Bioresource Technology 318 (2020) 124260

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

A simple downstream processing protocol for the recovery of lactic acid

from the fermentation broth
Sumit Kumar a, Neerja Yadav a, Lata Nain b, Sunil Kumar Khare a, *
a
Enzyme and Microbial Biochemistry Lab, Department of Chemistry, Indian Institute of Technology, Delhi, India
b
Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi, India

H I G H L I G H T S

• A simple and economical process for downstream of lactic acid has been developed.
• The pH of the extraction medium was critical during lactic acid purification.
• Optimized downstream process conditions resulted in a yield of 86% and 93% purity.
• The purified lactic acid was further characterized by FTIR and NMR.

A R T I C L E I N F O A B S T R A C T

Keywords: Lactic acid is one of the essential platform chemicals, and despite the availability of a range of downstream
Lactic acid processes, its effective recovery is still elusive. A phase partitioning process using n-butanol and a chaotropic salt
Downstream process ammonium sulphate was developed to recover lactic acid from the fermentation broth of Lactobacillus pentosus
Lactic acid bacteria (LAB)
SKL-18. During the optimization of various process parameters, the extraction medium’s pH was found to be
Solvent extraction
Fermentation broth
critical, with 2.5 being the best. The optimized process resulted in a lactic acid yield of 86% and found it to be
93% pure. The purity and characteristics of lactic acid were confirmed by FTIR and NMR spectra. This solvent-
based extraction procedure is an economical and straightforward downstream process for purifying lactic acid
produced from agro- and bakery-residues. The pure lactic acid can further be used for enzymatic synthesis of high
value-added product PLA, a biodegradable and biocompatible plastics.

1. Introduction vary in the range of 0.78–1.74 USD/kg lactic acid (Phanthumchinda

et al., 2018; Posada et al., 2012). The downstream process of lactic acid
Lactic acid is an essential chemical with widespread applications in accounts for 40–70% of the cost, and thus there is a need to develop a
food, textile, leather, cosmetic, and pharmaceutical industries. Chemical convenient and economic downstream strategy for lactic acid purifica­
synthesis and fermentation are two routes to produce commercial lactic tion (Joglekar et al., 2006; Li et al., 2016).
acid. The fermentation process has an advantage over the chemical Generally, due to the presence of media components, salts, and re­
process as it produces optically pure L-lactic acid, while the latter makes sidual sugars in the fermentation broth, the purification of lactic acid
a racemic mixture of DL-lactic acid. L-form of lactic acid is desirable in becomes a multistep process involving filtration, dialysis, and tedious
food and pharmaceutical applications as human beings metabolize it. column exchange procedures. A range of methods has been employed
Enantiopure L-lactic acid also serves as feedstock for the synthesis of for lactic acid recovery (Bishai et al., 2015; Komesu et al., 2017a;
polylactic acid (PLA), a biodegradable and biocompatible plastic (Alves Phanthumchinda et al., 2018). Precipitation is one of the methods to
de Oliveira et al., 2018; Nampoothiri et al., 2010). For all the end-use recover lactic acid from fermentation broth by adding calcium hydrox­
application, the purified form is requisite. Purification of lactic acid is ide. A significant disadvantage of this downstream process is the gen­
a costly process, and its economic analysis has shown that the cost can eration of a large amount of gypsum as waste, which poses

* Corresponding authors at: Enzyme and Microbial Biochemistry Laboratory, Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi
110016, India.
E-mail address: skkhare@chemistry.iitd.ac.in (S.K. Khare).

https://doi.org/10.1016/j.biortech.2020.124260
Received 26 August 2020; Received in revised form 8 October 2020; Accepted 10 October 2020
Available online 15 October 2020
0960-8524/© 2020 Elsevier Ltd. All rights reserved.
S. Kumar et al. Bioresource Technology 318 (2020) 124260

