fermentation

Article
Production and Purification of l-lactic Acid in Lab
and Pilot Scales Using Sweet Sorghum Juice
Agata Olszewska-Widdrat 1 , Maria Alexandri 1 , José Pablo López-Gómez 1 , Roland Schneider 1 ,
Michael Mandl 2 and Joachim Venus 1, *
1 Leibniz Institute for Agricultural Engineering and Bioeconomy, 14469 Potsdam, Germany;
aolszewska-widdrat@atb-potsdam.de (A.O.-W.); malexandri@atb-potsdam.de (M.A.);
pLopezGomez@atb-potsdam.de (J.P.L.-G.); rschneider@atb-potsdam.de (R.S.)
2 tbw research GesmbH, 1120 Wien, Austria; m.mandl@tbwresearch.org
* Correspondence: jvenus@atb-potsdam.de; +49-0331-5699-852




Received: 7 March 2019; Accepted: 19 April 2019; Published: 26 April 2019


Abstract: Sweet sorghum juice (SSJ) was evaluated as fermentation substrate for the production
of l-lactic acid. A thermophilic Bacillus coagulans isolate was selected for batch fermentations
without the use of additional nutrients. The first batch of SSJ (Batch A) resulted on higher lactic
acid concentration, yield and productivity with values of 78.75 g·L−1 , 0.78 g·g−1 and 1.77 g·L−1 h−1 ,
respectively. Similar results were obtained when the process was transferred into the pilot scale (50 L),
with corresponding values of 73 g·L−1 , 0.70 g·g−1 and 1.47 g·L−1 h−1 . A complete downstream process
scheme was developed in order to separate lactic acid from the fermentation components. Coarse and
ultra-filtration were employed as preliminary separation steps. Mono- and bipolar electrodialysis,
followed by chromatography and vacuum evaporation were subsequently carried out leading to
a solution containing 905.8 g·L−1 lactic acid, with an optical purity of 98.9%. The results of this
study highlight the importance of the downstream process with respect to using SSJ for lactic acid
production. The proposed downstream process constitutes a more environmentally benign approach
to conventional precipitation methods.

Keywords: fermentation; lactic acid; downstream; sweet sorghum juice; Bacillus coagulans

1. Introduction
Lactic acid (LA) is an organic acid which can be produced by lactic acid bacteria for many purposes,
starting from food preservatives and finishing at medicines [1]. The increasing demand for poly-lactic
acid (PLA), a biodegradable plastic, has also boosted lactic acid’s market [2]. Production of PLA
requires high optical purity of the biotechnologically produced LA [3]. To this end, LA production
requires many steps, starting from the pre-treatment of the specific feedstock, going through the
hydrolysis, fermentation and finishing at the separation and purification, the so called downstream [4].
The downstream process during biological manufacturing of LA is still an important challenge, further
complicated by the utilization of cheap and complex substrates. Efficient LA production has been
reported from various alternative substrates such as sugarcane, food waste, coffee pulp, acid whey,
molasses or avocado seeds to name a few [4–9]. Recently, there has been a lot of interest in lignocellulosic
materials as fermentation feedstocks [9,10]; nevertheless, other easier accessible substrates can be taken
into consideration for the production of LA.
Sweet sorghum is the most utilized crop for bio-based chemicals production in China, but it
has been also recognized worldwide as an interesting feedstock [11]. It has already been used for
the production of bioethanol or in two stage ethanol-methane production [12,13] and it is one of
the most common feedstocks for bio-butanol production [14]. Sweet sorghum juice (SSJ) has also

Fermentation 2019, 5, 36; doi:10.3390/fermentation5020036 www.mdpi.com/journal/fermentation

