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JOURNAL OF BIOSCIENCE AND BIOENGINEERING

Vol. 99, No. 2, 104108. 2005


DOI: 10.1263/jbb.99.104

2005, The Society for Biotechnology, Japan

Recovery of Lactic Acid by Repeated Batch Electrodialysis and


Lactic Acid Production Using Electrodialysis Wastewater
YOUNG-JUNG WEE,1 JONG-SUN YUN,2 YOON Y. LEE,3
AN-PING ZENG,4 AND HWA-WON RYU1*
School of Biological Sciences and Technology, Chonnam National University, 300 Yongbong-dong, Buk-gu,
Gwangju 500-757, Korea,1 BioHelix, Noan-myeon, Naju, Jeonnam 520-811, Korea,2 Department of
Chemical Engineering, 230 Ross Hall, Auburn University, Auburn, AL 36849-5127, USA,3 and
Biochemical Engineering Division, GBF-German Research Center for Biotechnology,
Mascheroder Weg 1, D-38124 Braunschweig, Germany4
Received 17 August 2004/Accepted 4 November 2004

Repeated batch electrodialysis for lactic acid recovery was investigated using lactic acid solution and fermentation broth. In both cases, lactate fluxes averaged more than 7.0 moles/m2 h, lactate recovery reached more than 99% for all the batch runs, and specific energy consumption per
unit lactate transported was lower than 0.25 kWh/kg-lactate. When electrodialysis wastewater
was used as a fermentation medium, supplemented with 100 g/l glucose, up to 92.4 g/l lactic acid
was produced with a productivity of 0.67 g/l h. In addition, when electrodialysis wastewater was
supplemented with 150 g/l whole-corn flour hydrolyzate and 5 g/l corn steep liquor, 2.5-fold and
1.8-fold increases in lactic acid productivity and maximum cell growth, respectively, were achieved,
as compared with lactic acid fermentation using electrodialysis wastewater supplemented with
glucose only.
[Key words: electrodialysis, lactic acid, Enterococcus faecalis, lactic acid fermentation, electrodialysis wastewater]

a high biological oxygen demand (BOD, >1000 ppm) (11).


Therefore, if the electrodialysis wastewater can be used for
subsequent fermentations, costs for lactic acid production
and wastewater disposal can be simultaneously reduced. To
our knowledge, although many studies of electrodialysis for
lactic acid recovery has been conducted (810, 1214), the
evaluation of lactic acid fermentation using electrodialysis
wastewater has rarely been investigated.
The aim of this work was to investigate the possibility of
using electrodialysis wastewater for subsequent lactic acid
fermentation. To this end, we first attempted to isolate lactic
acid from lactic acid solution and fermentation broth by a
repeated batch electrodialysis. The possibility of using a
10-compartment cell stack, equipped with commercial ionexchange membranes for the recovery of lactic acid, was
also tested using a laboratory scale electrodialyzer with the
lactic acid solution and fermentation broth.

Lactic acid has a long history of usage in the food industry as a preservative and acidulant, and now it has wide applications in the fields of food, pharmaceutical, medical,
and chemical industries (13). Lactic acid is generally considered as a fundamental chemical, due to its high reactivity
that stems from having both carboxyl and hydroxyl groups,
by which lactic acid can be converted into several potential
chemicals, such as propylene glycol, acetaldehyde, acrylic
acid, 2,3-pentanedione, propanoic acid, and polylactic acid
(PLA) (4, 5). Recently, demand for lactic acid has been
increasing considerably, due to its new application as the
monomer of PLA that can be used for the production of
biodegradable and compostable plastics (5, 6). In general,
lactic acid can be manufactured by either microbial fermentation or chemical synthesis. The chemical synthesis
produces both D- and L-lactic acids, but microbial fermentation is able to selectively produce an optically active L(+)or D(-)-lactic acid (5, 7).
Fermentation-derived lactic acid can be separated by
several recovery processes, such as calcium precipitation,
solvent extraction, and electrodialysis (2, 5). Among them,
electrodialysis has been found to be potentially useful in the
recovery of lactic acid from fermentation broth (5, 810).
However, the treatment of wastewater, generated after the
electrodialysis operation, may result in an increase in lactic
acid production costs, because this wastewater generally has

