PRODUCTION OF LACTIC ACID AND

ACETIC ACID

ACETIC ACID

Acetic acid (CH3 COOH,

molecular weight of 60.05).

Principal constituent of vinegar.

The first vinegar was spoiled wine,

Consisting the Latin word acetum

means sour or sharp wine.

Also known as : ethanoic acid,

ethylic acid, vinegar acid, and

methane carboxylic acid.

Glacial acetic acid is the pure

compound (99.8%), as

distinguished from the usual water

solutions known as acetic acid.

PROPERTIES

The boiling point :118°C.

Melting point of rhombic crystals : 16.6°C.

Glacial acetic acid is highly

corrosive to metals.

Acetic acid is soluble in alcohol,

miscible with water, glycerol, ether, acetone, benzene,

carbon tetrachloride,

Insoluble in carbon disulfide.

ACETIC ACID

ACETIC ACID PRODUCTION

Can be produced in the factories using the three major processes.

They are :

CHEMICAL REACTION :

Liquid- and vapor-phase oxidation of petroleum gases (with

catalyst),

Oxidation of acetaldehyde.

PRODUCTION FROM FOSSIL FUELS :

Acetaldehyde oxidation,

Hydrocarbon oxidation, and

Methanol carboxylation

BIOLOGICAL PROCESSES :

Aerobic process

Anaerobic process

ACETIC ACID BACTERIA

These Gram-negative bacteria belong to the family

Acetobacteriaceae, and to the alpha-subclass of Proteobacteria.

The recognized genera are: Acetobacter, Asaia, Acidomonas,

Gluconobacter, Gluconacetobacter, and Kozakia.

With the exception of Asaia, they produce large quantities of acetic

acid from ethanol, and can grow in the presence of 0.35% acetic

acid

ANAEROBIC PROCESS Produced by the two-step process.

FIRST STEP : Production of ethanol from a carbohydrate source (such as glucose).

Temperature : 30–32˚C using the anaerobic yeast Saccharomyces cerevisiae

C6 H12 O6 -> 2 CO2 + 2 CH3 CH2OH

SECOND STEP : Oxidation of ethanol to acetic acid.

Variety of bacteria can produce acetic acid,

Only members of Acetobacter used commercially (Acetobacter aceti at 27–37 ˚ C).

This fermentation is an incomplete oxidation because the reducing equivalents

generated are transferred to oxygen and not to carbon dioxide.

2 CH3 CH2OH + O2 -> 2 CH 3COOH + 2 H2O

Theoretical yield is 0.67g acetic acid / g of Glucose (100%).

Realistic yield of 76% (0.51g acetic acid / g of Glucose),

This process requires 2.0lb of sugar or 0.9lb of ethyl alcohol per pound of acetic acid

produced.

Complete aeration and strict control of the oxygen concentration during

fermentation are important to maximize yields and keep the bacteria viable.

ANAEROBIC PROCESS In the 1980s, emerged based on anaerobic fermentation using

Clostridia.

Commonly used Bacteria : Clostridium aceticum, C.thermoaceticum,

C. formicoaceticum, and Acetobacterium woodii

It is an obligate anaerobe, Gram-positive, spore-forming, rod-

shaped, thermophilic organism with an optimum growth

temperature of 55–60˚C and optimum pH of 6.6–6.8.

Clostridia can convert glucose, xylose, and some other hexoses and

pentose, fructose, lactate, formate, and pyruvate almost quantitatively

into acetate according to the following reaction:

C6H12O6 -> 3 CH3 COOH

Clostridium thermoaceticum is also able to utilize five-carbon sugars:

2 C5H10 O5 -> 5 CH3 COOH

Some acidogenic bacteria reduces the Co2 and 1-C into Acetate.

The anaerobic route should have a lower fermentation cost than the

aerobic process.

Theoretical yields : 3 mol of acetic acid is produced / mol of Glucose consumed

(i.e., 1g acetic acid per g glucose).

The overall reaction can be written as follows:

C6 H12 O6 + 2 H2O -> 2 CH3 COOH + 2 CO2 + 8H+ 8e –

2 CH3 COOH + 2 CO2 + 8H+ + 8e – -> CH3 COOH + 2 H2O

ENZYMES INVOLVED IN THE PRODUCTION OF AA : Tetrahydrofolate enzymes,

carbon monoxide dehydrogenase (CODH), NADP-dependent formate

dehydrogenase (FDH), and a corrinoid enzyme.

