III.

REVIEW ON PROCESS LITERATURE

Lactic acid can be synthesized industrially by two means either through chemically or by
microbial fermentation. However, the fermentation through microbes has some potential
advantages where pure lactic acid can be attained while, chemical synthesis of lactic acid always
give a racemixture. The commercial chemical production of lactic acid is based primarily on
lactonitrile. Hydrogen cyanide is added to the acetaldehyde in presence of a base to make
lactonitrile. The reaction ccurs at high atmospheric pressures in liquid phase. While production
of lactic acid by fermentation processes 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.

3.1 RAW MATERIAL PREPARATION

Fruit based industry produces large volume of wastes, both solids and liquids; these
wastes pose increasing disposal and pollution (high BOD or COD) problems and represent a loss
of valuable biomass and nutrients. However these carbohydrate rich wastes can be tuned as

valuable substrates for the commercial production of organic acids like lactic acid and thus can
be regarded as a viable option for meeting the growing demand for lactic acid Umesh (2014).
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 Gaffar (2014).
According to the journal by Jawad (2012), mango peel as a by-product of mango
processing industry could be a rich source of bioactive compounds, and enzymes such as
protease, peroxidase, polyphenol oxidase, carotenoids, vitamins C and E, dietary fibers, enzymes
and carbohydrate content of 20.8028.20% in dry weight samples of mango peel. Furthermore,
mango peels have been shown to be a rich source of flavonol O- and xanthone C-glycosides,
gallotannins and benzophenone derivatives. However, reports on the use of mango peels for the
production of industrially relevant metabolites such as lactic acid through fermentation processes
are rare. Thus, cultivation of microorganisms on these wastes may be a value-added process
capable of converting these materials, which are otherwise considered to be wastes, into valuable
products through processes with techno-economic feasibility.
According to the patent of Keikotlhaile (2010), pesticide residues have been found in
various fruits and vegetables; both raw and processed. One of the most common routes of
pesticide exposure in consumers is via food consumption. Most foods are consumed after passing
through various culinary and processing treatments. A few literature reviews have indicated the
general trend of reduction or concentration of pesticide residues by certain methods of food
processing for a particular active ingredient. However, no review has focused on combining the

obtained results from different studies on different active ingredients with differences in
experimental designs, analysts and analysis equipment.
The food crops treated with pesticides invariably contain unpredictable amount of these
chemicals, therefore, it becomes imperative to find out some alternatives for decontamination of
foods Bajwa (2014). The washing with water or soaking in solutions of salt and some chemicals
e.g. chlorine, chlorine dioxide, hydrogen peroxide, ozone, acetic acid, hydroxy peracetic acid,
iprodione and detergents are reported to be highly effective in reducing the level of pesticides.
Preparatory steps like peeling, trimming etc. remove the residues from outer portions. Various
thermal processing treatments like pasteurization, blanching, boiling, cooking, steaming,
canning, scrambling etc. have been found valuable in degradation of various pesticides
depending upon the type of pesticide and length of treatment.
Hydrolysis is a chemical process in which a molecule of water is added to a substance.
Sometimes this addition causes both substance and water molecule to split into two parts. In such
reactions, one fragment of the target molecule (or parent molecule) gains a hydrogen ion
(Wikipedia).
According to Kolusheva (2006), Starch hydrolysis is widely used process in various
industries. The production of low molecular mass products from starch substrate underlies the
sugar, brewing, spirits, textile, some food processing and other industries.
At present starch hydrolysis is carried out in two basic ways acidic and enzymatic. The
older and more traditional method is acidic hydrolysis which requires highly acidid medium (pH
= 1-2) obtained through mineral acids; high temperatures (150 230 C) and high pressure [1-4].
As a result of the thermal processing, acidic hydrolysis produces unnecessary byproducts which
contaminate the end-product-hydolysate. The enzymatic hydrolysis of starch is carried out under
milder conditions: lower temperatures (up to 100C), normal pressure, pH of the medium around
6-8 [2,5,6]. At the same time enzymatic hydrolysis is characterized by high reaction rate, high

