Process Biochemistry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

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

Review

A review on the current developments in continuous lactic acid

fermentations and case studies utilising inexpensive raw materials
⁎
José Pablo López-Gómez, Maria Alexandri, Roland Schneider, Joachim Venus
Leibniz Institute for Agricultural Engineering and Bioeconomy, Max-Eyth-Allee 100, Potsdam, Germany

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

Keywords: The numerous applications of lactic acid have made it one of the most important platform chemicals and, for
Lactic acid many years, its biotechnological production has been the topic of intensive research. Currently, most of the lactic
Continuous fermentations acid is manufactured in batch mode; however, continuous mode could offer various advantages, especially in
Cell recycle terms of productivity. This review provides a summary of the most recent studies on continuous lactic acid
Alternative substrates
production. Potential advantages of continuous over batch mode, the improvements that high cell densities can
represent, utilisation of low cost substrates and some economic aspects of continuous mode are discussed. The
review is complemented by 3 experimental case studies utilising inexpensive substrates: tapioca starch hydro-
lysate, molasses combined with rapeseed meal hydrolysate and acid whey. In all cases, continuous cultivation
was coupled with cell recycling using hollow-fibre membranes for L- or D-lactic acid production. Lactic acid
productivities higher than 5 g l−1 h−1 and substrate conversion yields above 80% were achieved for all the cases.
Final lactic acid concentrations were 50.3, 59.6 and 45.9 g l−1 for the tapioca starch hydrolysate, molasses and
acid whey respectively. These results support the claim that continuous cultivation using cell recycling is a
promising approach for high lactic acid productivities.

1. Introduction petrochemical-based plastics, is restrained by the current cost of lactic

In recent years, rising environmental and sustainability concerns
have led to a growing interest in the development of new and more
efficient processes that could reduce our dependency on fossil fuels.
Fermentation processes, which have been traditionally used for the
production of foods and feeds, are a clear example of this trend. The
wide variety of substrates that can be used and products that can be
formed have made microbial fermentations an intensively investigated
field.
Although it can be produced by chemical means, fermentation
processes account for around 80–90% of the global lactic acid pro-
duction [1]. Lactic acid is a highly versatile platform chemical with a
wide variety of applications that cover the food, pharmaceutical, cos-
metic, detergent and dairy industries. More recently, the market for
lactic acid has been expanding for the production of polylactic acid
(PLA). This biocompatible and biodegradable material has further
boosted the interest on lactic acid production and, with a continuously
growing market, it is estimated that global demand of lactic acid will
rise from 1220 kt in 2016 to 1960 kt by 2025 [1]. In spite of the pro-
mising forecast, work is still necessary to reduce manufacturing costs.
Particularly PLA production, which has to compete with the traditional
acid. It has been estimated that the production cost of lactic acid has to
be reduced by 50% for PLA to be competitive in the market [2,3].
Several aspects of lactic acid production have been widely in-
vestigated. The transition to more sustainable manufacturing methods
has taken the research from renewables to the investigation of residues
and waste materials as potential substrates for the production of lactic
acid. Simultaneously, in addition to the already known large number of
lactic acid producing organisms, the advent of new molecular biology
and genetic engineering tools have widened the options for strain de-
velopment and selection. Reviews covering different aspects of lactic
acid production such as: microorganism type, substrate, production
methods, downstream, application, etc. are available in the literature
[1,3–8]. However, there is not, to the best of the authors’ knowledge, a
recent review of the literature focused on the production of lactic acid
in continuous mode. The scope of this review is to present a summary of
the studies dealing with continuous cultivation for lactic acid produc-
tion, as an alternative to batch and fed-batch cultivations. Additionally,
experimental case studies, from our group’s own experience, are also
presented aiming to support the claim that high lactic acid productiv-
ities can be achieved during continuous mode using membrane cell
retention.

⁎
Corresponding author.
E-mail address: jvenus@atb-potsdam.de (J. Venus).

