An electrokinetic bioreactor: Using direct electric current for enhanced lactic acid fermentation and product recovery

Article abstract of DOI:10.1016/j.tet.2003.10.110

The microbiological production of organic acids by fermentation processes is growing in commercial importance. However, the removal of product and pH control are two main issues that limit the technical and commercial viability of such processes. A laboratory scale bioreactor combining conventional electrodialysis and bipolar membrane electrodialysis has been developed for in situ product removal and pH control in lactic acid fermentation. The electrokinetic process enabled removal of the biocatalytic product (lactic acid) directly from the bioreactor system, in a concentrated form, as well as enabling good pH control without generation of troublesome salts. Moreover, end-product inhibition of glucose catabolism was reduced, resulting in a greater generation of the end-product lactic acid. An automatic pH sensor and current application system was developed and successfully implemented for lactic acid fermentation in the electrokinetic bioreactor.

Full text of DOI:10.1016/j.tet.2003.10.110

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Tetrahedron 60 (2004) 655–661
An electrokinetic bioreactor: using direct electric current for
enhanced lactic acid fermentation and product recovery
Hong Li,a Roberta Mustacchi,a Christopher J. Knowles,a Wolfgang Skibar,b Garry Sunderland,b
Ian Dalrympleb and Simon A. Jackmana
,
*
aDepartment of Engineering Science, Oxford Centre for Environmental Biotechnology, University of Oxford and NERC Centre for Ecology
and Hydrology, Parks Road, Oxford OX1 3PJ, UK
bC-Tech Innovation Ltd, Capenhurst Technology Park, Capenhurst, Chester CH1 6ES, UK
Received 8 August 2003; revised 29 September 2003; accepted 17 October 2003
Abstract—The microbiological production of organic acids by fermentation processes is growing in commercial importance. However, the
removal of product and pH control are two main issues that limit the technical and commercial viability of such processes. A laboratory scale
bioreactor combining conventional electrodialysis and bipolar membrane electrodialysis has been developed for in situ product removal and
pH control in lactic acid fermentation. The electrokinetic process enabled removal of the biocatalytic product (lactic acid) directly from the
bioreactor system, in a concentrated form, as well as enabling good pH control without generation of troublesome salts. Moreover, end-
product inhibition of glucose catabolism was reduced, resulting in a greater generation of the end-product lactic acid. An automatic pH sensor
and current application system was developed and successfully implemented for lactic acid fermentation in the electrokinetic bioreactor.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
producing calcium lactate. Ammonium hydroxide and
sodium hydroxide have also been used for neutralization,
however the cost of sodium hydroxide for the neutralization
of the process is dominant in the operational cost in the
fermentation step.7 As a result of pH neutralization,
the product is an organic acid salt. After fermentation, the
process of separating the product from the medium and
converting the salt to an organic acid is complicated,
involving precipitation and acidification using a mineral
acid (sulfuric acid). These steps contain the major economic
hurdles for organic acid production. Moreover, the proces-
sing produces large quantities of an effluent containing high
concentrations of salts. For instance, in the case of calcium
carbonate neutralization, one ton of gypsum by-product will
be produced for every ton of lactic acid produced.2
Lactic acid has been produced commercially by fermenta-
tion since 1883.1 The major application of lactic acid is in
the food industry as an additive and preservative. Other
applications include use as a pharmaceutical intermediate,
lactate ester, which is an alternative solvent to glycol ether.
Lactic acid-derived polymers are becoming increasingly
important because of their application within drug delivery
systems and their biodegradable and thermoplastic nature
means that they can be produced as high volume
biodegradable plastics for packaging and other applications.
However, this potential can only be realised if the cost of
production is competitive on a global scale.2–5
Lactic acid is produced microbially from a variety of
feedstocks by fermentation using lactic acid bacteria
(Lactobacilli). In the conversion of glucose to lactic acid
by homofermentative Lactobacilli, two molecules of lactic
acid are produced for every molecule of glucose consumed.
pH control of this organic acid fermentation is crucial; in the
absence of such pH control, the final lactic acid concen-
tration may be less than half of that obtained with pH
control.6 Conventionally, calcium carbonate has been used
to neutralize the pH during lactic acid fermentation,
A low cost, environmentally sustainable process for organic
acid downstream processing is desirable. Many investi-
gations have been carried out into the separation, concen-
tration and purification of organic acids from fermentation
broth.1,2 Among these, conventional electrodialysis has
been developed for the separation, purification and concen-
tration of organic acid salts from a fermentation broth,
leading to economic product formation and low environ-
mental impact of the downstream processing. Conventional
electrodialysis is a membrane separation technology that
uses ion-exchange membranes under the influence of direct
current for separating and concentrating ions in solution. It
has been widely used for separating and concentrating
organic acid salts from fermentation broths.2,8 Bipolar
Keywords: Lactic acid; Electrokinetic; Electrodialysis; In situ product
recovery; Bipolar membrane.
*
Corresponding author. Tel.: þ44-1865-273032; fax: þ44-1865-274752;
e-mail address: simon.jackman@eng.ox.ac.uk
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.110

