fermentation

Article
Lactic Acid Production Using Sugarcane Juice as an Alternative
Substrate and Purification through Ion-Exchange Resins
Priscilla Zwiercheczewski de Oliveira , Luciana Porto de Souza Vandenberghe * and Carlos Ricardo Soccol

Centro Politécnico, Department of Bioprocess Engineering and Biotechnology, Federal University of Paraná,
Curitiba 81531-980, PR, Brazil; [email protected] (P.Z.d.O.); [email protected] (C.R.S.)
* Correspondence: [email protected]

Abstract: The commercial importance of lactic acid (LA) is due to its versatility, especially in the food
industry, and for being the precursor of poly-lactic acid, which demands a high-quality LA precursor.
The overall LA production process still has some bottlenecks related to costs; thus, alternative
substrates such as sugarcane juice may reduce the cost of the fermentation medium and provide a
favorable environment for the Lactobacillus pentosus strain, which continues to be explored. In this
context, this work presents the process of producing LA from sugarcane juice. The LA purification
method is also described using different ion-exchange resins, both in packed columns and in a stirred
tank. The fermentation kinetics showed the highest LA production of 113.74 g/L in 96 h, in which
a productivity of 1.18 g LA/L·h was reached. Among the purification techniques, the combined
use of Amberlite IR120 and IRA-67 resins under agitation in a stirred tank was the best condition,
and resulted in a final LA concentration of 189.11 g/L after 120 min, with 95% LA mass recovery.
This result demonstrates a simplified way to use ion-exchange resins safely and in a controlled
environment, and with process scale-up viability.

Keywords: sugarcane juice; Lactobacillus pentosus; lactic acid; recovery; purification; ion-exchange
resins

Citation: de Oliveira, P.Z.;

Vandenberghe, L.P.d.S.; Soccol, C.R.
Lactic Acid Production Using 1. Introduction
Sugarcane Juice as an Alternative
Lactic acid (LA) is the popular name for 2-hydroxypropanoic acid (C3 H6 O3 ), whose
Substrate and Purification through
molecule has a chiral carbon, which means that there are two optically active isomers: L(+)
Ion-Exchange Resins. Fermentation
and D(-)lactic acid. It has wide industrial application, mainly in the food industry; more
2023, 9, 879. https://doi.org/
recently, its application in the polymerized form of poly-lactic acid (PLA) has become indis-
10.3390/fermentation9100879
pensable in the cosmetic, pharmaceutical and biomedical industries [1]. The production
Academic Editors: Jie Bao and of those LA active forms depends on the production of the microbial strain, resulting in
Bin Zhang significantly different properties when polymerized. LA-producing bacteria (LAB) com-
Received: 29 August 2023
prise a group of bacteria that produce LA as the main metabolic product, such as the strain
Revised: 26 September 2023 Lactobacillus pentosus, which follows the homofermentative route [2]. Other LABs that are
Accepted: 28 September 2023 known for LA bioconversion with a high rate of production, namely between 99–222 g/L,
Published: 29 September 2023 are as follows: Sporolactobacillus inulinus, L. bulgaricus, L. plantarum, L. delbrueckii and L.
rhamnosus [1,3,4]. Several wastes and/or subproducts can be used as substrates to produce
LA, such as cheese whey, coffee mucilage, lignocellulosic hydrolysates, urban waste and
food waste [1,5,6]. Sugarcane juice is obtained from sugarcane, which is employed in the
Copyright: © 2023 by the authors. Brazilian sugar production and alcohol industry. Brazil is the main sugarcane producer,
Licensee MDPI, Basel, Switzerland. and its 2023/2024 harvest is expected to reach 637.1 million tons [7]. Sugarcane juice is
This article is an open access article extracted directly from sugarcane through a mechanical grinding process. It comprises
distributed under the terms and phenolic compounds and chlorophyll, a large amount of water (80%), sucrose (17%), glu-
conditions of the Creative Commons
cose (0.4%) and fructose (0.2%) [8,9]. From its solid fraction, nitrogenous substances, fats,
Attribution (CC BY) license (https://
pectins, organic acids and ash can be obtained [10]. Sugarcane juice’s composition varies
creativecommons.org/licenses/by/
according to the cultivar, harvest time, soil and climate [11]. After submerged fermentation,
4.0/).

