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Recent trends in lactic acid biotechnology: A brief

review on production to purification
Tayyba Ghaffar a, Muhammad Irshad a,*, Zahid Anwar a, Tahir Aqil b,
Zubia Zulifqar a, Asma Tariq a, Muhammad Kamran a, Nudrat Ehsan a,
Sajid Mehmood a

Department of Biochemistry, NSMC, University of Gujrat, Pakistan

Department of Botany, University of Gujrat, Pakistan

article info


Article history:

Lactic acid is one of the most important organic acid which is being extensively used

Received 8 February 2014

around the globe in a range of industrial and biotechnological applications. From its very

Received in revised form

old history to date, many methods have been introduced to improve the optimization of

11 March 2014

lactic acid to get highest yields of the product of industrial interests. In serious consider-

Accepted 15 March 2014

ation of the worldwide economic and lactic acid consumption issues there has been

Available online 13 April 2014

increasing research interest in the value of materials with natural origin, which are cheap,
abundant and easily available all around the year. Recent trends showed that lactic acid


production through fermentation is advantageous over chemical due to the environmental

Green biotechnology

concerns of the modern world. The eco-friendly processing and fermentable capability of

Lactic acid

many of the agricultural and agro-industrial based raw materials or by-products respec-


tively makes them attractive candidates in fermentation biotechnology to produce a value-

Product optimization

added product with multiple applications. In fact, major advances have already been


achieved in recent years in order to get pure lactic acid with optimal yield. The present
review work is summarized on the multi-step processing technologies to produce lactic
acid from different substances as a starting material potentially from various agroindustrial based biomasses. The information is also given on a purification through
schematic representation of the product of quality interests.
Copyright 2014, The Egyptian Society of Radiation Sciences and Applications. Production
and hosting by Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: 92 344 4931030.

E-mail addresses:, (M. Irshad).
Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications

Production and hosting by Elsevier
1687-8507/Copyright 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. All rights

J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s 7 ( 2 0 1 4 ) 2 2 2 e2 2 9


Background introduction

From the histological point of view lactic acid has a long history of uses for fermentation and was first discovered in 1780
by Swedish chemist, Carl Wilhelm Scheele, who isolated the
lactic acid from sour milk as an impure brown syrup and gave
it a name based on its origins: Mjolksyra. After nine years
around in 1789, Lavoisier named this milk component acide
lactique, which became the core origin of the current terminology for lactic acid. For a very long time until 1857 it was
being considered a milk component while later on that year
Pasteur discovered another phenomenon and postulated lactic
acid as a fermentation metabolite generated because of the
involvement of certain microorganisms. In support with Pasteurs discovery a French scientist Fremy produced lactic acid
by fermentation and this gave rise to first industrial production of lactic acid in the United States by a microbial process in
1881. From that time it has Wide applications in food, pharmaceutical, cosmetic and chemical industries etc (Narayanan,
Roychoudhury, & Srivastava, 2004). The worldwide demand
for lactic acid is estimated roughly to be 130 000 to 150 000 tons
per year (Randhawa, ahmed, & akram, 2012). However, the
global consumption of lactic acid is expected to increase
rapidly in the near future (Wee, Kim, & Ryu, 2006).


Common aspects in the synthesis of lactic

Lactic acid can be synthesized industrially by two means either

through chemically or by microbial fermentation. However,
the least one (fermentation through microbes) has some potential advantages e.g. pure lactic acid can be attained
whereas, chemical synthesis of lactic acid always give a
raceme mixture (Randhawa et al., 2012). The existence of L()-