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H. Li et al. / Tetrahedron 60 (2004) 655–661
electrodialysis involves water splitting within a bipolar
membrane, which is a laminate of cation and anion
exchange membranes, with the efficient generation of
protons and hydroxyl ions, thus producing acid and base
from the salt solution with high energy efficiency.9–11
Bipolar electrodialysis, integrating conventional electro-
dialysis with bipolar membrane water splitting, provides a
potentially attractive complement to organic acid fermenta-
tion, enabling the separation, purification and concentration
of salts and converting these salts into acid and base without
producing effluents containing high concentrations of salts
or gypsum, thereby avoiding discharge to the environment.
Another advantage of bipolar membrane electrodialysis is
that the base produced can be recycled and used for
neutralization of the fermentation process.2,10–12
of pH control in the fermentation chamber during electro-
dialysis. This can be partially achieved by electrodialytic
removal of the acid, however the pH remained lower than
optimal because some acid remained in the medium.15,18 In
order to maintain precise pH control, an additional pH
control system with addition of NaOH solution was
incorporated into the electrodialysis fermentor.18
Bipolar membrane electrodialysis has been used as a single
unit operation for industrial organic acid production,19
however, when coupled to fermentation, it is currently used
as a separate second downstream stage for acid formation
and base recycling.9,10 In this format it follows conventional
electrodialysis and enables recycling of base generated in
the fermentor, leading to the production of the organic acid
salt in the medium, which will also need to be converted into
acid in a downstream process.
There is the potential that separation and product recovery
can be applied in situ, during the fermentation process. As
lactic acid fermentation is product inhibited, the removal of
lactic acid produced in the fermentation medium will relieve
this inhibition.1 Studies of product removal following
fermentation have been carried out in a number of systems
by means of extraction,13 dialysis14 or conventional
electrodialysis15 as well as bipolar membrane electro-
dialysis.16 The in situ extraction of lactic acid is possible,
but it has yet to be demonstrated economically.1 Conven-
tional electrodialysis during fermentation with the cathode
located in the fermentation compartment damages the
bacterial cells if they come into direct contact with the
electrodes. Therefore, an ultrafiltration step must be used
before electrodialysis to prevent the cells from contacting
the electrode.17 A second issue for the microbiology is that
The aim of the current research is to combine the advantages
of both conventional electrodialysis and bipolar membrane
electrodialysis within the bioreactor configuration for lactic
acid fermentation. This electrokinetic membrane bioreactor
couples fermentation directly with both in situ lactic acid
separation and product concentration. The use of a
positively charged buffering system (bis-Tris) in the
fermentation and concentration chambers leads to the
migration of the negatively charged lactate ion alone into
the concentration chamber. There is in situ pH control
through removal of lactate and the direct use of hydroxyl
ions produced by bipolar electrodialysis. The potential
benefits of the bioreactor include relief of product
inhibition, and hence an increase in yield, plus separation
Figure 1. Electrokinetic bioreactor Each compartment has a circulation loop and reservoir for pH control, sampling and product recovery. Chambers A, MA, C
and M were recirculated at 120 ml/min using a peristaltic pump with Norprene tubing (Cole-Parmer Instrument Co. Ltd, London, UK). The anode and cathode
were connected to a computer-controlled DC power supply, with a pH sensor in chamber M connected to a computer interface.

