productivity ex-
mobilized Lb. acidophilus were reused in lactic acid fermentation of
hibited by MTAM-immobilized Lb. acidophilus may be due to the higher
Gracilaria sp. hydrolysate for eight straight batches, and the sugar
cell density in the unit space of the carrier. The same observations have
consumption and lactic acid production in repeated-batch fermentation
also been documented in many previous reports on the production of
of Gracilaria sp. hydrolysate were monitored. A gradual increase in
lactic acid by fermentation [44–46]. Overall, the immobilized cells
lactic acid concentration and sugar consumption was observed in the
exhibited excellent stability and metabolic activity over 2–7 cycles of
repeated-batch lactic acid fermentation.
Fermentative parameters, used to evaluate the kinetics of lactic acid
Table 3
production from Gracilaria sp. hydrolysate using free Lb. acidophilus in
Evaluation of kinetic parameters for lactic acid production by free cells in batch
batch fermentation and MTAM-immobilized Lb. acidophilus in repeated-
fermentation of MRS and MTAM-encapsulated Lb. acidophilus in repeated-batch
fermentation of MRS. batch fermentation, are shown in Table 4. The overall sugar con-
sumption (69–74%) of Lb. acidophilus in the lactic acid fermentation of
Batch Glucose CLA (g/L) r PLA (g/L∙h) YL/S (g/g sugar) Gracilaria sp. hydrolysate was much lower than that in the lactic acid
consumption
(%)
fermentation of commercial medium MRS. The reason for this could be
that the presence of inhibitors, such as furans, which were generated
Free cells 90.80d 35.47 ± 0.07c 0.74 ± 0.00c 0.95 ± 0.01a during acid hydrolysis of Gracilaria sp., may have retarded or inhibited
I 89.73e 34.94 ± 0.11d 0.73 ± 0.00d 0.93 ± 0.01a cell growth [28,54]. In this study, it was determined by using HPLC
II 99.93a 37.90 ± 0.22a 0.79 ± 0.00a 0.90 ± 0.00b
III 94.70c 37.12 ± 0.06ab 0.77 ± 0.00ab 0.93 ± 0.01a
analysis that 2.19 ± 0.40 g/L of 5-hydroxymethylfurfural was in the
IV 94.29bc 37.38 ± 0.15a 0.78 ± 0.00a 0.94 ± 0.01a hydrolysate of Gracilaria sp. (data not shown). Another possibility is
V 95.82b 37.29 ± 0.13a 0.78 ± 0.00a 0.94 ± 0.01a that sulfated galactose from galactans in red seaweed Gracilariai sp.
VI 95.94b 37.20 ± 0.52a 0.78 ± 0.01a 0.94 ± 0.02a [55,56] might not be utilized by lactic acid bacteria. Sulfated galactans
VII 95.82b 37.39 ± 0.10a 0.78 ± 0.00a 0.92 ± 0.00a
were reported abundant in red seaweeds, and the structural and sul-
VIII 94.53c 36.82 ± 0.14b 0.77 ± 0.00b 0.93 ± 0.03a
fation contents variations occur among the polysaccharides obtained
CLA (Lactic acid concentration); r PLA (Lactic acid production rate); YL/S (Lactic from different species and collected in different periods of the year or
acid yields). Data are expressed as mean ± SD (n = 3). Different letters in from different regions [56,57]. Chance and Mawhinney [58] indicated
column show significant differences (p < 0.05). that bacteria grew slowly on a carbon source of sulfated