stability of the enzyme towards the denaturing action of solvent, detergents, proteolythic nzymes,
and a decrease in the viscosity of the reaction medium at higher temperatures, etc.
Basic parameters which affect the hydrolysis process temperature, pH of the medium,
concentration of the substrate and concentration of the enzyme usually vary depending on the
source of the enzyme. Most often the hydrolysis with thermally resistant amylase is carried out
at a temperature 90-100C [2,6], concentration of the substrate in the suspensions varying from
20% to 35% [2,5,6,7], pH between 6 and 8, and enzyme concentration 0.03 1% [2,6,7].
Lactic acid production processes traditionally suffer from end-product inhibition. An
undissociated lactic acid passes through the bacterial membrane and dissociates inside the cell.
The inhibition mechanism of lactic acid is probably related to the solubility of the undissociated
lactic acid within the cytoplasmic membrane and the insolubility of dissociated lactate, which
causes acidification of cytoplasm and failure of proton motive forces. It eventually influences the
transmembrane pH gradient and decreases the amount of energy available for cell growth
(29,88). Therefore, to alleviate the inhibitory effect of lactic acid during the fermentation, it must
be removed selectively in situ from the fermentation broth Wee (2006).
In the preparation of mango peel before fermentation from the method by Jawad (2012),
The washed mango peels were cut into small pieces and blended with ultra pure water in the
ratio (1:1) (w/v) using an electrical food processor (Kenwood) at 25 C for 5 min. The humidity
of the washed mango peels to the air dried mango peels before staring the fermentation process
was calculated to be 1113%. While, the humidity of mango peels after mixing with water to be
in the fermentation suspension form was found to be 91.40%. The pH of the blended peel was
adjusted using 1 M of NaOH or HCl and thereafter, 30 mL was dispensed into conical flasks of
100 mL capacity.
In the method used by Umesh (2014), for the preparation of hydrolysates for
fermentation of fruit peel, the modified method of Puimput et. al., 2008 was used for substrate
hydrolysate preparation. About 8gram of each fruit peel waste was steam exploded in an

autoclave at 121C for 20min. Sterile water was added to the wet pretreated material to make the
volume of 200ml and boiled at 80C for 30 min followed by filtration with cheese cloth. Acid
hydrolysis of filtrate was carried out by autoclaving at 121C with concentration of 1% HCl v/v
for 30min. The pH of the hydrolysate after hydrolysis was adjusted with CaO to 6-6.8 and the
CaSO4 precipitate formed was removed by filtration with Whatman filter paper No.1.

3.2 REACTIONS
According to Gaffar (2014), 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 racemic mixture.
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 feedstocks that has no challenging food value Other biological agents capable of producing lactic
acid are also used such as strains of Rhizopus, Escherichia, Bacillus, Kluyveromyces and
Saccharomyces.

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 ccurs 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.
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.
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 35-45 C and at pH 5-6.5 by adding
a suitable base, such as ammonium hydroxide. 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.
3.2.2 According to the journal by Wee (2006)

Batch, fed-batch, repeated batch, and continuous fermentations are the most frequently
used methods for lactic acid production. Higher lactic acid concentrations may be obtained in
batch and fed-batch cultures than in continuous cultures, whereas higher productivity may be
achieved by the use of continuous cultures. Another advantage of the continuous culture
compared to the batch culture, is the possibility to continue the process for a longer period of
time.
3.2.3 According to the journal by Rao (2000)
Fermentation using lactic acid bacteria is widely used in food fermentation. The lactic
acid bacteria need to withstand varying environmental conditions including differences in
temperature, pH and salinity, depending on the specific application. An enhanced salt and pH
resistance of lactic acid bacteria is attractive in shrimp waste, fish, vegetables and seafood
fermentation.
3.2.4 According to Vijayakumar (2007)
Lactic acid bacteria are usually gram-positive, non-motile, non-spore-forming rods and
cocci. They lack the ability to synthesize cytochromes and porphyrins (components of respiratory
chains) and therefore cannot generate ATP by creation of a proton gradient. Since they do not use
oxygen in their energy production, lactic acid bacteria grow under anaerobic conditions, but they
can also grow in the presence of oxygen. They are protected from oxygen byproducts (e.g.
H2O2) because they have peroxidases and these organisms are aero tolerant anaerobes. They are
differentiated from other organisms by their ability to ferment hexoses to lactic acid. Lactic acid
bacteria can be divided into homo fermentative and hetero fermentative based upon the products
produced from the fermentation of glucose.
Homo fermentative organisms ferment glucose to two moles of lactic acid, generating a
net of 2 ATP per mole of glucose metabolized. Lactic acid is the major product of this