Fermentation 2023, 9, 879. https://doi.org/10.3390/fermentation9100879 https://www.mdpi.com/journal/fermentation

Fermentation 2023, 9, 879 2 of 11

calcium lactate is the main product created due to the use of sodium carbonate to control
the strong inhibition that the microorganism undergoes when the pH decreases throughout
the process. The conventional LA recovery process using fermentation broth requires
several steps, depending on the employed substrate. The most widespread and industrially
applied method consists of a precipitation method using sulfuric acid. Allied to the use
of activated carbon, this technique promotes a higher yield (around 60–70%) than other
recovery methods, but it still presents losses and leads to the production of undesirable
components, such as calcium sulfate [12–14].
Ion-exchange resins consist of a flexible polymeric base (phenolic, acrylic or polystyrene
divinylbenzene bases) with a loaded functional group. Such functional groups determine
the type of resin, the main ones being sulfonic acid, carboxylic and phosphoric groups,
which are strong and weak acid-exchange resins, respectively, and quaternary ammonium
and amino groups, which are strong and weak acid-exchange resins, respectively [15,16].
When acid resins are used in a medium in which the pH is greater than their pKa value,
they carry a negative charge and attract positive compounds, thus creating cationic resins.
Following the same reasoning, basic resins are anionic. Strong acid and basic resins have
approximate pKas of 1 and 14, respectively, while weak ones have approximate pKas of
4 and 10. Thus, strong resins are applied in most cases, since they are present an ionized
form most of the time [17]. The main advantages of this type of resin are its versatility and
adsorption capacity, in addition to its lower cost when compared to other types of resin.
However, it is sensitive to extreme temperatures [18]. The resins used in this study have
characteristics (Table 1) that meet different process requirements.

Table 1. Characteristics of Amberlite and Purolite resins.

Amberlite Amberlite Purolite Purolite

Properties
IR 120 IRA 67 C10SH A847S
Description Strong cationic Weak anionic Strong acid cationic Weak basic anionic
T max. 121 ◦ C ≤60 ◦ C 120 ◦ C 40 ◦ C
Styrene divinylbenzene Polyacrylic
Matrix Acrylic (gel) Polystyrene/divinylbenzene
(gel) gel/divinylbenzene
Functional group Sulfonic acid Polyamine Sulfonic acid Tertiary amine
Particle size 620–830 µm 500–750 µm 425–1200 µm 425–1200 µm
Humidity 53–58% ~60% 54–59% 56–62%

Considering its high availability during the year, and the low cost of acquiring sug-
arcane juice, it was used as a substrate by the bacteria L. pentosus to produce LA, with
a subsequent recovery step involving the use of activated carbon and precipitation with
sulfuric acid solution. In an alternative process, the use of ion-exchange resins for LA pu-
rification was also proposed, where the performances of resins manufactured by Amberlite
and Purolite were compared. The purification systems under agitation, carried out in a
tank with mechanical agitation, as well as the system using packed columns, were also
compared. The objective of this work was to develop the LA purification process from the
use of fermented and recovered broth, and present a feasible option to be transferred to an
industrial scale.

2. Materials and Methods

2.1. Material
The sugarcane juice used as the substrate in the LA production process was obtained
from a local market in Curitiba, Brazil, mechanically extracted from sugarcane, then sub-
jected to conventional filtration in order to remove suspended solids.
Ion-exchange resins from two brands, Amberlite and Purolite, were used. Amberlite
IR-120 and IRA-67 resins were purchased from the Merck group. Purolite C150SH and
A847S resins were supplied by the company.
 Ion-exchange resins from two brands, Amberlite and Purolite, were used. Amberlite
IR-120 and IRA-67 resins were purchased from the Merck group. Purolite C150SH and
Fermentation 2023, 9, 879 A847S resins were supplied by the company. 3 of 11