https://doi.org/10.1016/j.procbio.2018.12.012
Received 17 August 2018; Received in revised form 19 November 2018; Accepted 10 December 2018
1359-5113/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: López-Gómez, J.P., Process Biochemistry, https://doi.org/10.1016/j.procbio.2018.12.012
J.P. López-Gómez et al.
2. Advantages of continuous fermentation over batch
Currently, the vast majority of industrial lactic acid fermentations
occur in batch mode. However, this method involves long fermentation
times with low productivities and high capital costs [6,9]. Additionally,
the processes can suffer due to acidification of the medium and sub-
strate inhibition [10]. In comparison to batch processes, low interest
has been shown on the development of continuous lactic acid fermen-
tations. There is, however, a clear growing tendency in the annual
demand for lactic acid which underlines the need for more efficient
fermentations. Furthermore, the market opportunity of PLA can only be
seized with processes able to cope with the demand. Continuous fer-
mentation systems could potentially increase the lactic acid production
capacity while alleviating problems related to production costs.
In addition to the common limitations of a batch process, there are
some inherent characteristics of lactic acid production that drive the
development of continuous processes. In this type of systems, a constant
removal of the product means that problems related to product in-
hibition can be eliminated. Moreover, unlike in batch, a continuous
process can be run over longer periods of time without requiring stops.
From an economic point of view, continuous cultivations present the
benefit of high product titers, using only one inoculum. In spite of the
potential advantages and the presence of several studies on the litera-
ture, the application of the continuous lactic acid process in the in-
dustry has been scanty [11]. Possibly, the preference of batch systems is
due to the susceptibility to contamination issues and the level of com-
plexity of continuous fermentations. Additionally, since the separation
and purification processes account for most of the lactic acid production
operational costs [9] great efforts have been carried out targeting im-
provements on those steps. Nonetheless, as emphatically described by
Villadsen [12], the manufacturing of low-value products, e.g. lactic acid
or ethanol, can only be economically and industrially viable if run in
continuous mode.
In contrast to batch and fed-batch fermentations, it is possible to
maintain cell growth at a constant rate in continuous fermentations.
This characteristic is particularly important when, as in the case of
lactic acid, product synthesis is growth associated. In a continuous
fermentation reactor under steady state conditions, i.e. a chemostat, the
specific growth rate of a culture is equal to the dilution rate. Therefore,
it is possible to maximise productivity in a chemostat by adjusting its
dilution rate and, typically, productivities are higher in continuous
mode than in batch, as seen in Table 1 [7]. This feature was demon-
strated in a continuous fermentation, using L. delbrueckii subsp. lactis
QU 41, which showed that the productivity (18 g l−1 h−1) was 35-fold
higher than in batch mode and significantly higher than the results
reported in other studies [13]. Similarly, the productivity of lactic acid
resulted 1.5-fold higher in continuous mode and 3-fold higher in con-
tinuous mode with cell recycling, when E. mundtii QU 25 was used for
the fermentation of xylose [14]. The same work reports and compares
the results of other organisms for the production of lactic acid in
Table 1
Comparison of results for productivity (P), yield (Y) and lactic acid concentration, in batch and continuous mode, from other investigations.
Substrate Strain Batch
P Y
(g∙l−1∙h−1) (g∙g−1)
Xylose E. mundtii 2.08 0.90
Cassava starch L. plantarum 0.80 N/S
MRS medium L. delbrueckii 0.52 1.01
Defatted rice bran hydrolysate L. rhamnosus 5.20 0.95
Raw sugar (from sugarcane) S. laevolacticus 0.25 0.89
N/S: Not specified.
a
Continuous mode with cell recycling.
2
Process Biochemistry xxx (xxxx) xxx–xxx
continuous mode.
Improvements on volumetric productivities, when using continuous
mode instead of batch, have also been observed for other platform
chemicals, for example, cell recycling enhanced 6-fold the productivity
of butanol from the strain Clostridium saccharoperbutylacetonicum N1-4
using xylose as substrate [15]. On another example [16], Anaerobios-
pirillum succiniciproducens was cultivated in a continuous fermentation
combining an integrated membrane and an eletrodialysis system. The
results showed that under the best culture conditions, the succinic acid
productivity reached 14.8 g l−1 h−1, a value 20 times higher than the
one obtained in the batch experiments. The same pattern was observed
in a study [17], in which a genetically modified strain of Mannheimia
succiniciproducens was used in both batch and continuous fermentations
for the production of succinic acid. Results showed that productivities
in the continuous system were 3.6 times higher than in batch, reaching
a value of 38.6 g l−1 h−1. In addition, some efforts have been focused in
the development of mathematical models and system for the control
and regulation of this kind of processes [18] which could further en-
hance their performance [19].
Even when the productivities are similar, advantages of continuous
over batch and fed-batch processes have also been reported. Gao and
Ho [20], compared the production of lactic acid in both fed-batch and
continuous cultures of B. subtillis MUR1. Maximum productivities for
the same strain and medium in batch and continuous fermentations
were 16.1 g l−1 h−1 and 17.1 g l−1 h−1 respectively with the difference
that the maximum productivity was maintained only for 1 h in the fed-
batch experiment [20,21]. They concluded that, in contrast to fed-
batch, where maximum productivities can be sustained only for very
short periods, it is possible to maintain maximum productivities for
indefinite periods in a continuous culture. In another study [22], results
from continuous fermentation mode of E. faecalis RKY1, using glucose
supplemented by yeast extract as the substrate, were compared to those
from batch mode. Data showed that, at similar volumetric productiv-
ities, a reduction of 77% on the yeast extract concentration could be
achieved if continuous mode was used instead of batch.
3. High cell densities improve continuous fermentations
Continuous lactic acid fermentations can experience problems re-
lated to unutilised sugars and the wash out of cell cultures, complica-
tions that are intensified when dilution rates are increased [6]. Thus,
systems to achieve high cell densities have been proposed to solve the
aforementioned problems and increase productivities [6]. Commercial
production of lactic acid in high cell densities systems is most com-
monly carried out in fed-batch mode, however, cell recycling and cell
immobilisation methods have shown a very good potential for con-
tinuous fermentations [6,23]. Currently, there are various examples in
the literature in which different modes for cell recycling have been
tested (see Table 2) and that consistently show that continuous fer-
mentation can be enhanced by increasing cell densities. In their work,
Continuous Refs.
[LA] P Y [LA]
(g∙l−1) (g∙l−1∙h−1) (g∙g−1) (g∙l−1)
44.10 3.14 0.86 21.70 [14]
6.15 1.01 41.00a
N/S 3.79 1.08 18.96 [48]
8.00 1.21 20.00a
86.40 18.00 1.03 20.70 [13]
84.00 6.20 0.98 86.00 [25]
55.70 11.20 0.97 67.30 [29]
 J.P. López-Gómez et al.