fermentation. Hetero fermentative lactic acid bacteria ferment 1 mole of glucose to 1 mole of
lactic acid, 1 mole of ethanol, and 1 mole of CO2. One mole of ATP is generated per mole of
glucose, resulting in less growth per mole of glucose metabolized. Because of the low energy
yields, lactic acid bacteria often grow more slowly than microbes capable of respiration, and
produce smaller colonies of 2 3 mm.
Microorganisms for the lactic acid Production
There are large number of species of bacteria and some species of molds that possess the
ability to form relatively significant quantities of lactic acid from carbohydrates. Lactic acid
bacteria are important not only for the desirable reactions which they catalyze but also for the
undesirable activities which they promote.
Bacteria and fungi are the two groups of microorganisms that can produce lactic
acid.Although most investigations of lactic acid production were carried out with lactic acid
bacteria (LAB), filamentous fungi such as Rhizopus, utilize glucose aerobically to produce lactic
acid. Rhizopus species such as R. oryzae and R. arrhizus have amylolytic enzyme activity, which
enables them to convert starch directly to L(+)-lactic acid, but it also requires vigorous aeration
because R. oryzae is an obligate aerobe. In fungal fermentation, the low production rate, below P
= 3 g L1 h1 is probably due to the low reaction rate caused by mass transfer limitation. The
lower product yield from fungal fermentation is attributed partially to the formation of
byproducts.
Several attempts have been made to achieve higher cell density, lactic acid yield, and
productivity in fungal fermentation. Haung et al. produced lactic acid from potato starch
wastewater using R. oryzae and R. arrhizus. Park et al. produced lactic acid from waste paper by
using R. oryzae. Tay and Yang18 immobilized R. oryzae cells in a fibrous bed to produce lactic

acid from glucose and starch. Kosakai et al. cultured R. oryzae cells with the use of mycelial floc
formed by the addition of mineral support and poly ethylene oxide.
Garde et al. obtained lactic acid from wheat straw hemicellulose by using mixed culture
of Lactobacillus pentosus and Lactobacillus brevis. Yun et al. investigated the production of
lactic acid from single and mixed sugars using Enterococcus faecalis RKY1. The volumetric
productivity, cell growth and concentration of lactic acid were highest in glucose/fructose (mixed
sugar) than single sugar. Rivas et al. produced lactic acid from corn cobs by simultaneous
saccharification and fermentation using Lactobacillus rhamnosus.
Wee et al. reported the economical L(+)-lactic acid production from sugar molasses by
batch fermentation of Enterococcus faecalis. Kourkoutas et al. used immobilized Lactobacillus
casei cell on fruit pieces to produce lactic acid. Narita et al. reported the efficient production of
L(+)-lactic acid from raw starch by Streptococcus bovis 148. Chauhan et al. used the statistical
screening of medium components by Placket-Burman design for lactic acid production by
Lactobacillus sp. KCP01 using date juice. Patil et al. produced lactic acid from cane sugar using
mutant of Lactobacillus delbrueckii NCIM 2365. John et al. reported the solid state fermentation
for L-lactic acid production from agro wastes using Lactobacillus delbrueckii. Amrane and
Prigen designed a two-stage continuous reactor to produce lactic acid from lactose by using
lactobacillus helveticus and obtained high product concentration of lactic acid at very low
dilution rate.
Senthuran et al. explained lactic acid production by immobilized Lactobacillus casei in
recycle batch reactor. Fu and Mathews34 reported the lactic acid production from lactose by
Lactobacillus plantarum. Nolasco-Hipolito et al.35 explained the continuous production of L(+)lactic acid from hydrolyzed sago starch using Lactobacillus lactis. Amrane36 reported the
unstructured models for biomass formation, substrate consumption and lactic acid production
from whey using Lactobacillus helveticus.

Nancib et al. explained the joint effect of nitrogen sources and B-vitamin
supplementation of date juice on lactic acid production by Lactobacillus casei sub sp.
rhamnosus. Oh et al. used agricultural resources for the production of lactic acid by
Enterococcus faecalis. Schepers et al. reported the continuous lactic acid production in whey
permeate with immobilized Lactobacillus helveticus. Ohkouchi and Inoue40 studied the direct
production of L(+)-lactic acid from starch and food wastes using Lactobacillus manihotivorans
LMG 18011. Vasala et al. used high salt containing dairy products to produce lactic acid by using
Lactobacillus Salivarius ssp. Salicinius. Xu et al. reported the development of a continuous cellrecycle fermentation system for production of lactic acid by Lactobacillus paracasei. Xu et al.
used mixed culture of Lactobacillus sake and Lactobacillus casei for the production of lactic acid
from soybean stalk hydrolysate. Sakai et al.reported the production of lactic acid in pH-swing
open fermentation of kitchen refuse by selective proliferation of Lactobacillus plantarum.
Berry et al.produced lactic acid by batch culture of Lactobacillus rhamnosus in a
defined medium. Burgos-Rubio et al. reported the kinetic investigation of the conversion of
different substrates into lactic acid with the use of Lactobacillus bulgaricus. Hujanen and
Linko52 investigated the effects of culture temperature and nitrogen sources on lactic acid
production by Lactobacillus casei.
Bustos et al. used Lactobacillus pentosus for the production of lactic acid from vinetrimming wastes. The strains of amylase-producing Lactobacillus amylophilus were used often
for the direct conversion of starch into lactic acid. However, among the genus Lactobacillus,
Lactobacillus delbrueckii has appeared commonly in many investigation for the production of
lactic acid, Kutzanmanidis et al.45 used Lactobacillus delbrueckii NC1MB 8130 for lactic acid
production from beet molasses. Monteagudo et al. and Goksungur et al. also attempted to
produce lactic acid from beet molasses with Lactobacillus delbrueckii. Several amylolytic lactic