lactic acid which have high optical purity provides polylactic

acids with high crystallinity and high melting point (Oh et al.,
2005). One of the most expanding uses of lactic acid is its use
in polymerization of lactic acid to form polylactic acid (PLA), a
polymer of great interest because it can be produced from
renewable means which is biodegradable in nature. Fig. 1 illustrates the production of PLA using starch as a potential
starting substrate. Many PLA-based products are already
available in the market, where they are used to replace the
petroleum-based consumables (Ilmen, Koivuranta, Ruohonen,
Suominen, & Penttila, 2007). Lactic acid is the simplest hydroxy
acid which has an asymmetric carbon atom and is present in
two optically active forms. In humans and other mammals
only the L()- isomer is present, whereas the D()- and L()-both
enantiomers can synthesized using an appropriate bacterial
strains. Therefore, most of the worlds commercial lactic acid is
prepared by fermentation of carbohydrates by bacteria, using
homolactic microbes such as a variety of modified or optimized
strains the genus Lactobacilli, which especially produce lactic
acid. Commercially pure lactic acid can be synthesized by microbial fermentation of the following carbohydrates such as
glucose, sucrose, lactose, and starch/maltose derived from
feed-stocks such as beet sugar, molasses, whey, and barley
malt. The preference of feedstock entirely depends on its price,
availability, and on the respective costs of lactic acid recovery
and purification. Biomass of lignocelluloses is a low-cost and
extensively available renewable carbon source as an alternative to these conventional feed-stocks that has no challenging
food value (Pang, Zhuang, Tang, & Chen, 2010). Other biological
agents capable of producing lactic acid are also used such as
strains of Rhizopus, Escherichia, Bacillus, Kluyveromyces and
Saccharomyces (Maas et al., 2008).
Widely used method for the production of lactic acid is
Batch fermentation. Conditions for Fermentation are different
for each industrial method but are usually in the range of
45e60  C having a pH of 5.0e6.5 for Lactobacillus delbrueckii and

Fig. 1 e Production of Poly-L-lactic acid using starch as a substrate.


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43  C with a pH of 6.0e7.0 for Lactobacillus bulgaricus. The acid

synthesized is neutralized by calcium hydroxide or calcium
carbonate (Zhou, Causey, Hasona, Shanmugam, & Ingram,
2003). Fermentation takes 1e2 days under optimal lab conditions. The yield of lactic acid after the fermentation stage is
90e95 wt% based on the initial sugar or starch concentration.
Rate of fermentation depends mainly on the parameters such
as pH, temperature, initial substrate concentration and concentration of nitrogenous nutrients (Zhou et al., 2003). There
has been a great interest in solving the issues such as PLA
weakens at high temperature for the purpose to enhance the
use of this renewable plastic (Omay & Guvenilir, 2012). Hydrolysis reaction of methyl lactate is use to enhance the performance of batch reactive distillation to produce lactic acid
(Edreder, Mujtaba, & Emtir, 2010). Lactic acid bacteria are
conventionally particular microbes that have complex
nutrient necessities industrial wastes of food which have high
moisture and loaded in carbon source have been considered
as an eye-catching nutrient source for industrial production of
lactic acid (Randhawa et al., 2012). Product recovery is an
important step in lactic acid production that is associated to
separation and purification of lactic acid from fermentation
broth (Chakkrit, 2010). A conventional procedure for lactic acid
production by lactose fermentation involves the purification
steps that are necessary to attain the pure lactic acid. Alternatives to this industrialized procedure are being studied.
Numerous studies on lactic acid purification have been conducted by using several different techniques for separation
such as ion exchange, reactive extraction, membrane technology, distillation and electro-dialysis (Gonzalez, Alvarez,
lvarez, 2008).
Riera, & A


Chemical synthesis of lactic acid

The commercial procedure for chemical synthesis of lactic

acid is based on lactonitrile. Hydrogen cyanide is added to
the acetaldehyde in presence of a base to make lactonitrile.
The reaction occurs at high atmospheric pressures in liquid
phase. The crude of lactonitrile is recovered. Purification is
done by distillation. Then it is hydrolyzed to lactic acid,
either by concentrated H2SO4 or by HCl to produce the
resultant lactic acid and ammonium salt. After that lactic
acid is esterifies by methanol to produce methyl lactate
before purification through distillation, and then hydrolyzed
by water in the presence of acid catalyst to produce methanol and lactic acid. The chemical synthesis process produces a racemic mixture of DL-lactic acid. Following
reactions are involved in this process (Boontawan,
Kanchanathawee, & Boontawan, 2011).
(a) Addition of Hydrogen CyanideCatalyst
Hydrogen cyanide