Fermentation 2019, 5, 36 2 of 10

been used for the fermentative production of l-lactic acid. Wang et al. [15] used Bacillus coagulans
for repeated batch fermentations of acid hydrolysate of SSJ and their results showed a maximum
productivity of 2.90 g·L−1 h−1 and a yield of 0.943 g·g−1 . On the other hand, Lactobacillus rhamnosus
was used in batch fermentation coupled with a membrane separation in order to improve l-lactic acid
productivity [16]. In this case, repeated batch fermentations in a 7 L bioreactor were performed, in
which the yield and the productivity reached 0.954 g·g−1 and 17.55 g·L−1 h−1 respectively. B. coagulans
is a very promising candidate for lactic acid production due to its temperature resistance, robustness
and high LA productivity [17]. B. coagulans was also used as a platform organism in multi-substrate
utilization [6].
Many methods have been proposed in the literature for the separation and purification of lactic
acid from the fermentation broth [18]. The conventional process involves lactic acid precipitation
using calcium hydroxide. The recovery of lactic acid is usually performed by using an excess of H2
SO4 . This process generates high amounts of CaSO4 , as waste stream [3]. Consequently, the purity
of lactic acid decreases and together with chemicals used and waste streams produced, it is not an
overall environmentally benign process. Research is currently focusing on alternative methods for the
recovery of lactic acid from complex fermentation broths. Among the proposed methods, the most
promising ones so far seem to be ultra- and nanofiltration, electrodialysis, ion-exchange/adsorption,
reactive distillation and hybrid short path evaporation [3,19]. However, most of these methods have
only been tested in model lactic acid solutions or in well-defined media. Only a few studies so far have
performed downstream separation of lactic acid from complex fermentation media [6,13,14,20–22].
Combining these two approaches, the utilization of B. coagulans and SSJ created a perfect match
for the development of a downstream process, especially because all of the above-mentioned studies
showed only the utilization of SSJ for LA production, but none of those were performed at pilot scale
followed by separation and purification of LA. In this study, lab scale experiments were initially carried
out using SSJ as sole carbon and nutrient source. Subsequently, the process was transferred in pilot
scales, aiming to investigate the scalability of the proposed scheme. Finally, a downstream process was
applied in order to optimize purification steps with respect to SSJ. For this purpose, a set of filtration
systems was used, equipped with ceramic membranes. Afterwards electrodialysis was performed,
followed by decolorization and chromatography techniques in order to remove remaining ions. Finally,
vacuum distillation was employed in order to obtain a high concentrated lactic acid solution. To our
knowledge, besides the work published by the group of Wang et al. [14,15,23], SSJ has not been fully
investigated as a fermentation feedstock, or in pilot scales. Moreover, complete lactic acid separation
schemes from cheap renewable resources are scarce in the literature, so this work will contribute in the
development of efficient downstream processes.

2. Materials and Methods

2.1. Substrate
Sweet sorghum juice was produced by using a mechanical screw press (Kufferath Akupress A500)
for pressing fresh feedstock. Sweet sorghum feedstock consisted of biomass from two different species
(Sweet Chopper and Sugar Grace). Feedstock was cultivated in 2009, harvested in the last week of
September 2009 using a Class Jaguar harvester for chopping. Pressing of fresh feedstock was executed
on the same day, only some hours after cutting. The disintegrated chopped feedstocks, containing
approximately 20 mm or smaller solids, were continuously dosed into a mechanical screw press, at low
rotation speed (12–14 rpm). The exit of the screw press was guided by pneumatically controlled counter
cone in order to build up the necessary pressure (5 bar) to initiate the dewatering of the material.
The fractionation of sweet sorghum was done in a single step pressing to recover a pure juice without
adding any additional water. A course hydro sieve was used to separate fiber residues present in the
juice. Directly after, the sieve fresh juice was recovered in two different batches. The fresh juice had a
greenish color and it was slightly foaming. Fresh SSJ was immediately cooled after pressing and deep
Fermentation 2019, 5, 36 3 of 10