acid bacteria, such as Lactobacillus amylophilus Lactobacillus amylovorus and Lactobacillus

plantarum A6 can convert starch directly to lactic acid. The most common bacterium for the
industrial production of lactic acid is Lactobacillus delbrueckii, which is employed in
fermentations utilizing corn dextrose media. Other bacteria of industrial importance include
Lactobacillus bulgaricus, which utilizes lactose as a carbon source and finds use in lactic acid
production from whey media, and Lactobacillus pentosus, which is able to utilize the pentoses of
sulfite waste liquor.
3.2.5 According to the journal by Muller (2001)
Lactate is a common end product of fermentations. Some organisms, collectively called
the lactic acid bacteria, form large amounts of lactate. Lactic acid bacteria are subdivided
according to their fermentation products. The homofermentative species produce a single end
product, lactic acid, whereas the heterofermentative species produce other compounds, mostly
ethanol and carbon dioxide, along with lactate. These differences are due to the employment of
different pathways for glucose oxidation: in homofermentative organisms glucose breakdown is
via glycolysis according to glucose to lactate.
3.2.6 According to the journal by Umesh (2014)
The research work evaluates the fermentative utilization of fruit peel wastes (mango,
orange, banana and pineapple) as substrates for lactic acid production by employing
Lactoctobacillus plantarum as the starter culture. Thus the presentstudy highlights a methodology
for recycling, reprocessingand eventual utilization of fruit waste for beneficial uses rather than
their discharge to the environment which might cause detrimental environmental effects.
The optimal growth temperature, pH and NaCl concentration for the growth of
L.plantarum was found to be 37C,pH 6 and 2% NaCl. Analysis of the technological properties
of the culture was primarily done to evaluate its feasibility to be employed as a starter culture for

industrial fermentation. The acidification activity analyses showed thatthe isolated L.plantarum
strains exhibited medium acidification range of 0.65 to 0.71 pH (change in pH) after 6 hrs.
The highest lactic acid production was obtained from themango peels (10.08 g/L) ,
whereas the other substrates viz.orange, banana and pineapple peels produced 5.74 g/L,4.68 g/L
and 4.68 g/L of lactic acid respectively. Thus the present study highlights a methodology for
recycling, reprocessingand eventual utilization of fruit waste for beneficialuses rather than their
discharge to the environmentwhich might cause detrimental environmental effects.
3.2.7 According to the results of study by Rao
Four Lactobacillus species were studied for their ability to grow at high NaCl
concentrations and different initial pH values. Among these strains, Lactobacillus plantarum
strains 541 and A6 indicated to be the most salt tolerant. Both strains were able to ferment
glucose up to 8% salt and produce lactic acid even at 10% salt. For strain 541, the specific rate of
lactate production (qlac) and the yield of lactic acid relative to substrate (Yp/s) remained constant
up to 6% salt whereas the yield of biomass relative to substrate (Yx/s) decreased at higher salt
concentrations. In contrast, for strain A6, Yp/s decreased up to 6% salt whereas Yx/s did not vary
markedly. At 8% salt, decreasing performance in final biomass and lactic acid production was
observed. Both strains were able to reduce pH to values lower than 4.0 within 24h. A factorial
design was applied to study combined effects of salt and initial pH on the fermentation
parameters. It is shown that salt and pH do not interact significantly within the established
experimental domain, and that pH is the more dominant factor. Considering overall performance,
it is concluded that 4% salt and pH between 6.0-6.6 can be taken as appropriate conditions, for
the use of both strains as starter in processes where higher salt concentrations are preferred.