environmental and economic burdens. The membrane process of lactic method using n-butanol. Various parameters, such as pH of fermentation
acid recovery is limited by the high cost of membranes and the problem broth (pH 1.0–4.73), n-butanol volume (10–70 mL), ammonium sul­
of membrane fouling (Komesu et al., 2017b). Reactive extraction is one phate (0–50%, w/v), and lactic acid (0.22–3.0 M) concentration were
of the preferred choices, where an amine compound, along with a sol­ optimized to get purified lactic acid with the highest recovery. The pH of
vent, is used for lactic acid recovery (Krzyżaniak et al., 2013). Different the fermentation broth was set by using concentrated hydrochloric acid.
processes utilized for lactic acid purification have their advantages and The lactic acid concentration was varied by adding pure lactic acid to the
drawbacks (Komesu et al., 2017b). fermentation broth.
In the present study, lactic acid purification was attempted by
employing the phase partitioning method. This method takes into ac­ 2.5. Characterization of purified lactic acid
count the differential migration of lactic acid into the aqueous or organic
phase in the presence of a chaotropic salt, like ammonium sulphate Purified lactic acid was characterized by Fourier-transform infrared
[(NH4)2SO4] and solvent, n-butanol. A literature survey indicates that spectroscopy (FTIR) to identify the functional groups by pelleting it with
limited information is available on the downstream process of lactic acid KBr, and spectrum (4000–400 cm− 1) was recorded on Agilent (Model-
using n-butanol (Chawong and Rattanaphanee, 2011). Further study by Perkin Elmer IR version 10.6.0, USA). For Nuclear magnetic resonance
the same group extracted lactic acid using a mixed solvent electrolyte spectroscopy (1H NMR) studies, the purified lactic acid was dissolved in
system containing water, 1-butanol, and ammonium sulphate (Chawong deuterium oxide (D2O), and spectra were recorded at ambient temper­
et al., 2015). However, in these studies, lactic acid recovery was ature on Bruker 400 MHz (Germany). 1H chemical shifts were expressed
attempted by adding pure lactic acid in aqueous solution and not from in δ.
the complex fermentation broth. Therefore, given the simplicity of the
above purification protocol, lactic acid purification was attempted from 3. Results and discussion
fermentation broth in the current research.
Lactic acid is one of the essential platform chemicals, and its global
2. Materials and methods demand is consistently increasing due to application in the diverse field,
especially the synthesis of PLA (Alves de Oliveira et al., 2018) as a bio-
2.1. Chemicals based product using agro-industrial biomass wastes. The use of ionic
liquid in the pretreatment of lignocellulosic biomass and subsequent use
Lactic acid was procured from Sigma Aldrich (St. Louis, USA). The of ionic liquid-tolerant bacteria for fermentation is a significant
media components were bought from HiMedia Laboratory (Mumbai, advancement in this direction (Grewal and Khare, 2018; Yadav et al.,
India). All other chemicals used were of analytical grade. 2020). Despite the availability of a range of downstream processes,
significant recovery of lactic acid from fermentation broth is still elusive.
2.2. Microorganisms and growth conditions Therefore, the present study was focussed on developing an efficient and
simple setup process for lactic acid recovery from complex fermentation
The lactic acid was produced by Lactobacillus pentosus SKL-18 (iso­ broth.
lated from dairy plant floor wash NDRI, Karnal) previously isolated and
characterized in our lab. SKL-18 was maintained on DeMan, Rogosa, and 3.1. Downstream processing of lactic acid
Sharpe (MRS) agar slants at 4 ◦ C and was subcultured fortnightly. For
lactic acid production, the freshly grown mother culture (O.D. ≈ 0.8) Downstream processing of lactic acid produced by L. pentosus SKL-18
was used to inoculate the MRS medium at a 4% (v/v) inoculum level. was attempted using phase partitioning by using n-butanol as a single
The inoculated MRS broth was incubated at 37 ◦ C at 120 rpm for 48 h. solvent. The lactic acid bacteria produced 0.22 M of lactic acid in
After growth, the culture was centrifuged at 10,000 rpm for 10 min, and fermentation broth after 48 h of growth. The pH of the broth at the end
cell-free supernatant was used for lactic acid purification. of fermentation was 4.73. The extraction of lactic acid at a small scale
from 10 mL fermentation broth with n-butanol (30 mL) resulted in a
2.3. Downstream processing of lactic acid 20% recovery in the organic phase. Though recovered lactic acid was
pure as ascertained by the HPLC profile but recovery was significantly
The lactic acid produced was purified by using the phase partitioning less, and the majority of lactic acid was retained in the aqueous phase.
method. Fermented broth containing lactic acid (10 mL) was taken in a Optimization of various parameters such as pH of fermentation broth,
flask; ammonium sulphate (5 g) was added and dissolved completely. ammonium sulphate, and lactic acid concentration was performed to get
Further, 30 mL of n-butanol was added and vigorously vortexed for a better yield in the organic phase.
proper mixing before keeping it at shaking for 2 h at 30 ◦ C. The mixture
was then transferred to a separating funnel and allowed to stand till 3.1.1. Effect of pH on lactic acid recovery
aqueous and organic phases were distinct. After phase separation, the Partitioning of lactic acid was affected by the pH of the medium. At
upper organic phase was separated. The collected organic phase was lower pH lesser amount of lactic acid was retained in the aqueous phase,
vaporized at 50 ◦ C in a rotavapor. The obtained lactic acid in the dried and better recovery was obtained in the organic phase. The best re­
organic phase was dissolved in 5 mL Milli Q water and detected by high covery of lactic acid (63 ± 2.5%) was achieved in medium with pH 2.5.
performance liquid chromatography (HPLC). The recovery of lactic acid did not improve furthermore upon lowering
For lactic acid quantification, the samples were centrifuged at the pH of fermentation broth below 2.5. The pH significantly influenced
10,000 rpm for 10 min, and the clear supernatant was filtered through lactic acid extraction by the solvent because of its ionization behaviour.
0.2 µm syringe filters for lactic acid estimation by HPLC. The lactic acid Lactic acid is the primary form below its pKa of 3.86, and at pH above it,
was quantified on the Perkin Elmer HPLC system equipped with Aminex lactate exists predominantly in solution. Lactic acid extraction by the
HPX-87H ion exclusion column (7.8 × 300 mm) and refractive index solvent is best reported around a pH of 2. During the ultrasonic solvent
detector (RID) using 5 mM H2SO4 as mobile phase at a flow rate of 0.6 extraction of lactic acid using ethyl acetate, extraction yield improved
mL/min. considerably from 12% to 45%, when pH decreased from 5 to 2. But
yield didn’t improve further with a decrease of pH from 2 to 1 (Hu et al.,
2.4. Optimization of downstream process 2017). These findings are concomitant to our results. In another study, a
pH of 2 is reported to be best for lactic acid extraction using Aliquat 336
Lactic acid produced was purified by using the phase partitioning (Kyuchoukov et al., 2005). In contrast, during lactic acid extraction by n-