frozen (−20 ◦ C) on the same day. The screw press was able to generate typical fresh juice samples from
sweet sorghum, but sugar extraction was not optimized. The latter would include several fractionation3
Fermentation 2019, 5 FOR PEER REVIEW
steps to allow complete sugar extraction from feedstock. Deep frozen batches of juice were delivered
toMarch
ATB for
andfermentation experiments
June of the year 2010. The incomposition
March and June of two
of the the year 2010.
batches The composition
(Batch A and B) was ofanalyzed
the two
batches (Batch A and B) was analyzed in terms of sugar, nitrogen, phosphorus
in terms of sugar, nitrogen, phosphorus and ion content using methods described below. and ion content using
methods described below.
2.2. Microorganism
2.2. Microorganism
The B. coagulans A-35 strain used for all the experiments was isolated from alfalfa press residue
The B. coagulans
and characterized A-35
using strain
matrix used laser
assisted for all the experiments was
desorption/ionization isolated from
time-of-flight mass alfalfa press
spectrometry
residue
(MALDI-ToF MS) method. The strain was also identified by the Deutsche Sammlungmass
and characterized using matrix assisted laser desorption/ionization time-of-flight von
spectrometry
Mikroorganismen (MALDI-ToF MS) method.
und Zellkulturen GmbH.The strain was also
The strain identified
is available at by
thethe
ATBDeutsche Sammlung
collection and it is
von Mikroorganismen
being und Zellkulturen
stored as a cryo-stock GmbH. The
at −80 °C. Inoculum strain is available
preparation at the
was carried outATB collection
in shake flasksand
withit De
is
being stored as a cryo-stock at −80 ◦ C. Inoculum preparation was carried out in shake flasks with De
Man, Rogosa and Sharpe (MRS) broth (Merck, Darmstadt, Germany) with 0.67 g Everzit Dol (Evers
Man,
e.K., Rogosa
Hopsten, and Sharpe (MRS)
Germany) broth
dolomite as (Merck, Darmstadt,
buffer. The strain wasGermany)
incubatedwith 0.67
at 52 °CgforEverzit Dol
14 h in an(Evers
orbital
e.K., Hopsten, ◦
Germany) dolomite as buffer. The strain was incubated at 52 C for 14 h in an orbital
shaker at 100 rpm.
shaker at 100 rpm.
2.3. Fermentation
2.3. Fermentation
2.3.1.Laboratory
2.3.1. LaboratoryScale
ScaleFermentations
Fermentations
Lab-scalefermentations
Lab-scale fermentationsusingusingthethe different
different batches
batches of sweet
of sweet sorghum
sorghum juicejuice
werewere carried
carried out
out in 5 Lin
5 L BIOSTAT bioreactors (Sartorius AG, Goettingen, Germany), with 3
BIOSTAT bioreactors (Sartorius AG, Goettingen, Germany), with 3 L working volume. Temperature L working volume.
Temperature
and and
stirring were setstirring
at 52 ◦ Cwere set at
and 200 52 °C
rpm, andthe
while 200
pHrpm,
waswhile the pH was
continuously continuously
adjusted to 6 usingadjusted
20% (w/w) to
6 usingInoculum
NaOH. 20% (w/w)was
NaOH. Inoculum
2% (v/v) wasstudied
in all the 2% (v/v)cases.
in all Due
the studied cases.limitation,
to substrate Due to substrate limitation,
the experiments
the experiments were
were carried out only once.carried out only once.

2.3.2.Pilot
2.3.2. PilotScale
ScaleFermentation
Fermentation
Thepilot
The pilotscale
scalefermentation
fermentationwas
wascarried
carriedout
outin
inaa72
72LL BIOSTAT
BIOSTATUD UDbioreactor
bioreactor(B-Braun
(B-BraunBiotech,
Biotech,
Hessen,Germany),
Hessen, Germany),withwith50
50LLworking
workingvolume.
volume.The
Thesweet
sweetsorghum
sorghumjuice
juicewas
wasautoclaved
autoclavedatat121
121◦ C
°C
for 15 min, before inoculation. The fermentation was performed at 52 ◦ °C and 200 rpm,
for 15 min, before inoculation. The fermentation was performed at 52 C and 200 rpm, and the pH and the pH
wasmaintained
was maintainedatat66bybyadding
adding20%
20%(w/w)
(w/w)NaOH.
NaOH.Inoculum
Inoculumwaswasgrown
growninin11LLMRS MRSbroth
brothfor
for14
14hhatat
52 °C.
52 ◦ Atthe
C. At theend
endofofthe
thefermentation,
fermentation,the
thebroth
brothwas
wasinactivated
inactivatedatat90
90 °C
◦ for30
C for 30min
minininorder
ordertotobe
be
further processed.
further processed.

2.4.Downstream
2.4. DownstreamProcess
Process
AAschematic
schematicdiagram
diagramdescribing
describingthe
thedownstream
downstreamprocess
processisisshown
shownininFigure
Figure1.1.

Figure 1. Schematic diagram of the downstream process of lactic acid.

Figure 1. Schematic diagram of the downstream process of lactic acid.
2.4.1. Filtration and Softening
2.4.1. Filtration and Softening
A pre-filtration step (coarse filtration) was initially carried out using filter bags (Schwegmann
A pre-filtration
Filtrations-Technik step (coarse
GmbH, filtration) was
Grafschaft-Ringen, initiallywith
Germany), carried out pore
150 µm usingsize.
filter bags
The (Schwegmann
permeate stream
Filtrations-Technik GmbH, Grafschaft-Ringen, Germany), with 150 µm pore size. The permeate
stream was subsequently subjected to ultra-filtration at 1.5 bar using an UFI-TEC cross-flow
filtration system (UFI-TEC, Oranienburg, Germany) equipped with four ABB ceramic membranes
(UFI-TEC) with 0.1 µm pore size.
Cations removal (softening) was carried out using PUROLITE S950 acid chelating resin
(Purolite, Ratingen, Germany) packed in an expanded bed setting. Initially the pH value of the
Fermentation 2019, 5, 36 4 of 10