3.2.8 According to Parker Domnick Hunter | Process Filtration - Buyers Guide

(https://www.parker.com/literature/Domnick%20Hunter%20Process
%20Filtration%20Division/PAFD_literature/Life%20Sciences
%20Literature/Understanding%20bacterial%20and%20phage
%20contaminations.pdf)
Microbial fermentations are used for the production of a wide variety
of products including biopharmaceuticals, enzymes, amino acids and
antibiotics. Contaminations caused by bacteria or phage can have a
significant financial impact upon manufacturers as fermentation raw
materials must be replaced, additional downtime for root cause analysis
incurred and delays to the production schedule diminish facility productivity.
Despite much being written regarding the maintenance of aseptic conditions
for the duration of fermentations contaminations continue to occur. The
following article is intended as a practical guide to understanding why
contaminations occur in order to reduce the risk of their future occurrence.
Risk of contamination depends on the process Fermentation processes can vary greatly
in their scale, duration and complexity which relate to their purpose. Some fermentations are
more susceptible to contaminations these include those that utilize nutrient-rich medias; contain
slow growing organisms; take a protracted length of time; are performed under moderate
temperature and pH ranges.
A more complex process which uses multiple feeds and a high sampling frequency
correlate with an increased risk of contamination. The detrimental effects of these additional
operations on process robustness should be considered although they are frequently ignored as

process performance is easier to quantify than the risk of process failure. Once a contamination
has occurred the contaminating organism should be identified. Many manufacturers will be
aware of the organisms they typically encounter in their local environment. Contamination by an
organism that has been previously encountered might indicate a common source.
Contaminations in small-scale fermenters
Bench-scale fermenters are used in laboratories for research or development while largescale fermenters with capacities of hundreds of thousands of litres are used for the commercial
scale production of biomass, metabolites and enzymes. Small-scale fermenters are typically
designed for process flexibility rather than operating robustness. The ingress of contaminating
microbes in these fermenters can occur by the following routes: Silicone tubing used to deliver
feeds to the fermenter where insufficient care has been taken with tubing connections; Through
bottom entry stirrer seal assemblies where a single-mechanical seal has been used and is not
being continuously supplied with steam in order to prevent a hot spot; Improperly connected air
vent filters; Improper cleaning of the sparger; Poor tightening of the fermenter top lid screws.
Contaminations in production-scale fermenters Production fermenters are more heavily
engineered and typically hard-piped with contaminations being more frequently attributable to
tainted inocula or failures in the sterilization of liquids and gases entering the vessel. Failure to
inoculate fermenters with a pure culture of the producer organism is likely to lead to be
damaging to the process if the contaminating organism can compete with the producer under the
conditions within the vessel.
Contaminated inocula
The isolation of pure cultures is critical to avoiding frequent contaminations. Methods
for isolating pure cultures include dilution to extinction, pour plating and streak plating.
Developing production strains requires considerable time, effort and expense. Once the

development is complete the culture should be appropriately stored either at reduced temperature
or by storage in a dehydrated form. The quality of these stock cultures should be monitored in
order to ensure purity, viability and productivity by culturing in shake flasks and observing a
characteristic growth profile. The preparation of an inoculum from the stored stock of cells
requires manual operations during which the risk of contamination occurring is increased. Good
laboratory aseptic technique is required through the inoculum preparation process. Samples
should be taken throughout this process even though the results are unlikely to be available
before the inoculum reaches the production process. Detection of unwanted microbial species in
these samples facilitates root causes analysis when contaminations occur.
The contamination of fermenters has a significant financial impact on manufacturers.
The likely source of a contamination may depend on scale or the complexity of the fermentation
process being operated. The prevention of contaminations can be achieved by good equipment
design, the following of standard operating procedures and a detailed understanding of the
various sterilization processes that ensure a sterile barrier is maintained around the fermenter.
Filtration can be used for the sterilization of liquids and gases entering the fermenter. Prefiltration of air entering the fermenter can help protect against phage contaminations.
Researchers concluded that Both Lactobacillus plantarum strains can produce lactic acid
and reduce the pH to values lower than 4.0 at salt concentrations up to 8%. At high salt
concentrations uncoupling between growth and energy production indicates that lactic acid
production is still possible even in detrimental conditions for growth. Factorial designs indicate
for both L. plantarum strains, that salt and pH did not interact significantly within the
experimental domain and that pH was the most determining factor for glucose fermentation, in
the range of salt concentration tested.
Depending on the strains and parameters, maximum specific growth rate, cell biomass
and lactic acid production were at salt concentrations of either 1.3% or 2.5%. However since the

ability of both strains to grow without lag phase up to 4% salt, it is concluded that a 4% salt
concentration and initial pH between 6.0 and 6.6 can be a good compromise for their use. These
more drastic conditions would still allow an efficient lactic acid production to prevent growth of
spoilage microorganisms. Considering that either L. plantarum 541 or A6 present potential to be
used in processes containing high salt concentration, the choice of one of the two strains for raw
material processing will depend mainly on the type of carbohydrate to be added in. If glucose is
planned to be used as a growth substrate for acidification, strain 541 would be preferred to strain
A6.
Notwithstanding, for economical reasons the use of other alternative carbon sources,
containing complex carbohydrates such as starch or glycogen, may be preferred. For instance,
the ability of strain A6 to ferment glycogen contained in mussel processing wastes has been
reported (Pintado et al. 1999). The use of this type of wastes would present economical
advantages. Other types of seafood processing wastes might also be considered for lactic acid
production, which opens a broad range of applications to be investigated.