These enzymes are metalloproteins.

For example, CODH contains nickel, iron, and sulfur; FDH contains iron,

selenium, tungsten, and a small quantity of molybdenum; and the

corrinoid enzyme (vitamin B 12 compound) contains cobalt.

In most typical batch fermentations, cell concentration initially increases

exponentially and then decreases toward the end of the fermentation.

Acetate concentration also increases and then levels off.

High glucose concentration inhibits the initial growth of C. thermoaceticum.

However, after adaptation, the fermentation proceeds rapidly.

They appears to be a minimum ratio of nutrient concentration to glucose

concentration to produce acetic acid.

If glucose is still available but the nutrient is not, the microorganism will

produce by-products such as fructose.

Acetate production from glucose by C. thermoaceticum generates 5mol of

ATP/ mol of Glucose consumed.

This results in high levels of cell mass/ mol of Glucose consumed.

To maintain productivity, the cells must balance their ATP supply and

demand.

Since growth of C.thermoaceticum consumes more ATP than maintenance,

most of the acetic acid produced during the growth phase.

When cells use yeast extract as a source of amino acids, nucleotides and

fatty acids, they will need less ATP than if they have to synthesize thesecompounds using ammonium ions as the starting material.

Thus, assimilation of ammonium ions is important if cells are able to recycle

the ATP generated during production of acetic acid.

Ammonium sulfate (a cheaper nutrient) could partially replace yeast extract

without resulting in formation of by-products such as fructose.

Medium cost could be lowered further by substituting corn steep liquor foryeast extract.

INDUSTRIAL PRODUCTION OF ACETIC ACID

LET-ALONE METHOD

SURFACE FERMENTATION

SUBMERGED FERMENTATION

BATCH FERMENTION

LET-ALONE METHOD

Industrial fermentation processes have evolved from the

simple ‘let-alone’ method involving a partially filled open

container of wine exposed to air to the ‘field’

fermentation in which a series of casks are filled with wine

and inoculated in series by the vinegar produced in the

previous casks.

SURFACE FERMENTATION

The ‘trickling’ or ‘German’ process is a surface fermentation in which the

microbial population is attached to an appropriate support (usually beech wood shavings) and the product is trickled down while a large volume of

air is sparged up through the bottom of the tank.

This process was the basis for the manufacture of the trickling generator

that incorporates forced aeration and temperature control.

The partially converted solution collects at the bottom and is cooled,

pumped back up to the top, and allowed to trickle down until the

reaction is complete.

Ethanol conversion into acetic acid is 88–90%; the rest of the substrate is

used in biomass production or lost by volatilization.

ADVANTAGES : Include low costs, ease of control, high acetic acid

concentrations, and lower space requirements.

DISADVANTAGES : The costs of the wood shavings, long startup time, loss

of ethanol by volatilization, and production of slime-like material by the

Acetobacter (e.g., A.xylinum) are some of the disadvantages

SUBMERGED FERMENTATION

In 1949, Hromatkar and Ebner applied submerged fermentation techniques to

oxidation of ethanol to acetic acid.

The level of gas-phase oxygen is crucial to this process and thus, efficiency is based

on broth aeration with oxygen.

For industrial processes, 10–18% ethanol and 5 times the nutrients used for submerged fermentation are the starting conditions for fermentation.

When the concentration of ethanol reaches 0.4–2.4g/l , 50–60% of the solution is

removed and replaced with fresh substrate containing 10–18% ethanol.

There is usually ~80mg of dry bacterial solids per liter.

Theoretical yield is 1.7–2.1g acetic acid/Liter/Hour (in a Semi continuous).

Dead cells cause foaming; hence, mechanical defoaming techniques are

used to eliminate this problem.

Compared to surface fermentation, submerged fermentation results in higher

productivity, faster oxidation of ethanol, smaller reaction volumes, low personnel costs due to automation, fewer interruptions due to clogging by shavings, and

lower capital investment per product amount.

DOWNSTREAM PROCESSING & CELL SEPARATION

To isolate, purify, and concentrate the product often determines the economic

feasibility of the process.

The first operation is cell separation &Cell lysis, which can be done by cross-flow

microfiltration, Nano filtration and electro dialysis (Useful in concentrate the

glacial acetic acid).