H2 SO4 /
Sulphuric acid



Lactic acid


Lactic acid

Methyl lactate


(d) Hydrolysis by H2O

Methyl lactate


Lactic acid


Production of lactic acid by fermentation processes

Fermentation is an energy yielding process in which organic

molecules play role as both electron donors and electron acceptors. The molecule which is metabolized does not possess
its whole potential energy extracted from it. Therefore, lactic
acid bacteria are widely used as a cheap method for food
maintenance by fermentation and usually no or little heat is
required in fermentation (Boontawan et al., 2011). In batch
fermentation process the culture is first grown in a series of
inoculums vessels and after that transferred to the fermentor.
The size of inoculum is usually 5e10% of the liquid volume in
this fermentor. The fermentation is usually kept at 35e45  C
and at pH 5e6.5 by adding a suitable base, such as ammonium
hydroxide (Gonzalez, Alvarez, Riera, & Alvarez, 2007). Other
fermentations for lactic acid production are, fed-batch,
repeated batch, and continuous batch. But the higher concentration of lactic acid has achieved in batch and fed-batch
cultures than in others, whereas higher productivity has obtained by continuous cultures. Another advantage of the
continuous batch over batch culture is that the process can be
run for a long period of time (Vijayakumar, Aravindan, &
Viruthagiri, 2008). L() lactic acid is produced commercially
in fermentation processes by lactic acid bacteria or fungi such
as Rhizopus oryzae in submerged culture. Rhizopus sp. can
manufacture L() lactic acid from starch but the yield is very
low as compared to lactic acid bacteria. 85% yield of L() lactic
acid can be achieved using an airlift bioreactor under optimal
conditions. The mycelia are not suitable enough for lactic acid
as their morphology does not suit for fermentation because
they increase the viscosity of the medium. They wrap up
around the impellers and cause obstruction during sampling
and in overflow lines. Small pellets of mycelia of R. oryzae are
produced regulating the concentration of inoculated spores in
pre culture. But there is a problem with pellets that they have
insufficient mass transfer. Cotton like flock morphology can
be obtained by mineral supports (Narayanan et al., 2004). An
Overview of two production methods is given in the Fig. 2
(Vijayakumar et al., 2008; Wee et al., 2006).

Role of microbial cultures in lactic


(b) Hydrolysis by H2SO4


(c) Esterification

Ammonium salt

Lactic acid bacteria are generally gram-positive bacteria. They

are non-motile, have non-spore-forming rods and cocci. They
do not synthesize porphyrins and cytochromes there for why
they cannot generate ATP. Lactic acid bacteria grow under
anaerobic conditions as they do not use oxygen for their energy manufacture, but they are also capable of growing in the
presence of oxygen. They remain protected from byproducts

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of oxygen (e.g. H2O2) due to peroxidases they have and are

aero tolerant anaerobes. They are distinguished from other
organisms because of their capability to ferment hexoses to
lactic acid. They are divided generally in two categories according to their fermentation patterns, homo fermentative
and hetero fermentative bacteria (Vijayakumar et al., 2008).
Microorganisms which have capability to ferment cheap
raw materials rapidly, require small amount of nitrogenous
nutrients, provide high yields of required stereo specific lactic
acid under low pH and high temperature conditions, produce
small amounts of cell mass and slight amounts of other
byproducts are industrially desirable (Narayanan et al., 2004).
Most of the lactic acid production was carried out by using
microorganisms such as lactic acid bacteria (LAB), and filamentous fungi, e.g. Rhizopus. They utilize glucose in aerobic
conditions to produce lactic acid. Rhizopus species, R. oryzae
and R. arrhizus, can convert starch directly to L()-lactic acid
due to their amylolytic enzyme activity (Wee et al., 2006).
Fungi have drawn a great attention in Lactic acid production
such as R. oryzae. industrial fermentation has been improved
the yield of the desired product by using The immobilization
property of microorganisms which has been attractive for