was subsequently subjected to ultra-filtration at 1.5 bar using an UFI-TEC cross-flow filtration system
(UFI-TEC, Oranienburg, Germany) equipped with four ABB ceramic membranes (UFI-TEC) with
0.1 µm pore size.
Cations removal (softening) was carried out using PUROLITE S950 acid chelating resin (Purolite,
Ratingen, Germany) packed in an expanded bed setting. Initially the pH value of the permeate stream
was adjusted to 10 by adding 20% NaOH. The permeate was introduced in the column from below at a
flow of 6 bed volumes per h. Separation was complete when conductivity of the purified water was
below 1 mS cm−1 [6].

2.4.2. Electrodialysis
The filtrate obtained from the softening column was afterwards concentrated via monopolar
electrodialysis. A free lactic acid solution was achieved after bipolar electrodialysis, together with a
NaOH solution. Both electrodialyses were carried out in batch mode, under constant polarity and at a
temperature of 35 ◦ C. Monopolar electrodialysis was composed by a sheet flow stack having 11 cation
exchange membranes Type II (Fujifilm, Tilburg, the Netherlands) and 10 anion exchange membranes
Type II (Fujifilm), operating at 20 V and 3 A. Conductivity values of the diluate below 0.5 mS cm−1
indicated the end of the process [6].
The concentrated stream produced via monopolar electrodialysis was then introduced to bipolar
electrodialysis. Bipolar electrodialysis consisted also of 11 cation exchange membranes Type II (Fujifilm)
operating at 30 V and 5 A and 10 anion exchange membranes Type II (Fujifilm) operating at 20 V and
3 A. The process was finished when the conductivity of the salt stream was approximately 5 mS·cm−1 .
The acid stream was used for the following purification steps [6].

2.4.3. Decolorization and Chromatography

The removal of remaining cations and anions was carried out using cation and anion exchange
chromatography. The strong cation exchange resin RELITE EXC 08 (Resindion S.R.L., Binasco, Italy)
was initially applied followed by the weak anion exchange resin RELITE EXA 133 (Resindion S.R.L.).
The packed columns had a 2 L volume and flow was set at 6 bed volumes per h. At the end of the
process both columns were cleaned with water and regenerated. The strong acid resin PUROLITE
MN-502 (Purolite) was employed in order to remove the color impurities. The filtrate was finally
vacuum evaporated using a vacuum distillation plant (Büchi Labortechnik, Essen, Germany) at 55 ◦ C,
0.05 bar and 200 rpm.

2.5. Analytical Assays

The determination of sugar content and lactic acid concentration was carried out by HPLC
(DIONEX, Sunnyvale, CA, USA), coupled with a refractive index detector (RI-71, SHODEX, Yokohama,
Japan) and equipped with a Eurokat H column (300 mm × 8 mm × 10 µm, Knauer, Berlin, Germany),
eluted with 5 mM H2 SO4 at a flow rate of 0.8 mL·min−1 . An IonPac CS 16 column (250 mm ×
4 µm, DIONEX, Sunnyvale, CA, USA) was used for the analysis of cations in the sweet sorghum
juice and fermentation samples, operating at a flow rate of 1.0 mL·min−1 , at 40 ◦ C, with 30 mM CH3
SO3 H as mobile phase. The analysis of anions was carried out using an IonPac AS9-HC column
(250 mm × 4 µm, DIONEX, Sunnyvale, CA, USA), eluted with Na2 CO3 at a flow rate of 1.2 mL·min−1 ,
at room temperature.
Lactic acid optical purity analysis was carried out using HPLC (Knauer, Berlin Germany) coupled
with a Chiralpak® MA(+) column (Daicel, Tokyo, Japan, 50 mm × 4.6 mm × 3 µm), using 2 mM CuSO4
as mobile phase at a flow rate of 0.8 mL·min−1 . Detection was carried out with an ultraviolet detector.
Protein content was measured following the [24] standard method. Flow injection analysis (FIA)
was employed for the determination of total phosphorus (P) content, according to the international
standard [25].
Fermentation 2019, 5, 36 5 of 10

3. Results and Discussion

3.1. Optimization of the Fermentation Process

As shown in Table 1, the sweet sorghum juice mainly consisted of sucrose, fructose and glucose,
with sucrose being the predominant sugar with a concentration of more than 60 g·L−1 , for both batches.
The analytical composition of the substrate was very similar to the one presented by Di Cai et al.
regarding sugar content [26]. The sweet sorghum juice also contained nitrogen and phosphorus, giving
the possibility to be utilized as the sole nutrient source in lactic acid fermentations.