2.2. Microorganism
2.2. Microorganism
Lactobacillus pentosus was cultured in MRS broth (Man–Rogosa–Sharpe) at 35 °C for
24 h.Lactobacillus
After incubation, glycerol
pentosus was added
was cultured to the
in MRS culture
broth for storage at −20 °C,
(Man–Rogosa–Sharpe) ◦ C the
with
at 35 for
periodic renewal
24 h. After of the glycerol
incubation, ◦
culture. was added to the culture for storage at −20 C, with the
periodic renewal of the culture.
2.3. LA Production Using Sugarcane Juice as Substrate
2.3. LA Production Using Sugarcane Juice as Substrate
2.3.1. Inoculum
2.3.1. Inoculum
The growth medium and conditions were previously optimized by Oliveira et al. [19];
The growth medium and conditions were previously optimized by Oliveira et al. [19];
these were composed of sugarcane juice that was diluted to a concentration of 20 g/L of
these were composed of sugarcane juice that was diluted to a concentration of 20 g/L of
total reducing sugars and 25 g/L of yeast extract. Inoculum propagation was carried out
total reducing sugars and 25 g/L of yeast extract. Inoculum propagation was carried out
in a 125 mL Erlenmeyer flask with 75 mL of culture medium and 10% (v/v) of inoculum
in a 125 mL Erlenmeyer flask with 75 mL of culture medium and 10% (v/v) of inoculum
proportion, maintained at 30 ◦°C under agitation at 120 rpm, for 24 h.
proportion, maintained at 30 C under agitation at 120 rpm, for 24 h.
2.3.2.
2.3.2. LA
LA Production
Production Kinetics
Kinetics
The
The medium composition was
medium composition was previously optimized by
previously optimized by Oliveira
Oliveira et
et al.
al. [19]:
[19]: sugarcane
sugarcane
juice at a concentration of 230 g/L of total reducing sugars, 15 g/L of yeast
juice at a concentration of 230 g/L of total reducing sugars, 15 g/L of yeast extract, extract, 90
90 g/L
g/L
of CaCO 3, and inoculation proportion of 10% (v/v). The LA production kinetics were eval-
of CaCO3 , and inoculation proportion of 10% (v/v). The LA production kinetics were
uated in 125
evaluated in mL
125 Erlenmeyer
mL Erlenmeyer flasks containing
flasks 75 mL
containing of culture
75 mL medium,
of culture medium, incubated
incubatedat 30
at
°C,
30 andagitated
◦ C, andagitated at 120 rpmrpm
at 120 for for
168168
h. Samples were
h. Samples withdrawn
were withdrawn every 24 h24
every tohmonitor the
to monitor
concentration
the concentration of LA
of produced
LA produced (g/L), the pH
(g/L), theprofile and and
pH profile sugar consumption.
sugar consumption.

2.4. Conventional
Conventional LA
LA Recovery
Recovery
The LA recovery process using the fermented broth containing the form of calcium
lactate comprises several steps, as shown in Figure 1.

Figure 1. Steps
Steps of LA recovery.

The fermented
The fermented broth
broth containing
containing calcium
calcium lactate
lactate was
was heated
heated in
in aa water
water bath
bath at
at aa tem-
tem-
perature of ◦ ×gg(times
perature of 50 C, then
50 °C, then centrifuged
centrifuged at
at 1800
1800× (timesgravity)
gravity) for
for 20
20 min
min (Centribio,
(Centribio, model
model
80-2B). Then,
80-2B). Then, the
the centrifuged
centrifuged broth
brothwas
wasfiltered
filteredusing
usingaaBüchner
Büchnerfunnel
funnelandandWhatman
WhatmanNo. No.1
1filter paper.
filter paper.The
Theconditions described
conditions by by
described Oliveira et al.
Oliveira et [19] were
al. [19] usedused
were for clarification and
for clarification
precipitation processes. The activated carbon proportion was 15% (w/v)
and precipitation processes. The activated carbon proportion was 15% (w/v) considering considering the
fermented broth. Then, the solution was kept in a water bath at 50 ◦ C under agitation at
100 rpm for 25 min. After this time, the activated carbon was removed via centrifugation
(1800× g/20 min), and a clarified broth was obtained; this, in turn, passed through acid
precipitation with a sulfuric acid solution (4 mol/L) under magnetic stirring until complete
Fermentation 2023, 9, x FOR PEER REVIEW 4 of 12

the fermented broth. Then, the solution was kept in a water bath at 50 °C under agitation
Fermentation 2023, 9, 879 at 100 rpm for 25 min. After this time, the activated carbon was removed via centrifugation
4 of 11
(1800× g/20 min), and a clarified broth was obtained; this, in turn, passed through acid
precipitation with a sulfuric acid solution (4 mol/L) under magnetic stirring until complete
homogenization.
homogenization. TheThesolution
solutionwas
wasthen
then centrifuged
centrifugedagain
again to
to separate
separate the
the converted
converted LA
LA
from the calcium sulfate.
from the calcium sulfate.