Table 2
Recent studies on the production of lactic acid in continuous fermentation mode.
Microorganism Substrate Medium concentration T (°C) pH D (h−1) Working LA isomer [LA] (g∙l−1) Productivity Yield Configuration Refs.
(g∙l−1) volume (l) (g∙l−1∙h−1) (g∙g−1 or
%)

B. subtilis MUR1 Glucose 65 37 7.5 0.4 0.8 L(+) 44.1 17.6 62% Conventional [20]
85 0.05 77.1 5 50%
B. coagulans isolate A107 Tapioca starch 134.7 52 6.5 0.2 3 L(+) 50.3 10.1 0.80 Hollow fiber membrane cell- This
hydrolysate recycle study
B. coagulans isolate A40 Molasses 124.3, 153.9 and 52 6.5 0.1 3 L(+) 59.6 5.9 0.85 Hollow fiber membrane cell- This
168.71 recycle study
E. faecalis RKY1 KCTC 8890P Glucose 100 38 7 0.04 1 L(+) 90 3.72 0.9 Polysulfone membranes cell- [22]
recycle
E. mundtii QU 25 Xylose 50 43 6.2 0.15 4 L(+) 41 6.15 1.01 Hollow fiber membrane cell- [14]
recycle
L. coryniformis subsp. torquens Acid whey 90 30 6 0.2 3 D(-) 45.9 9.2 0.92 Hollow fiber membrane cell- This
recycle study
L. coryniformis subsp. torquens Wheat flour 260 30 5.7 Variable Variable N/S 20 2.7 N/S Adaptive control strategy [19]
Lactobacillus delbrueckii subsp. MRS medium 20 43 6 0.87 0.4 D(-) 20.7 18 1.03 Hollow fiber membrane cell- [13]
lactis QU 41 recycle
Lactobacillus sp. RKY2 Lignocellulosic 75 36 6 0.16 1 N/S 42 6.7 0.95 Hollow fiber membrane cell- [24]
hydrolysates recycle