Table 1. Chemical composition of two different batches of sweet sorghum juice.

Component Batch A Batch B

Sucrose (g·L−1 ) 66.78 68.87
Glucose (g·L−1 ) 24.87 23.43
Fructose (g·L−1 ) 13.46 17.51
Total nitrogen (mg·L−1 ) 1013.15 957.00
Total Phosphorus (mg·L−1 ) 422.40 355.00
Cl− (mg·L−1 ) 150.9 143.00
SO4 2− (mg·L−1 ) 189.67 253.00
Na+ (mg·L−1 ) 3.82 11.1
K+ (mg·L−1 ) 5183.8 4347.00
Mg2+ (mg·L−1 ) 333.33 295.00
Ca2+ (mg·L−1 ) 668.71 565.00

3.2. Lab Scale Fermentations Using SSJ as Sole Nutrient Source

Lab scale batch fermentations were carried out using batches A and B of sweet sorghum juice.
Initial sugar content did not differ significantly between the batches with approximately 105 g·L−1 for
A and 109 g·L−1 for B (Table 1). Additionally, the distribution of sugars was similar in both batches
with around 51–53% sucrose, 29–31% glucose and 16–17% fructose. Figure 2 shows the fermentation
profiles for the two batches tested. As seen in the figure, both fermentations had a similar behavior
with final LA concentrations of 78.75 and 72.71 g·L−1 for batch A and batch B respectively. There was
an apparent glucose repression over disaccharides and fructose, most noticeable in Figure 2A. Glucose
consumption occurred rapidly with a complete depletion after only 10 h of fermentation. During
the same period, the concentration of disaccharides did not show a significant reduction in batch
A, while fructose concentration barely changed in both fermentations. Once glucose was depleted,
the consumption of other sugars occurred at a faster rate and by 50 h, when the fermentations were
stopped, no residual sugars were present in batch B (Figure 2B). Only 11 g·L−1 of sugars remained in
batch A at 50 h and most likely, since the concentration values had not reached a plateau, residual
sugars concentration would have been lower if the fermentation had continued. In spite of that,
the yield for batch A was higher with a value of 0.78 g·g−1 compared to 0.68 g·g−1 for batch B. LA
production occurred faster during the first 10 h of fermentation, reaching about 30 g·L−1 in both cases
and a productivity of approximately 5 g·L−1 h−1 from the time that the exponential phase started
to the 10 h mark. The production rate slowed down after 10 h, possibly due to nutrients limitation.
Nonetheless, overall productivities were 1.77 and 1.63 g·L−1 h−1 for A and B which are promising
considering that the fermentations were carried out without the addition of any extra nutrients. It was
possible to overcome the limiting factors, such as lower sugar concentration when compared with
other studies done by Wang et al. [27].
 Fermentation 2019,
2019,5,5536
Fermentation2019, FOR
FOR PEER
PEER REVIEW
REVIEW 6
Fermentation 6 of 106

(A)
(A) (B)
(B)
Figure
Figure2.2.
Figure Fermentative
2. Fermentative production
Fermentative production of
production of lactic
of lactic acid
lactic acid (●)
acid () and
(●) and consumption
and consumption of
consumption of glucose
of glucose (○);
glucose (#); fructose ▼;
(○); fructose
fructose ▼;
H;
disaccharides (∆)
disaccharides(∆)
disaccharides by
(∆)by B.coagulans
byB.coagulans in
B.coagulansin two
intwo different
twodifferent batches
differentbatches (A,B)
batches(A,B) of SSJ.
(A,B)ofofSSJ.
SSJ.