MATERIALS AND METHODS


Microorganism Enterococcus faecalis RKY1 (7, 1517), a
homofermentative L(+)-lactic acid producer, was used in this investigation. Stock cultures were maintained at -20C in 5 ml vials
containing 50% (v/v) glycerol until use.
Inoculum preparation The medium for inoculum preparation (preculture medium) contained the following components
(g/l): glucose, 30; yeast extract, 10; and K2HPO4, 5. To prepare the
inoculum, cells from stock cultures were transferred to 15 ml of the
preculture medium in a 20 ml vial, and then incubated at 38C for
12 h. After three consecutive transfers, 2 ml of the final culture was

* Corresponding author. e-mail: hwryu@chonnam.ac.kr


phone: +82-62-530-1842 fax: +82-62-530-1869
104

VOL. 99, 2005

RECOVERY AND PRODUCTION OF LACTIC ACID

105

TABLE 1. Properties of ion-exchange membranes used


Properties
Type
Characteristics
Material
Electric resistance (0.5 M NaCl)
Burst strength
Thickness
Temperature range allowance
pH range allowance

Membranes
AMX
Strongly basic anion permeable
High mechanical strength (Na-form)
Styrene-divinyl benzene copolymer
2.03.5 W/cm2
450550 kPa
0.140.18 mm
040C
010

transferred to 40 ml of the preculture medium in a 50 ml vial, and


then incubated at 38C for 6 h in a shaking incubator (KMC8480SF; Vision Scientific, Daejon, Korea) set to 200 rpm, before
inoculation at 4% (v/v) in the fermentor.
Fermentation broth for electrodialysis To prepare a fermentation broth for electrodialysis, batch lactic acid fermentation
was performed in a 2.5-l fermentor (KF-2.5L; Kobiotech, Incheon,
Korea) containing 1 l of the fermentation medium, composed of
100 g/l glucose and 15 g/l yeast extract. The fermentation was
carried out at 38C and 200 rpm, and culture pH was automatically
controlled at 7.0 by adding 10 M NaOH. After the completion of
fermentation, cells were removed by centrifugation at 22,300 g
using a Supra 21K centrifuge (Hanil Science Industrial, Incheon,
Korea).
Electrodialysis The electrodialyzer consisted of a 10-compartment cell stack (TS-1-10; Tokuyama, Tokyo), a DC power supply (PE 1646; Phillips, Brussels, Belgium), three magnet pumps
(MD-15R; Iwaki, Tokyo), and three holding tanks made of acrylic
resin. The electrodialysis stack was separated into the feed, permeate, and electrolyte compartments by ion-exchange membranes.
The membranes used in these experiments were Neosepta anionexchange membrane (AMX; Tokuyama) and cation-exchange
membrane (CMX; Tokuyama), and the properties of these membranes are shown in Table 1. The effective surface area of each
membrane was 100 cm2. A platinum wire was installed at each end
of the stack to measure stack voltage. The anode and cathode were
made of platinum-plated titanium and stainless steel, respectively.
The lactic acid solution (Daejung Chemicals & Metals, Incheon,
Korea) and fermentation broth were used as feed solutions with 2 l
of the initial volume, and the initial volume of permeate solution
was 1 l. The electrode rinse solution of 3% (w/v) Na2SO4 with 2 l
of working volume was circulated through each electrode compartment. The flow rates of feed, permeate, and electrode rinse solutions were set to 0.8 l/min, and the operational temperature was
controlled at 32C using a counter current heat exchanger. Repeated batch electrodialysis was operated in a constant voltage
mode. Once feed conductivity decreased to lower than 10 mS/cm,
the entire feed solution was withdrawn from the holding tank, and
2 l of fresh feed solution was then fed into that holding tank. The
subsequent repeated batch electrodialysis was carried out by the
same method described above. After each experiment, membranes
were cleaned by circulating 0.05 M HCl solution for 20 min, followed by circulating 0.05 M NaOH solution, and then flushed with
distilled water until the pH of each solution was 7.0.
Batch fermentation using electrodialysis wastewater To
evaluate the possibility of using electrodialysis wastewater for
lactic acid fermentation, batch fermentation using electrodialysis
wastewater supplemented with 100 g/l glucose was performed on a
2.5-l fermentor (Kobiotech). In addition, whole-corn flour hydrolyzate and corn steep liquor were supplemented to the electrodialysis wastewater to enhance fermentation efficiency. Electrodialysis wastewater containing glucose or whole-corn flour hydrolyzate was sterilized by filtration through 0.22 mm nitrocellulose