3
Glucose 50 0.28 40 11.4 0.91
L. plantarum Glucose 50 37 6.8 0.06 3.6 N/S NA 2.42 of lactate 82.7% Electrodialysis with bipolar [61]
membrane
L. plantarum SW14 Cassava starch 20 30 5.4 0.2 0.4 Racemic 18.96 3.79 1.08 Conventional [48]
0.4 mixture 20 8.00 1.21 Hollow fiber membrane cell-
recycle
L. rhamnosus LA-04-01 Defatted rice bran 120 42 6 0.1 0.25 L(+) 86 6.20 (1st stage) 0.98 Cell immobilization using corn [49]
hydrolysate 2.18 (2nd stage) stover
Mixed culture Sludge from anaerobic 10 (glucose) 37 3.5 0.042 2 N/S 4.3 N/S 0.43 Anaerobic acidogenesis [62]
digestion
Mixed culture Glucose 25 35-60 5 N/S 2 N/S N/S 4.80 92% Upflow anaerobic sludge [63]
reactor
Mixed culture Whey N/S 55 3-5.5 N/S 2 N/S N/S 0-0.50 0-0.61 Low pH start-up on continuous [27]
mixed-culture
S. leavolacticus Raw sugar (from 75 37 6 0.17 1.5 D(-) 67 12 0.96 Membrane- integrated [28]
sugarcane) fermentation reactor
S. cerevisiae NBRC 10505 Raw sugar (from 75 30 4.5 0.17 1.5 D(-) 46.6-52.1 7.1-8.1 0.746 Membrane-integrated [11]
sugarcane) fermentation reactor
S. laevolacticus strain JCM 2513 Raw sugar (from 100 37 6 0.17 1.5 D(-) 64.4-69.5 11.20 97% Membrane-integrated [29]
sugarcane) fermentation reactor
Lactococcus lactis ssp. cremoris Whey-based medium 160 30 6 0.11 2 N/S 60 9.7 N/S Submerged polymeric hollow [60]
fibres
Bacillus coagulans Corn stover hydrolysate 58.1 (glucose) 50 5.5-6 0.167 0.6 N/S 30 (lactate) 3.69 0.95 Conventional [45]
17.9 (xylose)
Process Biochemistry xxx (xxxx) xxx–xxx
J.P. López-Gómez et al.
Table 3
Results from volumetric productivities and dilution rates obtained during conventional continuous fermentation and continuous fermentation with cell recycling
[24].
Substrate Concentration (g∙l−1)
Conventional
Glucose 50
75
Lignocellulose hydrolysate 50
75
Wee and Ryu [24], compared the results between continuous fermen-
tation with and without cell recycling when Lactobacillus sp. RKY2 was
used to ferment glucose or lignocellulosic hydrolysates. They demon-
strated that higher volumetric productivities were achieved using the
cell recycling system (using hollow-fibres) for both substrates and un-
derlined the possibility of working with higher dilution rates with cell
recycling as seen in Table 3.
Cell immobilisation has also been used to achieve high cell den-
sities. In a study [25], defatted rice bran hydrolysate was used as the
substrate for lactic acid production and corn stover bagasse was used
for the immobilisation of L. rhamnosus. Their results showed that in two
stages continuous fermentation system, at a dilution rate of 0.1 h−1 a
maximum concentration of 86 g l−1 of L(+)- lactic acid with a yield of
0.98 g g−1 was achieved with productivities of 6.20 g l−1 h−1 and
2.18 g l−1 h−1 for the first and second stages respectively. In their
study, Abdel-Rahman et al. [6] describe in depth the different methods
for increasing high cell densities in lactic acid production.
4. Utilisation of cheaper substrates for lactic acid production
The utilisation of alternative cheaper feedstocks or residues from
other processes has been targeted as a way to cut production costs and
there are several reports on the literature dealing with the topic
[1,6,10,26]. Unfortunately, the majority of previous studies of lactic
acid production in continuous mode have used synthetic media [27].
Commonly, productivities and lactic acid final concentrations tend to
be lower when pure mediums are substituted by cheaper options.
Furthermore, the pre-treatment of substrates, such as agro-industrial
residues or lignocellulosic materials, involves the utilisation of acids/
bases that need to be neutralised before the fermentation. In other
cases, the complexity of the alternative substrates causes problems of
inhibition during the fermentations. Additionally, these types of sub-
strates have usually undesired components that need to be removed
during the purification step (e.g. furfural, HMF, phenolic compounds,
acetic acid). All these aspects add to the production cost and are im-
portant limiting factors for the transition to alternative and cheaper
feedstocks.
Several potential feedstocks have been studied for the continuous
production of lactic acid (see Table 2). The raw sugar, from sugar cane,
was used in three separated studies [11,28,29] for the production of D-
Lactic acid. S. leavolacticus was used for 2 of the studies and a geneti-
cally modified strain of S. cerevisiae was used for the third one. Results
showed that S. leavolacticus was better in terms of productivity, yield
and final lactic acid concentration. On the other hand, S. cerevisiae, was
able to produce lactic acid at a pH of 4.5 (pH = 6 for S. leavolacticus),
which could be advantageous for the downstream processing and fer-
mentation control.
In a recent study, whey was used as the feedstock in a continuous
process with mixed cultures [27]. In the experiments, mixed cultures
were initially subjected to a pH of 3 during the beginning of the fer-
mentation and afterwards the pH was increased to 5.5. This experiment
was compared to a fermentation in which the pH was constantly
maintained at 5.5. The pH variation allowed the selection of species in
which B. coagulans became predominant. Results showed that the
Process Biochemistry xxx (xxxx) xxx–xxx
Productivity (g∙l−1∙h−1) Dilution rate (h−1)
Cell recycle Conventional Cell recycle
3.90 11.40 0.20 0.28
5.20 8.80 0.12 0.16
3.10 6.30 0.16 0.24
4.30 6.70 0.12 0.16
fermentation with pH variation produced a better yield and they con-
cluded that a start with low pH had a positive effect and that mixed
cultures could be used for the fermentation of whey.
5. General economic aspects of continuous lactic acid
fermentations
There are many aspects of the lactic acid production which im-
provement would benefit the processes and reduce production costs.
The main challenge is to achieve high lactic acid concentration, pro-
ductivities and yields while utilizing low cost substrates for the pro-
cesses [30] and strain selection plays a crucial role in this regard.
Currently, most of the lactic acid produced depends on expensive
substrates (e.g. glucose) and in general, the cost of raw materials can
account for around 40–70% of the total production costs [30]. Clearly,
the transition to the utilization of inexpensive substrates or residues
from other processes is an attractive alternative to reduce production
costs. On the downside, in most cases, a pre-treatment of these cheap
materials is required to make sugars available for the fermentation and
the addition of supplements is required. Furthermore, they are usually
constituted by a complex mixture of different sugars some of which are
not easily metabolized by every microorganism. Another problematic
aspect is the inability of the strains to grow at high substrate con-
centrations or the inhibitory effect that lactic acid causes in their
growth. Moreover, although the inhibitory effect of substrate and end
product is minimized when the strains are grown in continuous mode,
the productivities of the processes are still restrained by that factor.
Additionally, the requirement of maintaining a mild pH during the
fermentations is an important aspect which raises operational costs.
Firstly, this pH regulation means that a constant addition of base is
necessary which, together with the addition of nitrogen supplements,
can account for up to 14% of the operational costs [31]. Secondly, in
most commercial cases, the pH is regulated by the addition of lime,
which causes the conversion of lactic acid into calcium lactate which
has to be then acidified back into lactic acid increasing downstream
processing costs [32]. Either by the addition of bases or lime, the re-
quirement of maintaining mild pH conditions negatively impacts the
overall cost of the processes. All the previous challenges underline the
need of ‘better’ strains for the production of lactic acid and therefore,
considerable efforts have focused on the development of more robust
strains [32]. These strains should be able to work at high substrate and
lactic acid concentrations, able to consume the high complex mixture of
sugars inherent in the inexpensive substrates. Moreover, according to Li
et al. [33], the selected strain must antagonise possible contaminants,
and at the same time present stability during long-term cultivation and
resistance to high cell density and, possibly, shear stress. Strains with
these characteristics, in combination with the utilisation of inexpensive
substrates, could reduce the lactic acid production costs. As it was
mentioned before, an important advantage of continuous fermentations
lies in the fact that the process starts with one inoculum. Moreover,
when the strain has reached steady state conditions, high product titres
can be produced for many days, thus the sugars and the nutrients in the
substrate are being exploited to a highest possible extent, saving time
from potential lag phases.
4
J.P. López-Gómez et al. Process Biochemistry xxx (xxxx) xxx–xxx