2.5. LA
2.5. LA Purification
Purification through
through Ion-Exchange
Ion-Exchange Resins
Resins
Two LA
Two LA purification
purificationsystems
systemsusing
usingion-exchange
ion-exchangeresins
resinswere
weretested
tested(Figure
(Figure2),2),
andand
a
a protocol provided by the Purolite company was followed regarding the
protocol provided by the Purolite company was followed regarding the resins’ usage.resins’ usage.
When necessary, the resins initially underwent an activation procedure.
When procedure.

Figure 2.
Figure Diagram of
2. Diagram of the
the LA
LA purification
purification steps.
steps.

2.5.1. Resin Activation

2.5.1. Resin Activation
Amberlite resins: for anionic resins in Cl- form, sequential washes with 1N HCl–
Amberlite resins: for anionic resins in Cl- form, sequential washes with 1N HCl–de-
deionized water–1N NaOH–deionized water until pH 7. For cationic resins in H+ form,
ionized water–1N NaOH–deionized water until pH 7. For cationic resins in H+ form, se-
sequential washes with 1N HCl–deionized water until pH 7. Purolite resins: they are
quential washes with 1N HCl–deionized water until pH 7. Purolite resins: they are avail-
available in their active forms, needing successive washings with deionized water until the
able in their active forms, needing successive washings with deionized water until the
solution is clear.
solution is clear.
2.5.2. LA Purification Using Cationic and Anionic resins (Glass Column)
2.5.2. LA Purification Using Cationic and Anionic resins (Glass Column)
The glass column (450 mm × 20 mm) contained a minimum resin bed of 30 cm, and
eachThe glasswas
column column
packed (450using
mm deionized
× 20 mm) contained a minimum
water, avoiding resin bedofofbubbles.
the formation 30 cm, and
The
each column was
first column wasprepared
packed using deionized
with cationic water,
resins, andavoiding the column
the second formation
wasofprepared
bubbles. with
The
first column
anionic resinswas prepared
with a flow with cationic
rate of 0.2 BV/hresins,
for and the second column
LA percolation. was
Aliquots prepared
were with
withdrawn
anionic resins with a flow rate of 0.2 BV/h for LA percolation. Aliquots were
before percolation and at the end of the procedure to evaluate the pH and LA concentration. withdrawn
before percolation
After each sample and at the end
percolation, of the procedure
distilled water was to evaluate to
percolated thewash
pH and LA concentra-
the resins. For this
tion.
assay,After each sample
the efficiency percolation,
of the adsorption distilled
processwater was
(E) was percolated
calculated to wash the
considering resins.
the valuesFor
of
this assay, (C
the initial the
0 ) efficiency
and final of
(Ce the
) adsorption
concentration process
of (E)
adsorbate.was calculated considering the val-
ues of the initial (C0) and final (Ce) concentration of adsorbate.
C0 − Ce
E = 𝐶 − 𝐶 × 100 (1)
𝐸 = C0 × 100 (1)
𝐶
The adsorption capacity of the resin (q) was calculated considering the initial (C0 ) and
The adsorption capacity of the resin (q) was calculated considering the initial (C0) and
final (Ce ) concentration of adsorbate, the solution volume (V) and the resin mass (W).
final (Ce) concentration of adsorbate, the solution volume (V) and the resin mass (W).
C0 − Ce × V
q= (2)
W
Fermentation 2023, 9, 879 5 of 11

2.5.3. LA Purification Using Cationic and Anionic Resins Simultaneously (Stirred Tank)
Assays were carried out in a stirred tank (Tecnal TE-054MAG, with Tecnal TE-139
mechanical agitator) equipped with a digital controlling system for temperature and me-
chanical agitation, a total volume capacity of 2 L, and a propeller-type impeller for axial
flow. Samples were kept in contact with both resins, simultaneously, for 120 min, at a
temperature of 30 ◦ C and stirring at 100 rpm. After this time, conventional filtration was
performed to separate the resins and analyze the LA concentration.