industrial fermentation (Tanyldz, Bulut, Selen, & O
2012). Efforts have been made to improve the production of
lactic acid through metabolic engineering approaches (Wee
et al., 2006). Yeasts, such as Saccharomyces cerevisiae, are
more resistant to low pHs as compared to lactic acid bacteria.
genetically engineered yeast has been prepared for producing
lactic acid and has applied for large-scale production on
experimental basis (Ishida et al., 2005). Mixed culture of
Lactobacillus pentosus and Lactobacillus brevis were used by
Garde to produce lactic acid from wheat straw hemicelluloses.
Yun established the production of lactic acid using Enterococcus faecalis RKY1 from single and mixed sugars
(Vijayakumar et al., 2008). Escherichia coli were also used for the
production of L-lactate and D-lactate. JP203 (pta:Tn5 phoA_-lacZ
ppc:cat supE hsdS ara proAlacY galK rpsL xyl mtl), were reported
as the best E. coli strains for production of D-lactate, the strain
has numerous antibiotic resistance genes (kan and tet) (Zhou
et al., 2003). Enterococcus faecium No. 78 has been used for the
production of lactic acid in repeated batch fermentation
mode. It was separated from puto, which is a type of fermented
rice in the Philippines sago starch was used as the sole carbon
source which was enzymatically liquefied (Nolasco-Hipolito
et al., 2012). A Bacillus sp. strain 2e6 has been isolated for
production of L-lactic acid at 55uC from soil samples. It is a
good lactic acid producer because of its thermo-phillic characteristic and optically pure L-lactic acid can be formed by this
strain under open circumstance without any sterilization (Qin
et al., 2009).


Fig. 2 e A schematic representations of the two

manufacturing processes of lactic acid.


Raw material for lactic acid production

The commercial production of lactic acid using fermentation

technology mainly depends on the cost of raw material used.
Therefore, it is compulsory to select a raw material for industrial production of lactic acid with a number of characteristics such as low cost, rapid rate of fermentation, lowest
amount of contaminants, high yields of lactic acid production,
little or no formation of by-products and availability for whole
year (Randhawa et al., 2012).
E. faecalis has been used to hydrolyze Agricultural resources
such as wheat, barley, and corn by commercial amylolytic
enzymes RKY1 and fermented into lactic acid. Lactic acid
productivities obtained were at >0.8 g/L h although no added
nutrients were provided to those resources, using barley and
wheat (Oh et al., 2005). Attempts have been also made for
lactic acid production from sugar cane molasses as a cheap
raw substrate through fermentation by using indigenous
bacterial culture (L. delbrueckii). The conditions are optimized
for fermentation taking into consideration fermentation time,
substrate level and temperature as factors for main process.
Recently several authors have reported the valorization of
lignocellulosic materials by biotechnology elsewhere in
different sectors in addition with their suitable applications
(Anwar, Gulfraz, & Irshad, 2014; Asgher, Shahid, Kamal, &
Iqbal, 2014; Iqbal, Kyazze, & Keshavarz, 2013; Shahzadi et al.,
2014). Lignocellulosic biomass was used for the production
of lactic acid as an alternate to above conventional feed-stocks
(Fig. 3). Lignocellulose is composed of and cellulose and
hemicullolose that is made up of hexose and pentose sugar


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surrounded in phenolic polymer lignin matrix. the main procedure relies on hydrolysis by cellulolytic and hemicellulolytic
enzymes to obtain sugars from lignocelluloses which are
fermentable. A pretreatment, either chemical or mechanical
is required of the lignocellulose to reduce the size of particle,
to remove the lignin or to modify it and to improve the convenience of the polysaccharides for the purpose of enzymatic
hydrolysis (Maas et al., 2008). Supplementation of fermentation media is very necessary for the fast production of lactic
acid with adequate nutrients. Yeast extract is used as the
most general nutrient for production of lactic acid, but this
may cause of increase in production costs considerably. A byproduct from the process of corn steeping has been utilized
effectively for lactic acid production as an alternate. Since the
corn steep liquor is derived from corn, 85% of its nitrogen
content consists of amino acids, peptides and proteins (Wee
et al., 2006). For production of optically pure D-lactic acid
from raw glycerol, five technical schemes have been planned.
These were pretended and assessed economically based on
five fermentative scenarios by using engineered strains of E.
coli (Posada, Cardona, & Gonzalez, 2012).