CMX
Strongly acidic cation permeable
High mechanical strength (Cl-form)
Styrene-divinyl benzene copolymer
2.03.5 W/cm2
500600 kPa
0.160.20 mm
040C
010

filter paper (Millipore, Bedford, MA, USA).


The whole-corn flour hydrolyzate was prepared by enzymatic
treatment of whole-corn flour. The enzymes used here were Termamyl
120L (a-amylase, 120 KNU/g; Novo Nordisk A/S, Bagsvaerd, Denmark) and AMG 300L (glucoamylase, 250 AGU/g; Novo Nordisk
A/S). The milled corn flour was suspended in electrodialysis wastewater, and the pH of this suspension was adjusted to 6.0. a-Amylase (300 mg) was added to the suspension, and the mixture was
reacted at 95C for 30 min. The resulting solution was cooled to
lower than 60C, the pH was adjusted to 4.5, and 300 mg of glucoamylase was added. This saccharification step was aseptically carried out in a 2-l Erlenmeyer flask at 55C for 24 h with shaking at
200 rpm. The hydrolyzed solution was sieved through a 0.125 mm
screen, and then filtered through Whatman no. 1 filter paper. The
volume of this hydrolyzate was adjusted to 960 ml, which was then
used for the fermentation medium.
Analytical methods
Cell growth was measured by a UV160A spectrophotometer (Shimadzu, Kyoto) at a wavelength of
660 nm, which was then converted to dry cell weight by calculation using a calibration curve related to optical density and dry cell
weight. One unit of optical density corresponded to 0.8 g dry cell
weight. Lactic acid was analyzed using a Waters high-performance
liquid chromatography system (Millipore), equipped with a Waters
486 tunable absorbance detector set to 210 nm. An Aminex HPX87H ion-exclusion column (300 7.8 mm; Bio-Rad, Hercules, CA,
USA) was used with 5 mM H2SO4 as a mobile phase, at a flow rate
of 0.6 ml/min, while the column temperature was maintained at
35C. Glucose was enzymatically measured by the glucose oxidase-peroxidase method using a Glucose-E kit (YD Diagnostics,
Seoul, Korea).
During electrodialysis, stack voltage and conductivity were
measured using a digital multimeter (model 45; Fluke, Everett,
WA, USA) and conductivity meter (HI 9635; Hanna Instruments,
Woonsocket, RI, USA), respectively. The recovery of lactic acid
and current efficiency were calculated using the following equations:
C P , fVP , f C P , iVP , i
R = ----------------------------------------- 100
C F , iVF , i CF , fVF , f
F ( C C , fV C , f C C , i V C , i )
h = ----------------------------------------------------- 100
n Idt

where R is the rate of recovery of lactic acid (0 R 100), h is


current efficiency (0 h 100), F is the Faraday constant (96,485
C/mole), Cp, f is the final lactate concentration of permeate solution, Vp, f is the final volume of permeate solution, Cp, i is the initial
lactate concentration of permeate solution, Vp, i is the initial volume
of permeate solution, CF, i is the initial lactate concentration of feed
solution, VF, i is the initial volume of feed solution, CF, f is the final
lactate concentration of feed solution, VF, f is the final volume of
feed solution, n is the number of cell pairs, I is current, and t is
time.

106

J. BIOSCI. BIOENG.,

WEE ET AL.