3.3 SEPARATION
3.3.1 According to the journal by Vijayakumar (2007)
For the recovery of lactic acid, additional calcium carbonate is added to the medium, the pH is
adjusted to approximately 10, and the fermentation broth is heated and then filtered. This

procedure converts all of the lactic acid to calcium lactate, kills bacteria, coagulates protein of
the medium, removes excess calcium carbonate and helps to decompose any residual sugar in the
medium. Various processes are employed for the recovery and purification of the lactic acid. In
one procedure, the heated and filtered fermentation broth is concentrated to allow crystallization
of calcium lactate, followed by addition of sulfuric acid to remove the calcium as calcium
sulfate.
The lactic acid is then re-crystallized as calcium lactate, and activated carbon is used to
remove colored impurities. As an alternative to the latter step, the zinc salts of lactic acid are
sometimes prepared because of the relatively lower solubility of zinc lactate. In other procedure,
the free lactic acid is solvent extracted with isopropyl ether directly from the heated and filtered
fermentation broth. This is a counter current continuous extraction, and the lactic acid is
recovered from the isopropyl ether by further counter-current washing of the solvent with water.
In a third procedure, the methyl ester of the free lactic acid is prepared, and this is separated from
the fermentation broth by distillation followed by hydrolysis of the ester by boiling in dilute
water solution (the methyl ester decomposes in water).
The lactic acid is then obtained from the aqueous solution by evaporation of the water,
and the methanol is recovered by distillation. In a fourth procedure, secondary or tertiary alkyl
amine salts of lactic acid are formed and then extracted from aqueous solution with organic
solvents; the solvent is removed by evaporation, and the salt then is decomposed to yield the free
acid. An older procedure, not utilized commercially to any extent today, involves direct highvacuum steam distillation of the lactic acid from the fermentation broth, but decomposition of
some of the lactic acid occurs.
The fermentation broth is generally heated to 70 C to kill the bacteria and then acidified
with sulfuric acid to pH 1.8. The clarified lactic liquor is then ion exchanged and concentrated to
80 %. Smell and taste can be improved further by oxidative treatment with hydrogen peroxide.

The lactic acid obtained at this stage is suitable for some food industries. The lactic acid
produced from biological fermentation requires extensive purification operations. It is of
particular importance that the recovery processing equipment be resistant to the corrosive
action of the high concentrations of lactic acid that accumulate. Therefore, special stainless steel
equipment is most often employed for this purpose.
3.3.2 According to the journal by Taskila (2012)
Biotechnical production of lactic acid may be based on several alternative microorganisms. In addition to lactic acid bacteria filamentous fungi (e.g. Rhizopus spp.), other grampositive bacteria (e.g. Bacillus coagulans) and metabolically engineered yeasts have been used
also in industrial scale. The advantage of fungi is that they are active at and tolerate low medium
pH. Low pH reduces significantly the consumption of neutralizing agent (Ca(OH)2) in the
fermentation stage and subsequent formation of gypsum (CaSO4) in the product recovery stage.
The advantage of filamentous fungi, Bacillus spp. and yeasts compared to lactic acid
bacteria is their simple nutrient requirement in the fermentation medium. Filamentous fungi and
Bacillus spp. are better suited to lignocellulosic fermentation raw materials as they are in general
able to utilize pentose sugars in addition to hexoses. Anaerobic fermentation is generally
speaking more feasible and this favors yeasts and lactic acid bacteria. When optimized the
technical parameters such as product yield, RP and final product concentration are quite similar
for each of these production organisms.
3.3.3. According to Dunuwila (2003) Separation Process for Biobased Lactic Acid
All commercially viable, lactic acid-producing microorganisms presently require
neutralization during fermentation to ensure that the pH does not become low enough to kill the
microbes. To obtain the acid, a cation elimination process is necessary, wherein the base cation