3.3.
3.3. Pilot
3.3.Pilot Scale
PilotScale Fermentation
ScaleFermentation using
Fermentationusing SSJ
usingSSJ
SSJ
Due
Duetoto
Due the
tothe higher
thehigher
higher LALALA yields
yields
yieldsandand productivity
productivity
and productivityvalues values obtained
obtained
values in thein
obtained the
the previous
inprevious fermentations,
fermentations,
previous batch
fermentations,
Abatch
was A
batch A was
was selected
selected for pilotfor
selected for pilot
scale scale
scale experiments.
experiments.
pilot Fermentation
Fermentation
experiments. was
was carried
Fermentation wasoutcarried
using
carried out50using
out 50
50 L
L of batch
using L of
A batch
of and
batchtheA
A
and the
profile of profile
the of
process the
is process
shown inis shown
Figure 3. in
As Figure
in the 3.
lab As in
scale the lab scale
experiments,
and the profile of the process is shown in Figure 3. As in the lab scale experiments, the glucose experiments,
the glucose the glucose
concentration
concentration
decreased
concentration decreased
rapidly and was
decreased rapidly
totally
rapidly and
and was
was totally
consumed consumed
after
totally after
after 13
13 hh of
13 h of fermentation.
consumed of fermentation.
Together withTogether
fermentation. glucose,with
Together the
with
glucose,
glucose, the
consumption the ofconsumption
disaccharidesof
consumption of disaccharides
started rapidly with
disaccharides started rapidly
an apparent
started with
with an
rapidlydecrease anin apparent
its decrease
consumption
apparent decreaseratein its
after
in its
consumption
13 h. As in therate after
previous 13 h.
cases Asthe in the previous
consumption cases
of the
fructose consumption
was noticeable
consumption rate after 13 h. As in the previous cases the consumption of fructose was noticeable of fructose
only was
after noticeable
glucose was
only
only after
depleted. glucose
afterSugars
glucose was
were
was depleted.
completely Sugars
Sugars were
depleted.consumed completely
after
were consumed
60 h of fermentation.
completely consumed By after
the60
after hh of
end
60 fermentation.
of the fermentation,
fermentation. By
By
the −1 −1
LA end of
reached the
a fermentation,
maximum LA reached
concentration ofa maximum
approximately concentration
73 g·L of
the end of the fermentation, LA reached a maximum concentration of approximately 73 g∙L withaaa
with approximately
a yield of 73
0.70 g∙L
g·g with
and
−1
−1

yield
yield of
of 0.70
productivity
0.70 ofg∙g
g∙g1.47 g·Laa−1productivity
−1 and
−1 and h−1 .
productivity of
of 1.47
1.47 g∙L
g∙L−1 hh−1..
−1 −1

Figure 3. B.
Figure3.3.
Figure B. coagulans
coagulans fermentations
fermentations carried
carried out
out in
in pilot
pilot scale
scale (50
scale (50 L)
(50 L) with
L) with SSJ
with SSJ as
SSJ as sole
as solecarbon
sole carbonand
carbon and
and
nutrient
nutrient source.
source. Fermentative
Fermentativeproduction
production of
oflactic acid
lactic ()
acid and
(●) andconsumption
consumption of glucose
of glucose(#); fructose
(○);
nutrient source. Fermentative production of lactic acid (●) and consumption of glucose (○); fructose H;
fructose
▼;
disaccharides (∆).
▼; disaccharides
disaccharides (∆).
(∆).

SSJ
SSJ has been tested for the fermentative production of various products as shown in Table 2.
SSJ has
has been
been tested
tested for
for the
the fermentative
fermentative production
production of of various
various products
products as as shown
shown in in Table
Table 2.2.
Regarding
Regarding l-lactic acid production, the highest yield and productivity have been achieved by employing
Regarding LL-lactic
-lactic acid
acid production,
production, the the highest
highest yield
yield and
and productivity
productivity have have been
been achieved
achieved byby
B. coagulans strain coagulans
aemploying in repeated batch fermentations [15]. However, SSJ was supplemented with yeast
employing aa B. B. coagulans strainstrain inin repeated
repeated batch
batch fermentations
fermentations [15]. [15]. However,
However, SSJ SSJ was
was
extract and
supplemented soya peptone, in contrast to this study that SSJ was utilized as sole carbon and nutrient
supplemented with yeast extract and soya peptone, in contrast to this study that SSJ was utilized as
with yeast extract and soya peptone, in contrast to this study that SSJ was utilized as
source.
sole To the authors’ knowledge, To this is the first study in whichthisSSJ is tested in pilot scales for l-lacticis
sole carbon
carbon and
and nutrient
nutrient source.
source. To the the authors’
authors’ knowledge,
knowledge, this is is the
the first
first study
study inin which
which SSJ
SSJ is
acid production,
tested showingL-lactic
the industrial feasibility of the process.
tested in
in pilot
pilot scales
scales for
for L-lactic acid
acid production,
production, showing
showing the
the industrial
industrial feasibility
feasibility of
of the
the process.
process.
Fermentation 2019, 5, 36 7 of 10

Table 2. Comparison between this work and previous studies on lactic acid and other fermentation
products from SSJ.