RESULTS AND DISCUSSION


Repeated batch electrodialysis using lactic acid solution
Repeated batch electrodialysis using the lactic acid
solution was attempted to maximize the lactic acid concentration in the permeate compartment. The feed and permeate
solutions contained 40 g/l and 0.8 g/l lactic acid, respectively. The electrodialyzer was operated in the constant voltage
mode (15 volts). As shown in Fig. 1, the lactic acid concentration in permeate solution was 146.0 g/l after the fifth
batch run. The rates of recovery of lactic acid averaged 27.4
g/l h, during repeated batch electrodialysis. Current efficiency reached 75% and higher for all but the first batch
run, indicating generally good electrodialysis performance
(Table 2). Lactate flux averaged 7.1 moles/m2 h, and the
maximum lactate flux was 7.9 moles/m2 h in the third batch
run, which was 1.5-fold higher than that of conventional
batch electrodialysis (first batch run). A clear increase in
lactate flux was observed during repeated batch electrodialysis until the third batch run. The rate of recovery of lactic
acid reached 99% and higher for all the batch runs. Specific
energy consumption per unit lactate transported was lower
than 0.3 kWh/kg-lactate. In general, specific energy consumption averaged 0.24 kWh/kg-lactate. These results indicate that the repeated batch operation of electrodialysis is
more advantageous than conventional batch electrodialysis
in terms of electrodialysis performance.
Repeated batch electrodialysis using fermentation
broth
Repeated batch electrodialysis, which was proven
to be an efficient process for lactic acid recovery as described above, was applied to the recovery of lactic acid
from fermentation broth. This fermentation broth contained
approximately 76 g/l lactic acid and was used as feed solution. Permeate solution contained 0.8 g/l lactic acid. Electrodialysis was carried out in the constant voltage mode
(15 volts). The profiles of lactic acid concentration in feed
and permeate streams during repeated batch electrodialysis
are shown in Fig. 2. Lactic acid concentration in permeate
solution reached 166.3 g/l after the third batch run, indicating high performance electrodialysis. Several other studies
showed lower lactic acid concentrations. For example,
Nomura et al. (18) applied immobilized cell culture to electrodialysis and obtained 70 g/l lactic acid, and Boyaval et al.
(13) applied the cell recycle culture system to electrodialysis and obtained 85 g/l lactic acid. Current efficiency reached
70% and higher for all but the third batch run (Table 3),
which is slightly lower than that of the repeated batch electrodialysis using the lactic acid solution. Lactate flux averaged 7.2 moles/m2 h, and the maximum lactate flux was 7.8

FIG. 1. Time course of lactic acid concentration in feed and permeate solutions during repeated batch electrodialysis using lactic acid
solution. Symbols: closed circles, lactic acid in feed solution; open circles, lactic acid in permeate solution.

FIG. 2. Time course of lactic acid concentration in feed and permeate solutions during repeated batch electrodialysis using fermentation broth containing lactic acid solution. Symbols: closed circles, lactic acid in feed solution; open circles, lactic acid in permeate solution.

moles/m2 h in the second batch run, which was 1.2-fold


higher than that of batch electrodialysis (first batch run).
This result is quite similar to that observed in repeated batch
electrodialysis using the lactic acid solution. The rate of recovery of lactic acid reached 100% for all the batch runs.
Specific energy consumption per unit lactate transported
was lower than 0.3 kWh/kg-lactate, which generally averaged 0.25 kWh/kg-lactate. This value is slightly higher than
that of repeated batch electrodialysis using the lactic acid
solution, which might be due to the presence of some impurities in the fermentation broth.

TABLE 2. Performance during repeated batch electrodialysis using lactic acid solution
Batch number
1
2
3
4
5
Average

Lactate flux
(moles/m2 h)

Recovery
(%)

Current efficiency
(%)

Power consumption
(Wh)

5.2
7.4
7.9
7.8
7.4
7.1

97.9
100
100
100
100
99.5

73.4
78.6
80.8
78.9
75.2
78.4

24.2
24.2
25.2
25.8
25.8
25.0

Specific energy
consumption
(kWh/kg-lactate)
0.25
0.23
0.23
0.24
0.25
0.24

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107

TABLE 3. Performance during repeated batch electrodialysis using fermentation broth containing lactic acid
Batch number
1
2
3
Average

Lactate flux
(moles/m2 h)

Recovery
(%)

Current efficiency
(%)

Power consumption
(Wh)