needed to neutralize the acid during fermentation is replaced by protonation. Previously, several
strategies have been pursued by a number of researchers:
The gypsum process, wherein sulfuric acid is used to acidify the calcium salt of lactic
acid (calcium lactate is produced by neutralizing the fermentation broth with lime), results in
stoichiometric production of calcium sulfate (gypsum), which has low quality and limited
commercial value. Both the acid and the base are irreversibly consumed during the associated
chemical processes.
Electrodialysis, which uses a conventional concentrating electrodialysis step followed
by watersplitting electrodialysis to convert the salt to acid and base, is an alternative. Numerous
economic studies have indicated that this process is costly in both capital (membranes) and
operating costs (electrical power), and probably is not feasible for commodity chemical
production.
Extraction of lactic acid by polar organic solvents, water-soluble trialkyl amines, and
water-immiscible long-chain trialkyl amines in the presence of pressurized carbon dioxide have
been studied. None of these techniques is commercially viable because of long residence times
and large processing volumes.
The proposed separation process was designed to overcome the limitations of the
existing technologies. Furthermore, the technical challenges associated with the unit operations
of the proposed separation process were identified. Plausible solutions for investigation during
Phase II will be proposed.
3.3.4 According to efthilia Arvaniti, Michael Goldsworthy et al.
Microbial cells were removed using filtration or flocculation. Then lactic acid solvent
extraction or distillation is then used to purify the product. It can be purified further by use of
activated carbon and ion-exchange resins, other techniques include electrodialysis or purification
via intermediate ester formation.

3.3.5 According to Niju Narayanan et al.

The broth containing calcium lactate is filtered to remove cells, carbon treated,
evaporated and acidified with sulphuric acid to get lactic acid and calcium sulphate. The
insoluble calcium sulphate is removed by filtration; lactic acid is obtained by hydrolysis,
esterification, distillation and hydrolysis.
3.3.6 According to I.S Udachan et al.
Two-step process was used in separating and purifying lactic acid first by liquid-liquid
extraction followed by back extraction, liquid liquid extraction was done using trioctylamine as
extractant. Then to recover the lactic acid back extraction was used and the solution was reacted
with NaoH.

3.4 PURIFICATION
3.4.1 According to the journal by Vijayakumar (2007)
Lactic acid is sold in various commercial grades, and the better grades require that well-purified
substrates be utilized in the fermentation medium in order to reduce the levels of impurities
present during recovery which, without great difficulty, cannot be separated from the lactic acid.
Also, in this regard, the sugar should be depleted from the medium by harvest of the
fermentation. One of the commercial grades of lactic acid, crude or technical grade is a
colored product prepared for commercial usage at mass fraction in water of w = 22, 44, 50, 66

and 80 %. It is prepared by employing sulfuric acid to remove the calcium from the calcium
lactate derived from the heated and filtered fermentation broth, followed by filtration,
concentration, and refiltration to remove additional calcium sulfate. Thus, this grade of lactic
acid contains many of the impurities from the fermentation medium, and it finds many industrial
uses where purity of the product is not essential as, for example, in the deliming of hides in the
leather industry.
3.4.2 According to the journal by Ghaffar (2014)
The recovery of lactic acidmust be improved in order to reduce lactic acid losses and to increase
purity. 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
achievemore 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.
3.4.3 According to the journal by Komesu (2013)
The development of an efficient method of separation and purification of the lactic acid
from fermentation broth is very important, since these steps are responsible for 50 % of the

production costs. A considerable amount of researches have done a great deal of work on the
purification procedures.
Hybrid short path evaporation is an alternative separation process with potential for
recovery and concentration of thermally unstable molecules such as lactic acid. It has been
recognized as a potential method because of its low evaporation temperature and short residence
time that minimize thermal decomposition problems.
3.4.4 According to Evangelista (1994)
Lactic acid was purified by filtration, carbon treatment and evaporation. Activated
carbon is mixed with the crude extract to remove the colored components. The spent carbon is
then filtered out and the filtrate is sent to the evaporator where excess water is evaporated under
mild vacuum at moderate temperature (0.57 atm and 70C) to 37% calcium lactate concentration.
This preparation is then acidified with 63% sulfuric acid to precipitate calcium sulfate, which is
filtered out. The lactic acid is bleached a second time and then evaporated to 52 or 82%
concentration.
Lactic acid was purified by calcium lactate crystallization. The crude extract is bleached
with activated carbon and then acidified slightly before undergoing a second bleaching. Excess
water is evaporated under vacuum to obtain a density of 1.12 kg/m. At this concentration,
calcium lactate crystallizes upon cooling. The crystals may be redissolved, treated with sodium
sulfide to remove heavy metals, bleached, and recrystallized to improve purity.
Lactic acid was purified by liquid-liquid extraction The crude extract is filtered,
acidified with sulfuric acid and the resulting calcium sulfate precipitate is filtered out. The crude
lactic acid
is bleached with activated carbon and the heavy metals, calcium, and amino acids are removed
by ion exchange. Excess water is evaporated under vacuum to about 44% lactic acid
concentration before it enters the countercurrent extraction columns. The lactic acid is extracted