Fermentation Nitrogen Volume Yield Productivity

Product Microorganism Ref.
Type Source (L) (g·g−1 ) (g·L−1 h−1 )
Sporolactobacillus
d-lactic acid Fed-batch YE* 0.3 0.96 1.55 [4]
inulinus
Repeated YE & Soya
Bacillus coagulans 3 0.93 2.45 [15]
batch peptone
YE/Corn Bacillus
Batch and
steep coagulans/Lactobacillus 2 0.92 1.84 [27]
purification
l-lactic acid powder rhamnosus
Batch and Lactobacillus
YE 1 0.93 0.6 [5]
purification plantarum
Pilot scale and
- Bacillus coagulans 50 0.70 1.47 This study
downstream
YE & soya Corynebacterium
Lysine Batch 1 0.22 - [11]
peptone glutamicum
(NH4 )2
Saccharomyces
Ethanol-methane Batch SO4 /(NH2 )2 0.3/0.8 0.89 24.7 [13]
cerevisiae
CO/Urea
Acetone-butanol- Ammonium Clostridium
Batch 1.5 0.41 0.53 [26]
ethanol acetate acetobutylicum
* Yeast Extract.

3.4. Downstream Process of the Lactic Acid Produced in Pilot Scale

The utilization of lactic acid for high value applications requires high optical purity. Even though
no residual sugars were left in the medium after fermentation, residual proteins, phosphorus and
ions were still present in high concentrations (Table 3). Several steps were carried out in order to
separate lactic acid from the rest of the fermentation’s components. After the fermentation, a coarse
filtration step was employed in order to separate bigger particles that could possibly damage or
block the ultra-filtration membranes. Low LA losses of <10% were observed in this step, as well as
a slight decrease of all the other components of interest (total nitrogen and phosphorus, Cl− , SO4 2− ,
Na+ , K+ , Mg2+ , Ca2+ ). Ultra-filtration was then carried out, mainly in order to separate the biomass.
The majority of LA was found in the permeate stream (70.63 g·L−1 ), corresponding to a recovery rate of
92.1%. This step also contributed to a removal of approximately 37.2% of total nitrogen.
Among the alternative technologies that have been proposed in the literature for the downstream
separation and purification of lactic acid from complex fermentation broths, electrodialysis has been
proven as a promising alternative [7,28,29]. In this study, monopolar electrodialysis (MED) has been
investigated for the concentration of sodium lactate after ultra-filtration and bipolar electrodialysis
(BED) was subsequently employed in order to convert lactate to lactic acid. Before electrodialysis,
a softening step is necessary for the removal of divalent ions (mainly Mg2+ and Ca2+ ), which can cause
fouling of the electrodialysis membranes. As can be seen from Table 3, the concentration of Mg2+ and
Ca2+ after softening was 0.48 mg·L−1 and 6.5 mg·L−1 , respectively, values corresponding to removals of
99.8% and 98.4%. This stream was initially treated with monopolar electrodialysis membranes, which
generated two streams: the concentrate and the diluate. A volume of 20.6 L of concentrate stream
containing 180.1 g·L−1 of sodium lactate was obtained. Additionally, a considerable decrease of 69% in
total nitrogen as well as in total phosphorus (22% removal) was achieved after this step. A second
softening step was again required due to the high concentration of divalent anions in the concentrate
stream. Bipolar electrodialysis was then carried out, resulting in three streams: acid (18.9 L), salt (8.5 L)
and base (32 L). A recovery percentage of 85.6% of lactic acid was observed in the acid stream, whereas
only 13.19 g·L−1 was lost in the base stream.
The acid stream was further treated by cation and anion exchange resins for further removal of
residual ions. By applying chromatography, more than 90% of the monovalent ions were successfully
Fermentation 2019, 5, 36 8 of 10

removed. Before vacuum evaporation, a decolorization step was employed for the removal of residual
compounds contributing in the yellowish color of the stream. Finally a 2.9 L solution containing
905.8 g·L−1 of lactic acid was obtained, whereas the concentration of residual impurities was <1.5 g·L−1 ,
corresponding to an overall lactic acid purity of approximately 99.8% (w/w). Optical purity was slightly
lower in comparison to the end of the fermentation (99.8%), with a value of 98.9%. High lactic acid
purities are of major importance, as already highlighted, especially when the production of PLA is the
final goal. These processing steps resulted at high lactic acid purity; however, the overall lactic acid
yield (from the end of the fermentation until the final distillated solution) was 62.4% meaning that a
considerable amount is lost during the different treatments.