Optimization of lactic acid fermentation

A range of procedure variables have been optimized in the

production of lactic acid using wastes raw materials as a
substrate. It has been found that productivity is affected by
temperature, fermentation time and the level of substrate.
The highest yield has been obtained after 7 days of fermentation in media possessing 18% substrate level having a mean
value of 7.76  0.08 g/100 mL (77.6 g/L) at temperature of 42  C.
The maximum recovery was 78.30%.of lactic acid with respect
to initial whole sugar contents of the media (9.91  0.20 g/

100 mL) (Randhawa et al., 2012). R. oryzae NRRL 395 which

immobilized in polyurethane foam has been used in Lactic
acid production by using response surface methodology.
Three independent variables; pH, glucose concentration, and
rate of agitation has been explained by a 23 full-factorial
central composite design (Tanyldz et al., 2012). Maximum
production of lactic acid was achieved 93.2 g/L by using a
glucose concentration of 150 g/L, pH of 6.39 and rate of
agitation 147 rpm. Agitation rate and concentration of glucose
have found to be as limiting factors. So, any variation in these
parameters can alter the production of lactic acid. Lactic acid
production is not affected by the Initial pH due to neutralizing
agent added. Production of lactic acid under optimum conditions using immobilized whole cells was calculated about 55%
that is higher than the lactic acid production from suspension
culture systems (Tanyldz et al., 2012). In another study,
calcium alginate has been utilized to immobilize L. delbrueckii
bacteria by using pineapple waste. Various important factors
such as temperature, pH, calcium alginate concentration,
beads diameter and inoculum size have to be consider systematically for the successful production of lactic acid. Yeast
strains having heterologous L-lactate dehydrogenases can be
used for lactic acid production. As these microbes can survive
in acidic environments, it was identified that at low pH, cells
are stressed by lactic acid (Valli et al., 2006). Two low-cost
nitrogen sources such as yeast autolysate (YA) and corn
steep liquor (CSL) have been used for The production of D()
lactic acid with Lactobacillus LMI8 sp. To verify maximal lactic
acid production a central composite design was used. The
results of the experiments have evaluated by surface response
methodology. The assays were carried in Erlenmeyer flasks of
250 mL consisting of 100 mL of production medium which was
maintained by refrigerated incubation at a temperature of
37  1  C for 48 h at 200 rpm. CSL and interactions between CSL

Fig. 3 e Lignocellulosic substrates for lactic acid production.

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and YA was found to be affecting significantly Lactic acid

production. Maximum production of D() lactic acid calculated was 41.42 g/L e a central value, which corresponded to
5 g/L of YA and 15 g/L of CSL (de Lima, Coelho, Blanco, &
Contiero, 2009). Two response surface methodologies
including central composite designs have been applied successfully to assess the effect of corn steep liquor, cheese whey,
ammonium sulfate, and pH and temperature control on lactic
acid fermentation by using Lactobacillus sp. LMI8 which was
isolated from wastewater of cassava flour. In the first design,
ammonium sulfate and corn steep liquor were investigated as
cheap nitrogen sources in a combination with some other
components to replace yeast extract for economical production. The best results have been achieved with 15 g/L of corn
steep liquor, 55 g/L of lactose and 5.625 g/L of ammonium
sulfate. A second central composite design has been applied
After explaining the optimal nutritional conditions for production of lactic acid, to determine the level to which pH and
temperature affect the lactic acid production with the aim of
improving the fermentation production (Edreder et al., 2010).
Production of lactic acid by immobilized cells of Lactococcus
lactis IO-1 from glucose has been studied using cells immobilized either by encapsulation in microcapsules or entrapment
in beads of alginate in membrane of alginate. The process is
optimized by Taguchi method using shake flasks and then
(Sirisansaneeyakul et al., 2007).


Purification of lactic acid

The recovery of lactic acid must be improved in order to reduce

lactic acid losses and to increase purity (Gonzalez et al., 2008).
Purification or product recovery is an important step in production of lactic acid that is associated with separation and
purification of lactic acid form fermentation broth. Fermentation broth contains a number of impurities such as residual
sugars, color, nutrients and other organic acids, as part of cell
mass. These impurities must be removed from the broth in order