In spite of the challenges, techno-economic evaluations carried out
using continuous set-up for lactic acid production and inexpensive re-
sources, present promising results. In the study of Sikder et al. [34],
lactic acid production was performed using cell recycling with su-
garcane juice as substrate. When operating at a cell concentration of
22 g l−1, lactic acid titre, yield and productivity were 106 g l−1,
0.96 g g−1 and 53 g l−1 h−1 respectively. The cost of the entire up-
stream process accounted for about 36% of the total fixed capital cost.
Their study revealed that the main cost contributors were the raw
material and yeast extract, but the utilising of alternative substrates
could decrease the cost. The authors also concluded that coupling
continuous operation with microfiltration and nanofiltration for the
subsequent separation of lactic acid could further decrease the cost, due
to reduced raw material consumption.
6. Case studies
to 6.0 with the addition of 20% (w/w) NaOH, and the α-amylase pre-
paration BAN (Novozymes) was added, at a dosage of 1 ml per kg of
substrate (liquefaction). After 2 h, the temperature was set to 60 °C, pH
was adjusted to 4.2 with 4% HCl, and the glucoamylase-rich solution
Stargen™ 002 (Genencor) was added at a dosage of 1.4 ml per kg of
substrate (saccharification). After 20 h, the solution was collected, ul-
trafiltrated using 0.1 μm membranes and used for continuous fermen-
tations.
Rapeseed meal hydrolysis was also carried out at technical scale,
with 15% solids. The suspension was initially thermally treated at
119 °C for 30 min. Protein hydrolysis was performed at 50 °C, at pH
adjusted to 6.0 with 20% NaOH and stirring at 400 rpm. Once the
conditions were stabilized, 1.5 ml of Flavourzyme (Novo Nordisk) and
Neutrase (Novozymes) per kg and 0.70 ml/kg of Viscozyme
(Novozymes) were added. Hydrolysis lasted for 6 h, and the liquor was
afterwards ultrafiltrated with 0, 1 μm membranes (Table 4).

The following three case studies are experiences from our lab
showing how continuous fermentation can be successfully implemented
for the conversion of inexpensive raw materials to lactic acid.
6.1. Materials and methods
6.1.1. Substrates
Tapioca starch was kindly provided by Emsland Stärke GmbH
(Germany). Acid whey was supplied by the company Glanbia
Ingredients (Ireland). Sugar beet molasses were purchased by Pfeifer &
Langen GmbH (Germany). The composition of the substrates is pre-
sented in Table 4.
6.1.2. Microorganisms and cultivation conditions
For the case studies, two L- and one D-lactic acid producers were
tested. For L-lactic acid, two Bacillus coagulans strains (A107 and A40)
isolated from rapeseed meal by the Leibniz Institute for Agricultural
Engineering and Bioeconomy (Germany) were employed for continuous
fermentations using tapioca starch and molasses. The inoculum was
prepared in MRS broth (Merck, Germany) with dolomite EVERZIT Dol
0.5–2.5 mm (Evers, Germany) as buffer, at an orbital shaker set at
100 rpm, 52 °C for 13 h for strain A107 and 24 h for strain A40.
Lactobacillus coryniformis subsp. torquens (DSM 20005) was used for
D-lactic acid fermentation from acid whey. Inoculum was prepared in
MRS medium for 24 h at 30 °C and at 100 rpm in an orbital shaker.
6.2. Hydrolysis
Tapioca starch was hydrolyzed in two steps (liquefaction and sac-
charification) using 15% solids dissolved in distilled water in 55 l
technical scale. The suspension was heated up to 85 °C, pH was adjusted
6.3. Fermentation
Continuous fermentations were carried out in 5 l BIOSTAT bior-
eactors (Sartorius AG, Germany), with 3 l working volume, using a cell
retention module with hollow-fiber filters UMP-1147 (Pall
Corporation), with a pore size of 0.2 μm. A schematic representation of
the system can be seen in Fig. 1.
The experiment using tapioca starch was performed at 52 °C, with
stirring at 200 rpm at pH of 6.5, in the presence of 15 g l−1 yeast ex-
tract. Tapioca hydrolysate was sterilized separately at 121 °C for
15 min, whereas yeast extract was autoclaved at the same temperature
for 30 min. The continuous mode was operated under a constant dilu-
tion rate of 0.2 h−1. Two feeding solutions were utilized: feed 1 con-
tained tapioca starch hydrolysate with 134.7 g l−1 glucose and feed 2
contained 30 g l−1 yeast extract. The carbon and the nitrogen source
were fed to the reactor at a ratio of 50:50.
Fermentations with molasses were carried out using the same
system as discussed above, using a protein hydrolysate instead of yeast
extract and a total sugar concentration of 168.7 g l−1 in the feed 1.
During the process two more feeding solutions were prepared, with a
total sugar concentration of 153.9 g l−1 (feed 2) and 168.7 g l−1 (feed
3). Both substrates were supplied at a ratio of 50:50, at a dilution rate of
0.1 h−1.
D-lactic acid fermentations with acid whey as substrate were per-
formed using the same apparatus, but operational conditions were
adapted to the specific requirements of the strain. For L. coryniformis
subsp. torquens temperature was set at 30 °C, agitation at 200 rpm and
pH was controlled at 6.0 with 20% NaOH. For the batch mode, 1 l acid
whey was diluted with water in order to contain 90 g l−1 lactose, and
then inactivated at 100 °C for 15 min. Medium was also supplemented
with 20 g l−1 yeast extract, 2 g l−1 K2HPO4, 0.1 g l−1 MgSO4 and