6.6
7.8
7.4
7.2

100
100
100
100

70.5
70.0
67.9
69.5

53.7
53.9
53.7
53.8

Lactic acid fermentation using electrodialysis wastewater Electrodialysis wastewater, generated after the
electrodialysis of fermentation broth, might be expected to
have some moieties of nutrient sources that were not utilized during fermentation. Therefore, we attempted to investigate the possibility of using electrodialysis wastewater
as a fermentation medium for subsequent lactic acid fermentations. To evaluate the amounts of nutrient for lactic acid
fermentation in electrodialysis wastewater, batch lactic acid
fermentation was carried out using electrodialysis wastewater, supplemented with 100 g/l glucose. Figure 3 shows
the time course of the cultivation of E. faecalis RKY1 using
a 2.5-l fermentor containing 1 l of the fermentation medium.
Lactic acid concentration in Fig. 3 indicates the lactic acid
produced during fermentation. As shown in Fig. 3, although
the fermentation was completed after 138 h, lactic acid was
produced at up to 92.4 g/l with a productivity of 0.67 g/l h,
and the maximum dry cell weight reached 3.2 g/l after 60 h
of fermentation. Lactic acid productivity and cell growth,
obtained in this experiment, could be achieved by supplementation with about 3 g/l yeast extract to the fermentation
medium containing 100 g/l glucose, which agrees with the
previous results of our study about the influence of yeast extract concentration on lactic acid fermentation by batch culture of E. faecalis RKY1 (19). This means that electrodialysis wastewater should have some nutrient sources available
to E. faecalis RKY1. Therefore, it can be expected that,
once some nutrients are supplemented to electrodialysis
wastewater, fermentation efficiency should be considerably
enhanced.
To enhance fermentation efficiency, electrodialysis wastewater was supplemented with whole-corn flour hydrolyzate
and corn steep liquor. In this experiment, whole-corn flour

FIG. 3. Lactic acid fermentation using electrodialysis wastewater


supplemented with 100 g/l glucose. Symbols: circles, lactic acid;
squares, glucose; triangles, dry cell weight.

Specific energy
consumption
(kWh/kg-lactate)
0.25
0.25
0.26
0.25

hydrolyzate was prepared by the enzymatic treatment of


150 g/l whole corn, and 5 g/l corn steep liquor was used as a
nitrogen source. As shown in Fig. 4, 67% glucose (100 g/l)
was released from 150 g/l whole corn. Lactic acid concentration shown in Fig. 4 indicates the lactic acid produced
during fermentation. After 57 h of fermentation, lactic acid
was produced at up to 94.2 g/l with a productivity of 1.65
g/l h, and maximum dry cell weight reached 5.7 g/l. Lactic
acid productivity and maximum cell growth, obtained in this
experiment, were 2.5-fold and 1.8-fold higher, respectively,
than those of a former investigation presented in Fig. 3. On
the basis of glucose consumed, lactic acid yield was determined to be 0.93 g/g. This result suggests that electrodialysis wastewater is suitable as a supplement of the fermentation medium.
Tejayadi and Cheryan (20) reported previously that, in
lactic acid fermentation using whey and yeast extract, raw
material cost was 68% of the total production cost of lactic
acid. kerberg and Zacchi (21) also reported that, through
the simulation study of lactic acid fermentation process, operating costs including raw materials, neutralizing agents,
hydrolyzing enzymes, and membranes for electrodialysis
comprised approximately 80% of the total production costs
of lactic acid from wheat flour. In addition, the wastewater
treatment step should increase overall operating costs for
lactic acid fermentation. Therefore, the results presented in
this paper should provide helpful information for the reduction of production costs of lactic acid and for the decrease in
environmental pollution.

FIG. 4. Lactic acid fermentation using electrodialysis wastewater


supplemented with 150 g/l whole-corn flour hydrolyzate and 5 g/l corn
steep liquor. Symbols: circles, lactic acid; squares, glucose; triangles,
dry cell weight.

108

J. BIOSCI. BIOENG.,

WEE ET AL.

ACKNOWLEDGMENTS
This study was financially supported by Chonnam National
University in Prof. Ryus sabbatical year of 2002.