2.6. Analytical Methods

The pH measurement was performed using a potentiometer. The total reducing sugars
were determined via the reaction with dinitrosalicylic acid–DNS [20], which was adapted
for the determination of the total sugar content; this underwent acid hydrolysis using HCl.
L. pentosus is a homofermentative LAB, which prioritizes the production of 2 mols of LA
per mol of glucose. Therefore, the yield of the LA fermentation process was calculated
from the conversion ratio of the substrate (total sugars) into product (LA), considering
the maximum theoretical yield, in molecular weight, of the conversion of glucose into LA,
equivalent to 1.0. The LA solution obtained after the precipitation step was eluted in a
column (10 mL) of solid-phase extraction packed with 5 g of C18 resin, as a clean-up step
prior to chromatography analysis. The procedure consisted of activating the column with
methanol (8 mL), washing with MilliQ water (8 mL), sample elution (6 mL), and washing
with MilliQ water (20 mL). The samples were analyzed using HPLC equipment (Agilent
1260 Infinity), a Hi-Plex column (300 × 7.7 mm) at 60 ◦ C, an isocratic regime of mobile
phase H2 SO4 0.005 M, and a flow rate of 0.6 mL/min.

2.7. Statistics
All results are expressed as average ± standard deviation (in triplicate). Analysis of
variance (ANOVA) was used to assess the data significance, where p ≤ 0.05 was considered
statistically significant. The results were analyzed using the Software Statistica 5.0 (StatSoft,
Tulsa, OK, USA).

3. Results and Discussion

3.1. LA Production Kinetics
The L. pentosus strain showed good adaptation to the medium containing sugarcane
juice, liable to cell growth and product accumulation. The LA production kinetics were
monitored for 168 h; however, from 120 h onwards, a solid calcium lactate was observed,
making the recovery and analysis steps more difficult. Samples were withdrawn every
24 h, and in every sample, it was found that the LA concentration in the fermented broth
was very low, where the predominant form was the precursor calcium lactate. The best
production was reached in 96 h (1.18 g/L·h), accumulating 113.74 g/L of LA (Table 2).

Table 2. Data for LA production kinetics over 96 h.

Time (h) pH TS (g/L) LA (g/L) Productivity (g/L·h) YP/S LA Mass Recovery

0 6.53 230.67 ± 0.00 – – – –
24 6.52 146.62 ± 0.00 73.49 ± 50.78 3.06 0.28 28%
48 6.41 136.37 ± 0.00 71.73 ± 5.89 1.49 0.18 30%
72 6.33 161.15 ± 0.00 79.16 ± 7.42 1.10 0.29 33%
96 6.00 156.02 ± 0.00 113.74 ± 29.53 1.18 0.34 49%
TS: total sugars (g/L); LA (g/L): LA concentration after precipitation process; YP/S : conversion of substrate
to product.

LA concentrations produced from different substrates are found in the literature.

Karp et al. [21] reached a productivity of 1.13 g/L·h using L. agilis, and 138 g/L of LA
was produced after optimization using vinasse as a substrate enriched with soy molasses.
Bernardo et al. [22] used cheese whey and corn liquor as a fermentation medium for L.
Fermentation 2023, 9, 879 6 of 11

rhamnosus B103. At first, they obtained 57 g/L of LA and a productivity of 1.18 g/L·h;
then, LA production was increased to 106 g/L using the fed-batch operation. In optimized
synthetic medium, Wang et al. [23] immobilized L. pentosus ATCC 8041 cells in an optimized
hydrogel mix. The LA production through free cells was 90 g/L, with a productivity of
1.3 g/L·h; through immobilized cells, LA production reached 106 g/L and a productivity
of 2.2 g/L·h was obtained. Paulova et al. [24] achieved an LA productivity of 4.0 g/L·h
using chicken feather hydrolysate and L. casei. In addition, de la Torre et al. [25] achieved
a high LA productivity of 6.7 g/L·h using orange peel hydrolysate and L. delbrueckii. It
should be noted that in the present work, a high production of LA was achieved using
sugarcane juice directly as a substrate, without prior treatment or cell manipulation.
pH control is essential in lactic fermentation, promoting better LA production, yield,
and productivity. LAB have a certain sensitivity to changes in the pH of the fermentation
medium. When LA is produced, the pH decreases as LA has the ability to dissociate
and release protons [12]. According to the pH data obtained during kinetics, CaCO3 was
effective in controlling pH variations, supporting the variations that occur in the period
between cell maintenance and LA production, with the ideal range being between 6–6.5.
The relationship between a high sugar intake (Table 2) and low LA production in the
first 24 h of fermentation indicates the consumption of the available sugars for the energy
necessary for cell maintenance (sugar metabolism and pyruvate production). In turn, LAB
convert pyruvate into LA, contributing to the decrease in pH, and they may slow down
their growth, LA production and sugar consumption [12]. After 48 h, LAB may adapt to
the medium conditions via enzymatic regulation mechanisms, allowing them to retake
the metabolism and leading to the rapid formation of a solid block of calcium lactate. Li
et al. [14] describe a rapid increase in LA production on the third day of fermentation
using Lactobacillus strains. From the fourth day, the production begins to fall in a medium
composed of food waste. Cirlini et al. [26] also observed log phase cycles during the initial
48 h of fermentation in a screening study of Lactobacillus strains using elderberry juice as
the substrate. In this study, there was a clear formation of a solid block of calcium lactate
before 120 h when the considerable acidification of the medium occurred, causing analytical
difficulties. Therefore, kinetics ended at this time.