Table 4
Compositional analysis of the substrates utilized in the case studies.
Substrate Glucose Disaccharide (g∙l−1) Fructose/ Lactic acid Dry matter Nkjel PO43− SO42− NH4+
(g∙l−1) Galactose (g∙l−1) (%) (mg∙l−1) (mg∙l−1) (mg∙l−1) (mg∙l−1)
(g∙l−1)

Tapioca hydrolysatea 139.0 3.5b n.d. n.d. 13.7 113.0 6.0 36.1 0.0
Acid wheyc n.d. 258d n.d. n.d. 26.0 2288.0 683.0 2687.0 159.0
Molasses 56.6 505.0e 17.0f 29.7 84.8 – 23.1 7177.0 150.0
Rapeseed meal 5.4 1.7e 4.7f n.d 4.7 – 291.0 562.1 159.6
hydrolysatea

a
Analysis carried out after ultrafiltration with 0.1 μm membranes.
b
Maltose.
c
Analysis carried out after nanofiltration.
d
Lactose.
e
Sucrose.
f
Fructose.

5
J.P. López-Gómez et al.
Fig. 1. Schematic representation of hollow fibers membrane-integrated bioreactor system for cell-recycle continuous production of lactic acid.
0.05 g l−1 MnSO4. Two feeding solutions were supplied during con-
tinuous mode at a ratio of 50:50 and at a dilution rate of 0.1 h−1. Feed
1, contained acid whey permeate (90 g l−1 lactose) and feed 2 con-
tained 40 g l−1 yeast extract, 4 g l−1 K2HPO4, 0.2 g l−1 MgSO4 and
0.01 g l−1 MnSO4.
6.4. Analytical assays
The determination of lactic acid, the optical purity, and sugar
concentration was carried out via HPLC analysis as described by Neu
et al. [35].
Microbial growth was followed using direct measurements of dry
cell weight (DCW) and colony forming units (cfu). Dry cell weight
measurements were carried out by drying the cell biomass at 105 °C
until constant weight. The number of living cells was counted on petri
dishes containing either MRS agar (D-lactic acid strain) or Nutrient agar
(L-lactic acid strains), after incubation at the appropriate temperature
for each strain for 48 h and 24 h, respectively.
6.5. Continuous fermentation using tapioca starch hydrolysate
Cassava (Manihot esculenta spp. esculenta) root ranks number six
among the most important crops worldwide and it is mainly cultivated
in tropical and sub-tropical countries [36]. This root, also known as
tapioca, is rich in starch which can account for about 70–75% (w/w).
Tapioca starch is used for food and feed, as well as for the production of
biotechnological products such as bio-ethanol and succinic acid since it
is a relatively cheap raw material [37,38].
Bacillus coagulans strains have been recently shown to be efficient L-
lactic acid producers from various renewable resources like coffee pulp
[39], coffee mucilage [35], corn flour hydrolysate [40], excess sludge
[41], corn stover hydrolysate [42], acid hydrolysate of oil palm empty
fruit bunch [43] and sugarcane bagasse [44], in batch or fed-batch
cultivation mode. However, the fermentative production of lactic acid
from B. coagulans is not yet thoroughly investigated. Ahring et al., [45]
demonstrated continuous fermentations with a B. coagulans isolate
using clarified corn stover hydrolysate as substrate. Even though lactic
acid yield on biomass sugars was 0.95 g g−1, maximum productivity
was only 3.69 g l−1 h−1. In this case study, tapioca starch is proposed as
an inexpensive substrate for the potential industrial production of lactic
acid using a B. coagulans isolate.
The kinetics of the continuous fermentation is presented in Fig. 2.
Glucose consumption started rapidly, together with simultaneous lactic
acid production. Continuous mode started after 10 h of batch cultiva-
tion, when only 1.3 g l−1 of glucose were left unconsumed. A small
amount of maltose was also consumed by the time that glucose con-
centration in the medium became limiting. Lactic acid concentration at
the end of the batch phase was 53.8 g l−1, with an average yield and
productivity of 0.71 g lactic acid per g of total sugars in the substrate
and 5.38 g l−1 h−1, respectively. For the continuous cultivation a
6
Process Biochemistry xxx (xxxx) xxx–xxx
dilution rate of 0.2 h−1 was selected and kept constant until the end of
the process. The strain reached steady state conditions relatively fast,
and during this time no glucose was detected in the fermentation
medium. Lactic acid concentration presented an average value of
50.3 g l−1, and the concentration of maltose was less than 0.3 g l−1
after almost 27 h of fermentation. Maximum values in lactic acid titer,
yield and productivity were obtained after 17 h of cultivation and were
53.8 g l−1, 0.87 g g−1 and 10.77 g l−1 h−1, respectively, whereas the
average yield and productivity of the overall process were 0.80 g g−1
and 10.11 g l−1 h−1. The main drawback is the co-production of formic
and acetic acids from the strain. More specifically, formic acid
(1.3 g l−1) was detected after 17 h (27 h of fermentation) of continuous
cultivation, at the point that lactic acid yield and productivity reached
their maxima values. The highest value was observed after 56 h
(5.4 g l−1), and after this point, formic acid production started to de-
crease, until a final value of 1.6 g l−1, at the end of the process. Acetic
acid production followed a similar profile to formic acid. Its formation
was initiated after 29 h, reached 2.2 g l−1 after 50 h and then ceased.
Eventually, the strain started to consume both formic and acetic acids,
almost at the same time, leading to a final acetic acid concentration at
the end of the fermentation of only 0.5 g l−1. Formic and acetic acid
production from a B. coagulans strain has also been reported by Payot