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S. Kumar et al. Bioresource Technology 318 (2020) 124260

butanol, Chawong and Rattanaphanee (2011) reported improved re­

covery from 51.62% to 94.95% as pH decreased from 1.21 to 0.27.

3.1.2. Effect of ammonium sulphate concentration

Furthermore, partitioning and yield of lactic acid were not affected
by the addition of different ammonium sulphate concentrations. How­
ever, the addition of ammonium sulphate helped in better partitioning
as without ammonium sulphate aqueous phase got dissolved in butanol,
and the volume of the organic phase increased. Butanol and water are
miscible, and the addition of ammonium sulphate helped in salting out
of the water from butanol resulting in proper aqueous and organic phase
separation. Chawong et al. (2015) have reported a similar ammonium
sulphate role during lactic acid extraction. They had highlighted that
ammonium sulphate reduced mutual solubility between water and n-
butanol and was advantageous for lactic acid extraction during high
solvent-to-aqueous phase volume ratio.
Additionally, they have also reported improvement in lactic acid
extraction with an increase in ammonium sulphate concentration. This
finding is contrary to our result in which there was no effect on lactic
acid recovery upon varying ammonium sulphate concentration. How­
Fig. 1. Schematic flow sheet of the lactic acid recovery process from the
ever, analysis of the results of Chawong et al. (2015) revealed that the
fermentation broth.
improvement of lactic acid extraction upon an increase in ammonium
sulphate concentration was only meager. At 3.0 M lactic acid concen­
as a leftover in large amounts. The methodology developed in this study
tration extraction percentage improved scantily from 84.72% to 89.08%
is devoid of any such drawbacks and thus deemed simple, economical,
by increasing ammonium sulphate concentration from 0 to 5 g. The
and efficient.
salting out phenomenon is also reported to enhance lactic acid recovery
Tong et al. (2004) undertook the production of lactic acid by
efficiency from corn stover hemicellulose-derived liquor using trioctyl­
Lactobacillus rhamnosus (ATCC 10863), which was purified using
amine/octanol and inorganic salt (NaCl), resulting in improvement of
Amberlite IRA 92 (weak anion exchange resin) and achieved 96.2%
extraction efficiency by 32.2% with 3.85 times higher distribution co­
purity and 82.6% yield. Yu et al. (2015) had used a molecular distillation
efficient. Moreover, salting out also helped to remove residual sugars
technique following calcium precipitation and solvent extraction to
and most of the salts (82.8%) during the recovery of lactic acid (Lan
purify lactic acid from fermentation broth. The optimization of various
et al., 2019).
molecular distillation parameters was undertaken by response surface
methodology, which yielded 95.6% pure lactic acid with a 74% recovery
3.1.3. Optimization of lactic acid concentration and n-butanol volume
(Yu et al., 2015). The membrane-integrated separation process resulted
Varied lactic acid concentrations (0.22–3 M) were employed to
in 99.5% purity and a 76% yield of lactic acid from a fermentation broth
observe its effect on lactic acid recovery. Lactic acid concentration of
(Lee et al., 2017). Thus comparison of these results reveals that the ef­
0.5 M was best to achieve 86 ± 2.4% recovery in the organic phase. The
ficiency of a more straightforward one-step process of lactic acid
impact of the volume of extractant n-butanol was also studied for opti­
extraction by n-butanol is comparable with a costly multistep process
mum lactic acid extraction. Butanol volume of 30 mL was optimum as
that had been in practice.
increasing volume more than this resulted in organic phase dissolution
This simple downstream process was also successfully used to purify
resulting in lesser aqueous phase volume. Sequential extraction three
lactic acid (LA) produced by utilizing various bakery wastes in our lab.
times (3x10 mL butanol) was better compared to one-time extraction
Bread waste was found to be best used for LA production by solid-state