3.2. LA Recovery
At the end of fermentation, the calcium lactate present in the fermented broth precipi-
tated; then, this broth was heated to 50 ◦ C in a water bath to remove excess CaCO3 and
bacterial cells through a centrifugation step. The supernatant was subjected to clarification
using activated carbon in an environment with controlled temperature and agitation; this
was in order to remove pigments and organic substances. After centrifugation, the clarified
supernatant was subjected to precipitation using a 4 mol/L H2 SO4 solution, thus obtaining
the LA. The best results for LA production were found in 96 h (Table 2), with 49% of LA
recovered; this percentage is compatible with the average found in the literature, from
50% [27]. These values related to the precipitation process demonstrate the need for a later
step in LA purification, namely, obtaining a molecule with a high level of purity.

3.3. LA Purification through Ion-Exchange Resins

3.3.1. LA Purification Using Cationic and Anionic resins (Glass Column)
The initially treated sample contained 144.91 g/L of LA, which was converted using
sulfuric acid at pH 1. The results of the purification procedures with cationic and anionic
resins packed in columns are shown in Table 3.
Amberlite’s resins showed better results than Purolite’s resins, with final samples of
purified LA of 62.24 g/L and 36.35 g/L, respectively. It is possible that this fact is due to the
Amberlite resins being supplied in an inactive form. In this way, there is a guarantee that
the resins are pure without contamination, and there is greater control of their activation
through sequential washes and pH monitoring. Purolite resins are supplied in their active
form, with a pre-washing step being part of the protocol to remove chemical residues and
Fermentation 2023, 9, 879 7 of 11

dust. This is a positive point considering the facility and that it reduces the time required
to prepare the experiments; however, the quality control is reduced.

Table 3. Data for purified samples in packed columns with ion-exchange resins.

Resins pH LA (g/L) LA (g/L)—w H2 O

Purolite
C10SH 1–1 53.92 ± 5.10 15.78 ± 1.12
A847S 1–6 36.35 ± 2.85 18.42 ± 2.44
Amberlite
IR 120 1–1 34.98 ± 3.40 20.83 ± 3.10
IRA 67 1–8 62.24 ± 5.40 14.97 ± 1.88
C10SH: Purolite cationic resin; A847S: Purolite anionic resin; IR 120: Amberlite cationic resin; IRA 67: Amberlite
anionic resin; pH of input sample–pH of output sample; LA concentration (g/L) after elution; residual LA
concentration (g/L) in the resin-washing water.

Regarding efficiency (Table 4), considering that the sample was in subsequent contact
with cationic and anionic resins, on average, the rates obtained were very close between
the manufacturers, Amberlite (66.68%) and Purolite (68.85%). The average recovery in
LA mass was also close between Purolite (25%) and Amberlite (27%), but these were very
low rates. The proper use of resins requires studies of different parameters and different
adaptation possibilities. It is known that ion-exchange resins have high selectivity, being
able to reach a high LA yield and generate a minimum amount of waste. Cationic resins
showed a good adsorption capacity for both manufacturers, Amberlite (165 mg/mL) and
Purolite (136 mg/mL), respectively. For the anionic resins, the results were low due to the
previous treatment in cationic resins.

Table 4. The LA mass recovery rates, efficiency and adsorption capacity of resins, in packed columns.