et al. [46], when molasses were employed as fermentation substrate in
continuous mode. Genomic analysis carried out in the study of Su and
Xu [47], indicated that B. coagulans can utilize hexoses using an
homofermentative pathway, but by-products like acetic acid and suc-
cinic acid could be also formed. In other lactic acid producers, like L.
plantarum, formate and acetate production are connected to limiting
glucose concentrations in the fermentation medium [1]. This fact could
provide a possible explanation for our experiment, since the production
of both by-products was also observed when no glucose was detected in
the substrate.
As it is shown in Fig. 2, high cell densities were achieved (almost
55 g·l−1 of DCW) and the living cells (5.0–9.0 cfu x 1012 per l) were
quite stable throughout the process. In this point it is important to stress
that there was no membrane fouling during the process.
Comparing the obtained result with the cited publications presented
in Table 2, the attained lactic acid productivity was similar to the values
obtained when pure glucose was utilized as fermentation substrate
[13,22]. Cassava starch was also investigated as fermentation substrate
by Bomrungnok et al. [48] in conventional continuous fermentation
mode and coupled with hollow-fiber membranes (Table 2). Even
though final lactic acid concentration resulted similar for the two
strategies (18.96 g·l−1 and 20 g·l−1, respectively), productivity was 2-
fold higher (Table 2). Li et al. [49], tested defatted rice bran hydro-
lysate-another starch-rich material- for continuous fermentations using
immobilized cultures of L. rhamnosus. The authors achieved high lactic
acid productivities (6.2 g·l−1 h−1) and concentrations (86 g·l−1) when
operating at a dilution rate of 0.25 h-1.
J.P. López-Gómez et al.
6.6. Continuous fermentation using molasses
Molasses are one of the main by-products derived after sugar ex-
traction, and their high sugar content combined with their low market
price, have attracted the interest as renewable substrate for bio-
technological applications [50].
The B. coagulans isolate, strain A40 was selected for this case study,
due to its ability to utilize sucrose via homofermentative pathway. The
initial sugar concentration was almost 60 g·l−1, and sucrose assimila-
tion started without any evident lag phase (Fig. 3). Continuous mode
started after 8 h of batch cultivation, when the sugar concentration in
the substrate was 2.2 g·l−1. At this phase, the strain had already pro-
duced 45 g·l−1 of lactic acid, with a yield of 0.63 g·g−1 on total sugars
and a productivity of 5.63 g·l-1 h-1. The dilution rate was kept at 0.1 h−1
during the entire process. The total sugar concentration in the feeding
solution was 124.3 g·l−1.
After 18 h (10 h of continuous cultivation), steady state conditions
were achieved, in which no sugars were left unconsumed in the medium
and the average lactic acid production was approximately 49 g·l−1. The
average yield at this specific time frame (18–31 h) was increased to
0.83 g·g−1, whilst the average productivity presented a value of 4.89 g·l-
h . After 31 h, a new feeding solution was prepared with 153.9 g·l−1
1 -1
of total sugars since the strain was able to consume all sugars in steady
state, which was utilized until the 80th h of fermentation. Average
lactic acid concentration increased to 61 g·l−1 and approximately
1 g·l−1 of sucrose was detected in the medium. During this period,
average yield slightly increased to 0.87 g·g−1 and the productivity to
6.1 g·l-1 h-1.
Between 80–103 h of fermentation, the feeding solution contained
Fig. 3. Continuous fermentation using molasses with the strain B. coagulans A40. Sucrose (◼), glucose (○), fructose (▲), lactic acid (●), DCW (◊), cfu per L (♦).
7
Process Biochemistry xxx (xxxx) xxx–xxx
Fig. 2. Continuous fermentation using tapioca starch hydro-
lysate with the strain B. coagulans A107. Maltose (◼), glucose
(○), lactic acid (●), formic acid (×), acetic acid (*), DCW (◊),
cfu per L (♦).
168.7 g·l−1 of sugars, aiming to assess the feasibility of the strain to
tolerate higher sugar concentrations. Apparently, either the sugar
concentration or other potential inhibitory compounds present in mo-
lasses resulted in sugar accumulation and decreased the yield
(0.83 g·g−1) [50]. The last feeding solution contained again 153.9 g·l−1
of total sugars. Both yield and productivity were stabilized at the same
values as before (0.86 g·g−1 and 6.23 g·l-1 h-1), and the residual sugars
in the medium were less than 5 g·l−1. In total, this continuous fer-
mentation lasted for 167 h, and the average values of lactic acid con-
centration, yield and productivity were 59.1 g·l−1, 0.85 g·g−1 and
5.91 g·l-1 h-1, respectively. Even though this process lasted longer
(170 h) that in the case study using tapioca hydrolysate, membranes
were not blocked even with a high DCW (20 g·l−1).
Sugar molasses have been mainly evaluated in batch mode using
Lactobacillus strains [51–54] and E. faecalis [55,56]. Wee et al. [55]
highlighted the importance of yeast extract concentration in these fer-
mentations, concluding that 20 g·l−1 of yeast extract resulted in the
highest lactic acid productivity (5.3 g·l-1 h-1). Göksungur and Güvenç
[57] compared batch and continuous cultures of L. delbrueckii for lactic