Table 3. Compositional analysis after every step of the downstream process (DSP) of the fermentation
broth, obtained from the pilot scale fermentation, using SSJ as a feedstock.

V LA Ntotal Ptotal Cl− SO4 2− Na+ K+ Mg2+ Ca2+

DSP Step
(L) (g·L−1 ) (mg·L−1 ) (mg·L−1 ) (mg·L−1 ) (mg·L−1 ) (mg·L−1 ) (mg·L−1 ) (mg·L−1 ) (mg·L−1 )
End of
57.6 73.4 780 316 141.3 349 17033 3793 263 499
fermentation
Coarse Filtration 57.5 72.5 844 298 104 259 16831 3665 263 494
Permeate UF 54.4 70.6 560 260 102 231 16427 3514 251 474
Softening 1 61.7 62.5 449 211 87.4 190 15837 2694 0.48 6.5
Concentrate MED 20.6 180.1 415 492 281 536 44111 7363 7.05 26.7
Softening 2 21.1 166.8 323 465 277 555 41349 6989 1.53 12.0
Acid stream BED 18.9 159.7 221 435 265 556 492 73.4 2.74 39.0
Salt stream BED 8.5 n.d. 139 67.0 1.66 17.6 1161 86.2 2.19 49.8
Base stream BED 32.0 13.2 73.5 20.3 39.2 111 27779 41.36 0.98 24.1
Cation exchange 19.5 133.4 125 367 248 494 10.9 0.87 0.13 6.12
Anion exchange 21.0 128.1 109 <8 2.36 169 10.4 0.97 0.21 8.86
Decolorization 23.0 111.6 70.1 <9 2.63 147 10.7 0.63 0.17 1.07
Concentrate LA
2.9 905.8 415.7 24.9 <0.05 966 81.0 2.16 <0.025 <0.5
stream

Even though the production of lactic acid from renewable resources has been studied extensively,
its separation and purification from complex fermentation broths has been seldom investigated. Among
the separation steps used in this study, filtration and chromatography are the most studied processes
so far in the literature. The weak base resin Amberlite IRA-67 was tested by Moldes et al. [30] for the
recovery of lactic acid from Eucalyptus wood hydrolysate. The same resin was evaluated by Garrett et
al. [31] in an extractive fermentation using B. coagulans in corn stover hydrolysate. Their results showed
that the resin was able to maintain the pH of the fermentation for more than 108 days. More than
99% of lactic acid was recovered, but the authors provided no data on the resulting purity. The resin
Amberlite IRA-92 has been investigated for lactic acid recovery from paper sludge [23]. Flow rate and
sample volume load were optimized resulting at lactic acid recovery yield and productivity of 82.6%
and 96.2%, respectively. However, there were not insights on the individual ions that might be still
present in the fermentation broth.
Nanofiltration has been employed as the primary separation step of lactic acid from residual sugars
and other fermentation components from different broths such as coffee mucilage, sugarcane bagasse
hydrolysate, food waste, acid whey as well as sugar bread and crust bread hydrolysates [5,13,14,18].
The optimization of each processing step could enhance the recovery yields of the biotechnologically
produced lactic acid. Nanofiltration and tailor-made resins could be some options for a more
cost-effective and economically viable downstream of l-lactic acid.

4. Conclusions
The results of this study indicate that sweet sorghum juice is a promising substrate for the l-lactic
acid production when a thermophilic B. coagulans strain is employed. However, the lower concentration
of other important nutrients for growth, such as proteins and phosphorus, could be responsible for
the decreased lactic acid yield and productivity when batch B was used (0.68 g·g−1 and 1.63 g·L−1 h−1
respectively). Since batch A resulted in the highest lactic acid production, the same substrate was
Fermentation 2019, 5, 36 9 of 10

tested at a pilot scale fermentation, leading to similar results as in the lab-scale. The effective lactic acid
downstream enabled to reach 99.8% (w/w) product purity, what indicates that the purification process
based on ultra-filtration, electrodialysis, chromatography and distillation was effective.

Author Contributions: J.V. conceived of idea and the concept. Data curation A.O.-W. and R.S.; Supervision J.V.;
Writing-original draft, A.O.-W., M.A., J.P.L.-G. and M.M.; J.V. supervised the development of the work. All authors
discussed the results of the experiments and contributed to the final manuscript.
Acknowledgments: We kindly acknowledge support provided by Ökoenergie Utzenaich GmbH for the production
and harvest of sweet sorghum.
Conflicts of Interest: The authors declare no conflict of interest.

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