(30 mL butanol). Recovery improved from 73 ± 1.2 to 82 ± 1.3% for 1.0
fermentation (SSF). The produced LA was effectively purified with 85%
M lactic acid concentration, and it was concluded that three times
recovery using the above-mentioned novel downstream process. Circu­
sequential extraction results in better recovery for higher lactic acid
lar dichroism (CD) spectra revealed that the purified LA was of L-form as
concentrations. At lower concentrations (0.2–0.5 M), the three-time
desirable in food and pharmaceutical applications (Sadaf et al., un­
extraction process did not improve the lactic acid recovery. In study
published data, 2020).
by Chawong et al. (2015) three-time, consecutive extraction has been
shown to enhance lactic acid yield by 11.6% leading to a recovery of
94.92%. 3.2. Characterization of lactic acid
The process of lactic acid extraction under optimized conditions is
illustrated in Fig. 1. Under optimized conditions through three-time Further to characterize purified lactic acid, FTIR and NMR spectra
sequential extraction, the purified form of lactic acid was obtained were recorded. FTIR spectroscopy was used to characterize the lactic
from the MRS broth production medium. HPLC data confirmed the pu­ acid after purification to identify the purified product’s functional
rity of lactic acid (93 ± 2.1%) and 86 ± 1.8% recovery. Glucose was the group. FTIR spectra pattern of pure lactic acid from Sigma and purified
impurity present in the purified lactic acid. The MRS medium used for one from MRS broth production was similar. In the FTIR spectra of lactic
lactic acid production contains a substantial amount of glucose (2%, w/ acid sample purified from MRS broth, the –OH bond stretching was
v), and some impurities remained in purified lactic acid. The obtained observed at wavenumber 3161 cm− 1, the stretching vibration of the
yield and purity of lactic acid was quite comparable to other studies. –C–– O bond at 1728 cm− 1 and the –C–O– bond at 1127 cm− 1. The
Compared to the different processes, the present method is more spectra also showed asymmetric stretching vibration of the –COO– bond
straightforward as lactic acid is recovered in one-step phase partitioning at 1510 cm− 1, and the deformation of the –OH bonds at 1450 cm− 1. The
involving butanol and ammonium sulphate. Furthermore, the butanol –C–H and –CH3 bond stretching present in the molecule at wavenumber
used is recovered during rotary evaporation and can be used over again. 2985 and 2883 cm− 1. The methyl group’s asymmetric bending defor­
Moreover, other procedures have their inherent drawbacks, such as mation at 1402 cm− 1 was also observed in the extracted lactic acid. The
membrane processes have the problem of membrane fouling; calcium peak at 1230 cm− 1 was due to the C–O– asymmetric vibrations linked
hydroxide precipitation has the disadvantage of a generation of gypsum with asymmetric CH3 rocking vibrations. The comparison with the

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S. Kumar et al. Bioresource Technology 318 (2020) 124260

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the National Agricultural Science Fund (NASF), ICAR, Govt. of India for
Tong, W.-Y., Fu, X.-Y., Lee, S.-M., Yu, J., Liu, J.-W., Wei, D.-Z., Koo, Y.-M., 2004.
carrying out this study. Author SK is grateful to the Council of Scientific Purification of l(+)-lactic acid from fermentation broth with paper sludge as a
and Industrial Research (CSIR, Govt. of India) for Senior Research As­ cellulosic feedstock using weak anion exchanger Amberlite IRA-92. Biochem. Eng. J.
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providing the instrumentation and infrastructure facility for the study. liquids and lignocellulosic by-products for bio-based lactic acid production.
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Appendix A. Supplementary data Yu, J., Zeng, A., Yuan, X., Zhang, X., Ju, J., 2015. Optimizing and scale-up strategy of
molecular distillation for the purification of lactic acid from fermentation broth. Sep.
Sci. Technol. 50, 2518–2524.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2020.124260.