acid production from beet molasses. The authors reported a 2.3-fold
increment in productivity when operating in continuous mode. More-
over, the authors attempted to replace yeast extract with cheaper
sources (malt sprouts, corn steep liquor, cotton oilcake, soy flour and,
urea). Their results showed that yeast extract was the most adequate
nitrogen source, but malt sprouts and corn steep liquor were also pro-
mising alternatives.
J.P. López-Gómez et al.
Fig. 4. Continuous fermentation using acid whey with the strain L. coryniformis subsp. torquens. Lactose (◼), lactic acid (●), acetic acid (*), DCW (◊), cfu per L (♦).
6.7. Continuous fermentations with acid whey as substrate
Acid whey is an important by-product stream generated during
making of acid-coagulated dairy-products, such as yogurt and cream
cheese [58]. In contrast to sweet whey, that can be processed and then
sold as protein powder, there is not yet an efficient process for the
handling of acid whey.
An alternative to the valorization of acid whey could be the pro-
duction of D-lactic acid. In this case study, the strain L. coryniformis
subsp. torquens was selected as an efficient D-lactic acid from lactose,
and the kinetics of the continuous fermentation is presented in Fig. 4.
Initial lactose concentration was approximately 82 g·l−1 and 1.4 g·l−1
of lactic acid were still present in the substrate after nanofiltration. The
batch phase lasted longer in comparison to the previous case studies
that a B. coagulans strain was employed. Continuous mode started after
39 h of cultivation, when residual lactose was almost 2 g·l−1 and D-
lactic acid concentration had a value of 45.1 g·l−1. Dilution rate was set
at 0.2 h−1 for the entire process. For the batch phase, lactic acid yield
and productivity were 0.47 g·g−1 and 1.2 g·l-1 h1, respectively.
Initially, lactose was accumulating in the medium, reaching a con-
centration of 28 g·l−1 after 45 h of fermentation (or 6 h of continuous
cultivation). Steady state conditions were achieved after approximately
57 h, when residual lactose was between 4-5 g·l−1, and the average
lactic acid concentration was 46 g·l−1. Lactic acid yield on total sugars
was considerably enhanced, having an average value of 0.92 g·g−1,
almost 50% higher in comparison to the batch phase, whilst average
productivity presented a value of 9.2 g·l-1 h-1. Acetic acid was also
produced during the process, but its concentration did not exceed
2 g·l−1. The produced D-lactic acid presented an optical purity of more
than 99%, indicating that acid whey is a promising alternative substrate
for D-lactic acid production. The summary for the results obtained for
the three case studies can be seen in Table 2.
Continuous acid whey fermentation for D-lactic acid production has
not been studied so far. However, batch or fed-batch cultivations for
lactic acid production with different Lactobacillus strains have proven to
be a promising way to valorize this abundant waste stream [59,60].
Ramchandran et al. [60], utilized submerged polymeric hollow fibres
for lactic acid production from a whey-based medium using the strain
Lactococcus lactis spp. cremoris. The authors reported similar pro-
ductivities (9.7 g·l−1 h−1) to the ones achieved in this case study
(Table 2).
7. Conclusions
This review has provided an outline of the recent investigations and
developments on the production of lactic acid in continuous mode.
Although habitually produced in batch mode, the production of lactic
8
Process Biochemistry xxx (xxxx) xxx–xxx
acid in continuous processes presents numerous advantages. Unlike in
batch processes, problems related to product inhibition are eliminated
in continuous lactic acid fermentation. Moreover, the possibility of
maintaining cell growth at its maximum rate allows enhancing pro-
ductivities in continuous process. Furthermore, results showed that, in
general, integrated cell recycling systems can lead to high lactic acid
productivities and higher substrate conversions yields, thus increasing
the efficiency of the upstream process. From an economic point of view,
a continuous fermentation can be beneficial since the process can be
run for longer periods. These longer processes mean that less inoculums
and fewer stops are required, reducing operation costs. There are still
however, challenges that need to be addressed in order to add value to
the processes. One of the most important aspects is the development of
new more robust strains. These new organisms would be able to work at
lower conditions of pH, be more resistant to the substrate and lactic
acid as well as being able to consume the variety of sugars present in
inexpensive substrates.
The case studies for L- and D-lactic acid production using in-
expensive substrates (tapioca starch hydrolysate, molasses combined
with rapeseed hydrolysate, acid whey) reinforce the statement that
long-term stability of cell recycle systems is feasible. Lactic acid pro-
ductivities higher than 5 g·l−1 h−1 and substrate conversion yields
above 80% were achieved for all the cases.
In conclusion, the recent results and developments suggest that a
transition in the production of lactic acid from batch to continuous
mode is not only feasible but would also report benefits in terms of
productivities and production costs.
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