Resin Recovery Efficiency Adsorption Capacity (mg/mL)

Amberlite IR 120 19% 75.86% 165
Amberlite IRA 67 35% 57.50% 32.7
Purolite C10SH 30% 62.79% 136
Purolite A847S 20% 74.91% 21

Several types of ion exchangers have been used for LA recovery, and investigations
on two-step separation techniques, including the use of anionic and cationic resins, are
constant in the literature. Bishai et al. [28] observed that pH directly influences ion exchange.
From pH 2–3, the LA did not bind with anionic resins. No binding with the resins was
observed when the LA reached its pKa (3.85). The authors also observed that a decrease
in purity can be attributed to the release of other contaminating anionic groups in the
medium, which can be competitive for ion exchange. Msuya et al. [29] assembled an
ion-exchange resin system similar to the system proposed in this work (Section 2.5.2),
comprising a cation-exchange resin column connected to an anion-exchange resin column.
The authors used the weak anionic resin Indion FFIP (Ion Exchange India Ltd., Mumbai,
India) and the strong acid cationic resin Indion 225H (Ion Exchange India Ltd., Mumbai,
India), also using water for washing. As a result, a higher LA concentration (108 g/L)
was obtained, representing a concentration of 4.5 times the initial concentration (24 g/L)
before the process. Arcanjo et al. [30] used an initial LA solution (120 g/L) for recovery
in Amberlite IRA-67 and IRA-96 resin, where the total LA recovery was 25.8% (31 g/L).
They also tested a binary solution containing LA (345 g/L) and glycerol (230 g/L). LA
recovery from this solution was lower than the value obtained for the pure LA solution.
Furthermore, the authors concluded that LA and glycerol molecules are similar, which may
interfere in the selectivity of ion-exchange resins.
Fermentation 2023, 9, 879 8 of 11

Zaini et al. [31] also started LA purification using a strong acid cationic resin (IRA
120) to convert the lactate salt into LA. Due to the functional groups of sulfonic acid,
theoretically, the hydrogen ions (H+ ) that are linked to the functional group of the resin
will bind to the sodium lactate obtained at the end of the fermentation, and convert it into
LA. Subsequently, the non-dissociated form of LA obtained via the IRA 120 was kept in
contact with the resin IRA-67 (Cl− ) for the removal of anions. The pH of the effluent was
around 5.5–6.0, indicating that the organic acids present in the fermentation broth (LA and
acetic acid) were adsorbed by the resin. After washing the column with water, the LA
was desorbed with 0.5M HCl. The first acid to be eluted was acetic acid, followed by LA;
thus, competition between organic acids for binding to the functional groups of the resins
was observed.
According to the literature data survey, weak base resins are more efficient than strong
base resins for LA adsorption. This is mainly because weak base resins have greater
resistance to oxidation and organic fouling. Furthermore, the selectivity of the resin for
most organic acids is higher due to the elevated number of aromatic rings in styrene
resins, making it hydrophobic. Anion-exchange resins generally have different levels of
affinity towards the nutrients and compounds available in the medium, which could be
the main reason for the inhibitory effects observed towards each different type of anion-
exchange resin. The presence of this type of competitive adsorption can therefore reduce
the adsorption capacity of the resin by blocking available sites for LA adsorption [15].

3.3.2. LA Purification Using Cationic and Anionic Resins Simultaneously (Stirred Tank)
In this study, the two resins (cationic and anionic) were kept in contact with the
LA sample in a stirred tank, and the obtained data are listed in Table 5. After 120 min,
conventional filtration was performed. The initial sample (450 mL) contained 144.91 g/L of
LA, which was converted using sulfuric acid, with pH 1.

Table 5. Data for purified samples in stirred tank with ion-exchange resins.

Resins LA (g/L) pH LA (g/L)—w H2 O Recovery

Purolite
C10SH and
144.91–42.38 1–4 32.15 23%
A847S
Amberlite
IR 120 and
144.91–189.11 1–3 24.05 95%
IRA 67
C10SH: Purolite cationic resin; A847S: Purolite anionic resin; IR 120: Amberlite cationic resin; IRA 67: Amberlite
anionic resin; LA concentration (g/L) of initial and final sample; pH of initial sample–pH of final sample; residual
LA concentration (g/L) in the resin washing water.