to achieve more pure lactic acid. To recover and purify the L()lactic acid produced from the microbial fermentation media
economically and efficiently, ion exchange chromatography is
used among the variety of downstream operations. It is
extremely selective and gives product recovery at very low cost
within a short period of time. The other purposes were to
analyze the end product purity, to check adsorption or desorption behaviors of lactic acid and to examine the applicability of
this method for industrial usage. Process strengthening and
monomer grade lactic acid has been achieved in high purity by
advancement of a new membrane-integrated technology. It has
lesser the processing steps, chemical requirement and energy
expenditure. The fastidious modular design provides a great
elasticity in action of the system which the modern industrialized sector is looking for dreadfully in this era of shrunken profit
edge. With the optimized participation of nanofiltration and
microfiltration membrane modules in a steady production system, a logically high change of 76e77 L/m-2 h-1 has gained for a
bigger than 95% pure L() lactic acid (Pal & Dey, 2012). Commercial production of pure lactic acid has also been carried out
in many areas using strong-acid cation-exchange resins as solid
catalysts (Zhang, Ma, & Yang, 2004). Surface active molecules
such as enzymes and proteins are separated in aqueous solution
by a simple and low cost method known as foam separation.
Applicability and efficiency of foam separation technique has
been studied by lactic acid broth, yeast extract and spent
brewers residual beer was used to examine the partial purification of products and recovery of important components from
industrial waste stream (Kurt, 2006). Investigation has been
carried out to check the usability of nanofiltration in a definite
process of lactic acid production based on old bipolar electrodialysis operations. DK nanofiltration membrane was used for
recovery rate and purification of lactic acid efficiency. Magnesium and calcium ions are removed by nanofiltration efficiently
from a sodium lactate fermentation broth before its conversion
and concentration by electrodialysis (first level of potential
integration). Maximum removal of impurities and lactic acid
recovery has been achieved at maximum pressures of transmembrane. Phosphate and Sulfate ions are also partially

Fig. 4 e Substantial purification of lactic acid from fermentation broths by several membrane-based unit operations


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removed (40% rejection). Lactic acid can also be extracted from

aqueous solution using n-butanol as an extractant. Factors such
as pH, mixing time, initial concentration of lactic acid, and volume ratio between the organic and the aqueous phase affect the
extraction of lactic acid. Degree of lactic acid extraction and
distribution coefficient increases when the pH of aqueous solution is decreased. The pH effect is considerably marked when
the pH of the aqueous solution is less than 1. Initial concentrations of lactic acid and organic-to aqueous volume ratio appear
to have positive effect on the degree of extraction and distribution coefficient. As the n-butanol is miscible partially in water, so
integration of aqueous phase into organic phase in the extraction has a great organic-to-aqueous volume ratio (Chawong &
Rattanaphanee, 2011). Lactic acid can be separated and substantially purified from fermentation broths by several
membrane-based unit operations as shown in the Fig. 4.



Due to the growing demand of L() lactic acid for a wide range
of applications in addition with the production of biodegradable plastic (PLA), it is necessary to make improvement in the
conventional fermentation-based lactic acid production processes with efficient and sustainable method. Membrane
based hybrid reactor system have proved successful in this
goal without generating any negative environmental crash
(Fig. 4). The lactic acid production is significantly influenced by
fermentation time, temperature and substrate levels. Lactic
acid bacteria, regarded generally as safe, that produce lactic
acid optimal under conditions at 30  C and pH 5 in the
customized MRS broth containing 2% yeast extract and 2%
glucose (Adesokan, Odetoyinbo, & Okanlawon, 2009). Similarly, Biswas (2005) has also reported that under controlled pH
condition, there is a significant increase in the level of production of lactic acid when compared to uncontrolled conditions. Recently, lignocellulosic materials have received a great
attention as possible feed stocks to substitute the edible
starch material. Low cost cellulosic materials such as industrial wastes, agricultural waste and forestry waste are recommended as cost effective feed stocks for large scale
fermentation (Tang, Bu, Deng, Zhu, & Jiang, 2012). The separation and purification of lactic acid from the fermentation
broth are major components of the production expenditure
(Matsumoto, Panigrahi, Murakami, & Kondo, 2011). However,
there is still a big need for the researches to be carried out in
order to produce lactic acid biotechnologically and commercially within the lowest cost, lowering the cost of the raw
materials and improvement of high-performance microorganisms producing lactic acid (Vijayakumar et al., 2008).


Adesokan, Y., Odetoyinbo, B., & Okanlawon, B. (2009).

Optimization of lactic acid production by lactic acid bacteria
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Dissertation, Master of Science. Patiala, India: Thapar Institute
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