by diisopropyl ether in the first countercurrent extraction column. The extracted aqueous solution
still contains 20% of the total lactic acid in the crude lactic acid, which can be concentrated
further for technical applications. The acid is recovered from the solvent by countercurrent
extraction into water in the second countercurrent extraction column. the remaining solvent is
boiled off from the aqueous solution and the acid is concentrated by evaporating the excess water
to obtain food-grade lactic acid. The product is relatively free from ash but may contain other
impurities from raw materials.
Lactic acid was purified by esterification and distillation Crude lactic acid is fed into a
heated reactor where it reacts with methanol under the influence of small amounts of sulfuric
acid.
The molar ratio of lactic acid to methanol is kept at 1:1.5. The vapors distilling from the reactor
consist of methyl lactate, methanol, and water, with traces of lactic acid. This mixture is
introduced into the middle of a fractionating column. Methanol, the most volatile component,
rises to the top of the column, and is collected, condensed to a liquid and returned to the reactor.
The bottom fraction contains methyl lactate, lactic acid and water, which are collected in a kettle.
Hydrolysis of the methyl lactate takes place in the fractionating column and is completed in the
kettle. The methanol is boiled off and sent back to the reactor via the fractionating column.
3.4.5 According to Sun et al.
They used two reactors with a rectifying column carried out recovery of lactic acid
from the fermentation broth. Ammonium lactate obtained by fermentation was used directly to
produce butyl lactate by reacting with butanol for 6 h, and the esterification yield of ammonium
lactate was 87.7 % cation exchange resin which was modified by SnCl2 replaced sulphuric acid
as a catalyst, and neutral ammonium lactate replaced former lactic acid as a starting material
butyl lactate was rectified, and the purified butyl lactate was sequentially hydrolyzed into lactic

acid in presence of the cation exchange resin in the H+ form as a catalyst for 4 h, and the
hydrolysis yield was 89.7 % and the purity of recovered lactic acid was 90 %.
3.4.6 According to J. Vijayakumar et al. (2007)
Calcium carbonate is added to adjust the pH of the fermentation broth to 10 and it is
heated and filtered, This procedure converts all of the lactic acid to calcium lactate and was
allowed to crystallized followed by addition of sulfuric acid to remove the calcium as calcium
sulfate. The lactic acid is then re-crystallized as calcium lactate, and activated carbon is used to
remove colored impurities.
Lactic acid is solvent extracted with isopropyl ether directly from the heated and filtered
fermentation broth. This is a counter current continuous extraction, and the lactic acid is
recovered from the isopropyl ether by further counter-current washing of the solvent with water.
Methyl ester of the free lactic acid is prepared, and this is separated from the
fermentation broth by distillation followed by hydrolysis of the ester by boiling in dilute water
solution The lactic acid is then obtained from the aqueous solution by evaporation of the water,
and the methanol is recovered by distillation.
Secondary or tertiary alkyl amine salts of lactic acid are formed and then extracted from
aqueous solution with organic solvents; the solvent is removed by evaporation, and the salt then
is decomposed to yield the free acid.
The fermentation broth is generally heated to 70 C to kill the bacteria and then acidified
with sulfuric acid to pH 1.8. The clarified lactic liquor is then ion exchanged and concentrated to
80 %.
3.4.7 According to Eyal et al.
A preferred process for the lactic acid products from the mixture containing free lactic
acid and the dissolved lactate salt comprises of following steps: lowering down of the pH of
fermented broth, use of hydrophilic membrane and the volatile amine weak base to separate
lactic acid from the fermented broth, regeneration of lactic acid from salts of weak amine base.

3.4.8 According to garde, Arvid et al.

The first separation process is a microfiltration process for removal of cells, colloids and
particles. Electrodialysis process lactate and other ionic material are concentrated. Water is
removed from the diluate circuit of the electrodialysis by an reverse osmosis unit to insure a
satisfactorilyconductivity. In the nanofiltration process divalent cations are retained and
removed. By adding an inorganic acid before nanofiltration lactate is converted to lactic acid.
The lactic acid is extracted through anion-exchange membranes, driven by a
combination of diffusion and electrical migration. An alkaline solution on the other side of the
membranes
collects the lactate ions. Hydroxide ions flows back into the fermentation broth, cleaning the ionexchange membranes from organic buildup of fouling and replacing the removed lactate ions.