6
C.-C. Chen, et al. Process Biochemistry xxx (xxxx) xxx–xxx

Table 4 seaweed biomass and may provide an alternative approach to cell im-
Evaluation of kinetic parameters for lactic acid production by free cells in batch mobilization for cell-free lactic acid production.
fermentation of Gracilaria sp. hydrolysate and MTAM-encapsulated Lb. acid-
ophilus in repeated-batch fermentation of Gracilaria sp. hydrolysate. Acknowledgements
Batch Substrate CLA (g/L) r PLA (g/L∙h) YL/S (g/g sugar)
utilization This work was supported by the Center of Excellence for the Oceans,
(%) National Taiwan Ocean University from The Featured Areas Research
Free cells 69.20c 25.73 ± 0.25e 0.54 ± 0.01e 0.92 ± 0.03a
Center Program within the framework of the Higher Education Sprout
I 69.69c 25.80 ± 0.35e 0.54 ± 0.01e 0.90 ± 0.02ab Project by the Ministry of Education (MOE) in Taiwan (NTOU-RD-AA-
II 69.20c 25.84 ± 0.10de 0.54 ± 0.00de 0.92 ± 0.02a 2019-1-02011-2). This work was supported by the grants from the
III 69.44c 26.16 ± 0.08cde 0.55 ± 0.00cde 0.92 ± 0.01a Ministry of Science and Technology (Taiwan, R.O.C., MOST 105-2221-
IV 70.55bc 26.68 ± 0.15bc 0.56 ± 0.00bc 0.92 ± 0.00a
E-019-075 and 106-2221-E-019-065) and from Shu-Jih Education
V 72.23ab 26.48 ± 0.21bcd 0.55 ± 0.00bcd 0.89 ± 0.01b
VI 72.69ab 26.94 ± 0.41b 0.56 ± 0.01b 0.90 ± 0.01ab Foundation (TIARF106A004).
VII 73.65a 27.76 ± 0.64a 0.58 ± 0.01a 0.92 ± 0.01a
VIII 74.05a 27.58 ± 0.58a 0.57 ± 0.01a 0.90 ± 0.01ab References

CLA (Lactic acid concentration); r PLA (Lactic acid production rate); YL/S (Lactic
acid yields). Data are expressed as mean ± SD (n = 3). Different letters in
column show significant differences (p < 0.05).
monosaccharides. Lactic acid fermentation using free Lb. acidophilus
cells showed a sugar consumption of 69.20%, CLA of 25.73 ± 0.25 g/L,
r PLA of 0.54 ± 0.01 g/L∙h, and YL/S of 0.92 ± 0.03 g/g with Gracilaria
sp. hydrolysate. For immobilized cells, the CLA increased from
25.80 ± 0.35 g/L in the first batch to 27.76 ± 0.64 g/L in the seventh
batch, and the YL/S ranged from 0.89 g/g to 0.92 g/g, respectively. The
highest sugar consumption of 74.05% and the fastest r PLA of
0.58 ± 0.01 g/L∙h was obtained in batch VII. MTAM-immobilized cells
showed better efficacy than free cells in terms of sugar consumption
and lactic acid production in all Gracilaria sp. hydrolysate batches, al-
though in some cases there were no statistical differences.
Although the mechanisms of immobilization on microbial activity
remains elusive, some possible explanations for enhanced metabolites
production have been made in the previous studies. Some reports have
suggested that cell immobilization might re-arrange the intracellular
pools of metabolites, thus increasing the productivity of a certain me-
tabolite [13,59,60]. For example, Sawai, et al. [61] indicated that
continuous fermentation by Sporolactobacillus using the membrane-in-
tegrated fermentation reactor system significantly enhanced both vo-
lumetric productivity and optical purity (99.8%) for D-lactic acid. It has
been reported that the expressions of metabolic enzymes were en-
hanced in immobilize cells as compared to the suspended micro-
organisms [62,63]. In addition, the immobilized cells exhibited better
tolerance against changing environments and more resistant against
toxic substances during fermentation. Locally higher cell density might
be an advantage for immobilized cells on cell growth in the presence of
inhibitors. Nevertheless, increased resistance of immobilized cells
against toxic substances could be attributed to changes in cell mem-
brane composition and therefore cell permeability [64]. In this study,
the MTAM-immobilized cells showed excellent stability in fermentation
over eight consecutive batches (data not shown) while product lactic
acid yields remained relatively high. These results indicate that MTAM
is a potential immobilization technology for lactic acid bacteria that
could be applied to repeated-batch lactic acid fermentation of red alga
Gracilaria sp. hydrolysate.
4. Conclusion
Here, we provide a novel cell immobilization technique for lactic
acid production by taking advantage of the properties of PLLA-MTAM, a
renewable, low-cost, and easily manipulated microporous membrane.
PLLA-MTAM was evaluated in terms of cell encapsulation efficiency,
immobilization technique, and stability, and our data indicate that
MTAM immobilization enhanced lactic acid bacteria in the production
of lactic acid. This technology showed great potential for repeated-
batch lactic acid fermentation of both commercial medium and
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