REFERENCES
1. Davison, B. E., Llanos, R. L., Cancilla, M. R., Redman,
N. C., and Hillier, A. J.: Current research on the genetics of
lactic acid production in lactic acid bacteria. Int. Dairy J., 5,
763784 (1995).
2. Litchfield, J. H.: Microbiological production of lactic acid.
Adv. Appl. Microbiol., 42, 4595 (1996).
3. Hofvendahl, K. and Hahn-Hgerdal, B.: Factors affecting
the fermentative lactic acid production from renewable resources. Enzyme Microb. Technol., 26, 87107 (2000).
4. Varadarajan, S. and Miller, D. J.: Catalytic upgrading of
fermentation-derived organic acids. Biotechnol. Prog., 15,
845854 (1999).
5. Datta, R., Tsai, S. P., Bonsignore, P., Moon, S. H., and
Frank, J. R.: Technological and economic potential of
poly(lactic acid) and lactic acid derivatives. FEMS Microbiol.
Rev., 16, 221231 (1995).
6. Vink, E. T. H., Rbago, K. R., Glassner, D. A., and Gruber,
P. R.: Applications of life cycle assessment to NatureWorks
polylactide (PLA) production. Polym. Degrad. Stabil., 80,
403419 (2003).
7. Yun, J. S., Wee, Y. J., and Ryu, H. W.: Production of optically pure L(+)-lactic acid from various carbohydrates by
batch fermentation of Enterococcus faecalis RKY1. Enzyme
Microb. Technol., 33, 416423 (2003).
8. Boniardi, N., Rota, R., Nano, G., and Mazza, B.: Lactic
acid production by electrodialysis part I: experimental tests. J.
Appl. Electrochem., 27, 125133 (1997).
9. Ishizaki, A., Nomura, Y., and Iwahara, M.: Built-in electrodialysis batch culture, a new approach to release of end
product inhibition. J. Ferment. Bioeng., 70, 108113 (1990).
10. Hongo, M., Nomura, Y., and Iwahara, M.: Novel method
of lactic acid production by electrodialysis fermentation.

Appl. Environ. Microbiol., 52, 314319 (1986).


11. Harrison, R. G., Todd, P., Rudge, S. R., and Petrides, D. P.:
Bioseparations science and engineering, p. 319372. Oxford
University Press, New York (2002).
12. Nomura, Y., Yamamoto, K., and Ishizaki, A.: Factors affecting lactic acid production rate in the built-in electrodialysis fermentation, an approach to high speed batch culture. J.
Ferment. Bioeng., 71, 450452 (1991).
13. Boyaval, P., Corre, C., and Terre, S.: Continuous lactic acid
fermentation with concentrated product recovery by ultrafiltration and electrodialysis. Biotechnol. Lett., 9, 207212
(1987).
14. Madzingaidzo, L., Danner, H., and Braun, R.: Process development and optimization of lactic acid purification using
electrodialysis. J. Biotechnol., 96, 223239 (2002).
15. Ryu, H. W., Kang, K. H., Pan, J. G., and Chang, H. N.:
Characteristics and glycerol metabolism of fumarate-reducing
Enterococcus faecalis RKY1. Biotechnol. Bioeng., 72, 119
124 (2001).
16. Yun, J. S. and Ryu, H. W.: Lactic acid production and carbon catabolite repression from single and mixed sugars using
Enterococcus faecalis RKY1. Process Biochem., 37, 235240
(2001).
17. Wee, Y. J., Yun, J. S., Park, D. H., and Ryu, H. W.: Biotechnological production of L(+)-lactic acid from wood hydrolyzate by batch fermentation of Enterococcus faecalis.
Biotechnol. Lett., 26, 7174 (2004).
18. Nomura, Y., Iwahara, M., and Hongo, M.: Lactic acid production by electrodialysis fermentation using immobilized
growing cells. Biotechnol. Bioeng., 30, 788793 (1987).
19. Oh, H., Wee, Y. J., Yun, J. S., and Ryu, H. W.: Lactic acid
production through cell-recycle repeated-batch bioreactor.
Appl. Biochem. Biotechnol., 105108, 603613 (2003).
20. Tejayadi, S. and Cheryan, M.: Lactic acid from cheese whey
permeate. Productivity and economics of a continuous membrane bioreactor. Appl. Microbiol. Biotechnol., 43, 242248
(1995).
21. kerberg, C. and Zacchi, G.: An economic evaluation of
the fermentative production of lactic acid from wheat flour.
Bioresour. Technol., 75, 119126 (2000).