The stirred tank test showed an excellent result (189.11 g/L) when using the Amberlite
IR 120 and IRA 67 resins simultaneously in a controlled environment; meanwhile, the
Purolite resins showed a lower performance (42.38 g/L). Usually, ion-exchange resins
present high rates of LA purification, due to the selectively active groups. Cationic and
anionic resins packed in columns achieve between 91 and 98% purification [32]. Under
the optimized conditions of batch experiments, anionic resins present between 85 and 90%
purification [33,34]. The simultaneous use of cationic and anionic resins is not reported. The
purification of samples by means of ionic resins in stirred tanks presents good prospects.
The system is temperature-, agitation- and time-controlled, without other concerns re-
garding the flow rate oscillation; for example, packing glass columns are also prepared
with each resin. Also, with agitation using a propeller-type impeller, the sample is not
compacted, as the liquid is directed towards the base of the tank, leading to axial circulation,
better homogenization and better mass transfer; this demonstrates the adequacy of the
sensitive processes, and their ability to provide good heat distribution. In this way, the
flow of washing residues is reduced, and the separation between the resins can be carried
Fermentation 2023, 9, 879 9 of 11

out using the difference in density for subsequent regeneration, in addition to the use
of a smaller volume of solution. This is a much simpler procedure, aimed at removing
the residual sulfuric acid and the residual cation for the LA purification, scaling up the
process, and jointly using both resins (cationic and anionic). Table 6 briefly compares the
costs of each method for LA recovery and purification, in terms of the products used at
a lab scale. The total prices are close between the methods applied. Regarding the resins,
Purolite has the lowest cost; however, the difference between Purolite resins and Amberlite
resins is USD 82.88, which shows better results. At larger quantities, these products may
be less expensive. Also, an economic analysis including the energy consumption could be
considered for the next study.

Table 6. Brief price description of products used in LA recovery and purification methods.

Products Price * (USD) Total (USD)

Activated carbon (1 kg) 75.60
Conventional recovery 225.30
H2 SO4 4 M (1 L) 149.70
HCl 1 N (1 L) 51.86
Solutions for resins 94.66
NaOH 1 N (1 L) 42.80
Amberlite IR 120 (500 g) 71.20 Amberlite resins 161.50
Amberlite IRA 67 (1 kg) 90.30 Resins + solutions 256.16
Purolite C10SH (1 L) 86.64
Purolite resins 173.28
Purolite A847S (1 L) 86.64
* Prices were obtained directly from the supplier companies (Sigma Aldrich Brasil, Ltda. São Paulo-SP, Brazil,
Purolite do Brasil, Ltda, São Paulo-SP, Brazil) at a lab-scale.

4. Conclusions
LA was efficiently produced using sugarcane juice, an alternative substrate, which
was favorable to the cell multiplication of LAB L. pentosus. It was observed that the optimal
production conditions were reached after four days of fermentation, with satisfactory
productivity. The employed substrate does not need to undergo any pretreatment, which
means that it can be used directly in submerged fermentation conditions with simple
adjustments. In order to purify LA in a single step, both anionic and cationic exchange
resins were used together in contact with the sample in a stirred tank. This method
is simple and stable in terms of flow rates, time, and temperature. It does not require
successive washings with chemical solvents, which reduces the generation of effluents. It
allows the use of higher sample volumes in less time, and the resins’ recovery is facilitated
through density difference using water as a solvent. This study also provides insights into
scalability, and the results at the bench scale show great potential. The usage of combined
ionic exchange resins in contact with larger volumes of samples in a stirred tank could be
an option for industrial processes. This is feasible considering the operational volumes
and controlling conditions. This method can be used for transitioning scales, and future
studies concerning the adsorption and efficiency capacities of these resins when operating
simultaneously are envisaged. The purified LA must be then carefully analyzed, showing
good characteristics for polymerization.

Author Contributions: Conceptualization, P.Z.d.O. and L.P.d.S.V.; methodology, P.Z.d.O. and L.P.d.S.V.;
formal analysis, P.Z.d.O. and L.P.d.S.V.; investigation, P.Z.d.O.; writing—original draft prepara-
tion, P.Z.d.O.; writing—review and editing; Review, L.P.d.S.V., P.Z.d.O. and L.P.d.S.V.; supervision,
L.P.d.S.V.; project administration, L.P.d.S.V.; funding acquisition, L.P.d.S.V. and C.R.S. All authors
have read and agreed to the published version of the manuscript.
Funding: National Council for Scientific and Technological Development: Process306336/2022-7;
Coordenação de Aperfeicoamento de Pessoal de Nível Superior: CAPES-PROEX.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Fermentation 2023, 9, 879 10 of 11

Data Availability Statement: Not applicable.

Acknowledgments: The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil
(CNPq) for the research funding.
Conflicts of Interest: The authors declare no conflict of interest.

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