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 20.80–28.20% 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 ºC) 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 100ºC), 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 90-100ºC [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 11–13%. 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 121°C for 20min. Sterile water was added to the wet pretreated material to make the

volume of 200ml and boiled at 80°C for 30 min followed by filtration with cheese cloth. Acid

hydrolysis of filtrate was carried out by autoclaving at 121°C 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 world’s 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 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 ºC 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.”

“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.” Wee (2006)

“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.” Rao (2000)

“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.” Moreover, “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.” Vijayakumar (2007)

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 L–1 h–1 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

cell-recycle 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 vine-

trimming 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.”

“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.” Muller (2001)

“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.”

Furthermore, “The optimal growth temperature, pH and NaCl concentration for the growth of

L.plantarum was found to be 37°C,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 ΔpH (change in pH) after 6 hrs.”

Additionally, “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.” Umesh (2014)

“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.” Rao (2006)

“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.” Moreover, “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.” Furthermore, “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.” Parker Domnick Hunter | Process Filtration - Buyers Guide

Contaminations in small-scale fermenters

“Bench-scale fermenters are used in laboratories for research or development while large-

scale 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. Pre-

filtration 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

“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.” Furthermore, “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).” Moreover, “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 high-vacuum steam

distillation of the lactic acid from the fermentation broth, but decomposition of some of the lactic

acid occurs.” Additionally, “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.” Vijayakumar (2007)

“Biotechnical production of lactic acid may be based on several alternative micro-organisms. In

addition to lactic acid bacteria filamentous fungi (e.g. Rhizopus spp.), other gram-positive

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.”

Furthermore, “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.” Taskila (2012)

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:” Furthermore, “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.” Moreover,

“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.” Additionally, “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.” Also, “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.” Dunuwila (2003)

Separation Process for Biobased Lactic Acid

”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.” efthilia Arvaniti, Michael Goldsworthy 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.” Niju Narayanan 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.” I.S Udachan et al.

3.4 PURIFICATION

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. Vijayakumar (2007)

“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.” Moreover, “To recover and purify the L(þ)-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.” Ghaffar (2014)

“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.” Furthermore, “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.” Komesu

(2013)

“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 70°C) 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.” Furthermore, “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.” Moreover, “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.” Additionally, “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.” Evangelista (1994)

“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 %.” Sun et al.

“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.” Additionally, “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.” Furthermore, “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.” Moreover,

“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.” Also, “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 %.” Vijayakumar et al. (2007)

“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.”

Eyal 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.”

Furthermore, “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 ion-exchange membranes from organic buildup of fouling and replacing the

removed lactate ions.” garde, Arvid et al.