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H. Li et al. / Tetrahedron 60 (2004) 655–661
657
and concentration of lactic acid and direct use of hydroxyl
ions produced by water splitting to neutralize pH without
the requirement for additional acid or base. pH control is
coupled to the power supply to enable lactic acid to be
removed as it is produced.
supply (PL-P, Thurlby Thandar Instruments Ltd.
Cambridgeshire, UK). Within bipolar electrodialysis, pH
control was achieved through water splitting and migration
of hydroxyl ions into chamber M and through electro-
migration of lactate across the anion exchange membrane
into chamber MA.
An initial experiment was conducted using buffer and lactic
acid alone (i.e. no bacteria or glucose) to ensure that lactic
acid migrated across the anion exchange membrane and
could be recovered in compartment MA. The initial
concentration of lactic acid in the buffer solution was
79.3 mM (10 g lactic acid in 1.4 l). Electromigration was
observed, and a mass balance of 95% recovery of lactic acid
was maintained as it migrated from chamber M across the
membrane into chamber MA (Fig. 2). The current efficiency
under these conditions was approximately 100%.
2. Results
2.1. Bioreactor design and electromigration of lactic acid
in the electrokinetic bioreactor utilising bipolar
electrodialysis
The electrokinetic bioreactor consisted of four chambers,
labelled A, MA, M and C (Fig. 1). These chambers were
separated by membranes, and the complete system was
sealed by means of double O-rings between chambers and
the system was clamped together. Chambers A and C were
adjacent to the anode and cathode, respectively. Chambers
A, MA, and C had a total capacity of 100 ml, however they
were each connected to separate reservoirs, which enabled a
total volume of 500 ml solution to be recirculated through
each chamber. The reservoir for chamber MA contained a
pH control system for monitoring and control of pH.
Chamber M had a total capacity of 1400 ml. A cation
exchange membrane (Nafion 450, DuPont Fluoroproducts,
Fayetteville, USA) separated chambers A and MA, an anion
exchange membrane (AMH, Tokuyama Co. Ltd., Tokyo,
Japan) separated chambers MA and M, and a bipolar
membrane (BP-1, Tokuyama Co. Ltd., Tokyo, Japan)
separated chambers M and C. Chamber M was stirred at
250rpm using a magnetic stirring bar. The anode was
fabricated from titanium-plated steel, and the cathode was a
stainless steel plate. They were both square and had a
working surface area of 11£8.5 cm2. Chamber A and its
reservoir contained 500 ml 0.1 M phosphate buffer, initially
at pH 6.5. Chambers MA and C (including their reservoirs)
each contained 500 ml 0.1 M bis-Tris buffer, starting pH 8.5
and 6.5, respectively. Chamber M contained 1400 ml 0.1 M
bis-Tris buffer, pH 6.5, 20% (w/v) glucose. Harvested
bacterial cells were resuspended in this medium to a density
as indicated in the text for each bioreactor run. The anode
and cathode were connected to a DC programmable power
2.2. Lactic acid fermentation in the electrokinetic
bioreactor with the application of current part way
through the fermentation process
The bioreactor configuration and buffers in chambers A,
MA, C and M were the same as described in Section 2.1.
When glucose (20 g/l) was added to harvested cells and the
fermentation was allowed to proceed in the absence of an
applied current, lactic acid accumulated in chamber M
giving a total of 81 mmol after 86 h, with some diffusion
into compartment MA to a total of 93 mmol in the reactor
(Fig. 3). After 86 h, the current was applied and lactic acid
began to migrate into chamber MA. The amount of lactic
acid in M remained constant to 106 hours and thereafter
decreased significantly to approximately 10 mmol after a
further 20 hours, whilst the amount of lactic acid in MA
increased to a final level of 130 mmol after 48 h of applied
current. In the control bioreactor, lactic acid production was
very similar to that in the electrokinetic bioreactor up to
86 h (Fig. 4). However, after 86 h, there was very little
further increase in lactic acid production, such that total
lactic acid production in the electrokinetic bioreactor was
Figure 3. Lactic acid production in the electrokinetic bioreactor, with
application of current after 86 h and no pH control. The configuration of the
electrokinetic bioreactor and operating conditions are described in Sections
2.1 and 2.3, respectively. Starting glucose concentration was 111 mM
(20 g/l) and applied current density was 1.8 mA/cm22. The pH in chamber
M was not externally controlled. Lactate in chambers M (A) and MA (V)
was determined by ion chromatography. Harvested cells were used at a
concentration of 0.87 g dry cell weight/l in both bioreactors. Values are the
means of three determinations^SEM.
Figure 2. Lactic acid migration in the electrokinetic bioreactor with bipolar
electrodialysis Bioreactor configuration and buffer content were as
described in Section 4.1. Lactate (79.3 mM) migrated from chamber M
(K) into chamber MA (V) giving total lactic acid in the bioreactor
(MþMA) (A) as determined by ion chromatography. The current density
was fixed at 0.86 mA cm22. pH in chamber MA was controlled at pH 8.5 by
addition of 4 M NaOH.

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H. Li et al. / Tetrahedron 60 (2004) 655–661
Figure 4. Total lactic acid production in the electrokinetic and control
bioreactors without pH control. Lactic acid, measured as lactate, in the
electrokinetic (O) and control (W) bioreactors was determined by ion
chromatography. Bioreactor configurations, operating conditions and
harvested cells used were the same as in Figure 3. Values are the means
of three determinations^SEM.
Figure 6. pH levels in the electrokinetic and control bioreactors during
fermentation and applied current density for the electrokinetic bioreactor
without pH control. pH in the EK bioreactor (O) and control bioreactor (W)
was determined using a microelectrode. Bioreactor configurations,
operating conditions and harvesting of cells used were the same as in
Figure 3.
approximately 34% higher in comparison with the pro-
duction in the control bioreactor (Fig. 4). The average lactic
acid production rate over 180 h was 0.86 mmol/h/g dry cell
weight for the electrokinetic bioreactor and 0.64 mmol/h/g
dry cell weight for the control bioreactor. The increased
lactic acid production also correlated to differences in
glucose consumption in the electrokinetic and control
bioreactors (Fig. 5). The total mass balance of glucose to
lactic acid conversion was 57% for the electrokinetic
bioreactor and 46% for the control bioreactor at the end of
the fermentation.
suitable value. Manual pH control was achieved by
increasing the applied current when the pH fell below pH
6.5, and decreasing or switching off the current when the pH
increased above pH 6.5. The pH in the control bioreactor
was controlled at pH 6.5 using a pH controller and addition
of 4 M NaOH. The lactic acid production rate was
0.78 mmol/h/g dry cell weight in the electrokinetic
bioreactor and 0.51 mmol/h/g dry cell weight for the control
bioreactor (averages calculated over a period of 197 h). The
production of lactic acid in the electrokinetic bioreactor,
proceeded at a constant rate to 100 h and continued to
increase to 197 h, giving a maximum production of
168 mmol, whereas in the control reactor lactate production
stopped after 100 hours, with a maximum production of
112 mmol (Fig. 7). pH was maintained at pH 6.5 in both
bioreactors. The increased production of lactic acid in the
electrokinetic bioreactor may be due to the removal of lactic
acid from chamber M into chamber MA and due to the
different conditions of the pH control systems (Fig. 8).
Moreover, a higher concentration of lactic acid was
achieved in chamber MA (approximately double) at the
end of the fermentation compared to that in chamber M
As a result of lactic acid production, the pH in both
bioreactors had decreased to approximately pH 4 after 86 h.
After the current was switched on, the pH in chamber M in
the electrokinetic bioreactor increased dramatically and
finally stabilized at approximately pH 12 (Fig. 6).
2.3. pH controlled lactic acid fermentation in an
electrokinetic bioreactor
Having show that the application of current could recover
the pH in the fermentation chamber, leading to increased
lactic acid production, it was necessary to control the
applied current such that the pH could be maintained at a
Figure 7. Lactic acid production within an electrokinetic bioreactor with
manual pH control. Lactic acid levels in the electrokinetic bioreactor (O)
and control bioreactor (W) were determined by ion chromatography. The
pH in the electrokinetic bioreactor (þ) was controlled by manually
adjusting the current according to changes in pH in chamber M. The pH in
the control bioreactor (S) was controlled using a conventional pH
controller and addition of 4 M NaOH. Both bioreactors were controlled
at pH 6.5. Harvested cells were used at 0.79 g dry cell weight /l in both
bioreactors. Values are the means of three determinations ^SEM.
Figure 5. Glucose content in the electrokinetic and control bioreactors
without pH control. Glucose content in the electrokinetic (O) and control
(W) bioreactors was determined by a glucose oxidase-kit (Section 4.5).
Bioreactor configurations, running conditions and harvested cells used were
the same as in Figure 3. Values are the means of three determinations^
SEM.