Solvent extraction with distillation is the preferred method for chemically derived acetic acid, whereas freeze concentration is used for vinegar,

Furthermore, if the acetate is required in the free acid form, there will be

additional cost to convert the salt form produced in the anaerobic fermentation

into the free acid form.

Liquid–liquid extraction has been used to recover acetic acid from the chemical

manufacture of cellulose acetate, vinyl acetate, and other acetate products

Extraction efficiency is high when the organic acid is present in the un dissociated

(acid) form (i.e., at a low pH).

USES OF ACETIC ACID

LACTIC ACID

LACTIC ACID

Lactic acid (LA) is

Catabolic products of Primary metabolism by microbes

Also produced by many higher organisms including man who produces the acid in

the muscle during work.

LA products are formed from carbohydrate fermentation that are derived

from pyruvic acid via the EMP, PP, or ED pathways.

Products such as ethanol, acetic acid, 2, 3-butanediol, butanol, acetone,

lactic acid and xylitol production can also be produced as by products.

A lactic starter is a basic starter culture with widespread use in the dairy industry.

For cheese making of all kinds, lactic acid production is essential, and the

lactic starter is employed for this purpose.

1780—Scheele identified lactic acid as the principal acid in sour milk.

PROPERTIES OF LACTIC ACID :

Lactic acid is a three carbon organic

acid :

One terminal part of an acid or

carboxyl group,

Other terminal carbon atom is

part of a methyl or hydrocarbon

group

Central carbon atom having an

alcohol carbon group.

Soluble in water, but insoluble in other

organic solvents.

Low volatility

THE LACTIC ACID BACTERIA (LAB)

They are formicates group, non-spore forming, Rods or cocci shaped.

Carnobacterium Oenococcus Enterococcus Pediococcus

Lactococcus Paralactobacillus Lactobacillus Streptococcus

Lactosphaera Tetragenococcus Leuconostoc Vagococcus

Lacks porphyrins and cytochromes.

Do not carry out Electron transport phosphorylation and hence

obtain energy by substrate level phosphorylation.

Grow anaerobically but are not killed by oxygen. (as is the case with many

anaerobes).

They obtain their energy from sugars and are found in environments where

sugar is present.

They have limited synthetic ability and hence are fastidious, requiring, when

cultivated with the addition of amino acids, vitamins and nucleotides.

LACTIC ACID BACTERIA INTO TWO MAJOR GROUPS HOMOFERMENTATIVE GROUP : Produce lactic acid as the sole product of the

fermentation of sugars.

Glucose almost exclusively into lactic acid.

It converts the D-glyceraldehyde 3-phosphate to lactic acid.

Via : the Embden-Meyerhof pathway (i.e. glycolysis).

Since glycolysis results only in lactic acid as a major end-product of glucose

metabolism, two lactic acid molecules are produced from each molecule of

glucose with a yield of more than 0.90 g/g (30,31).

Only the homofermentative LAB are available for the commercial production of

lactic acid.

HETEROFERMENTATIVE GROUP : Besides lactic acid also produce ethanol, as well as CO2. Uses the enzyme – Aldolase.

Aldolase : Key enzyme in the EMP pathway and spits hexose glucose into

three-sugar moieties.

Catabolize glucose into ethanol and CO2 as well as lactic acid.

It receive five-carbon xylulose 5 phosphate from the Pentose pathway.

The five carbon xylulose is split into glyceraldehyde 3-phosphate (3-carbon),

which leads to lactic acid.

And the two carbon acetyl phosphate which leads to ethanol.

USE OF LACTIC ACID BACTERIA FOR INDUSTRIAL PURPOSES

The desirable characteristics of lactic acid bacteria as industrial microorganisms

include :

Ability to rapidly and completely ferment cheap raw materials,

Minimal requirement of nitrogenous substances

Produces high yields of the much preferred stereo specific lactic acid

Ability to grow under conditions of low pH and high temperature, and

Ability to produce low amounts of cell mass as well as negligible amounts of

other by products.

CHOICE OF A PARTICULAR LACTIC ACID

BACTERIUM

LACTIC ACID BACTERIUM IS ABLE TO FERMENT

Lactobacillus delbreuckii subspecies

delbreuckii

Sucrose

Lactobacillus delbreuckii subspecies

bulgaricus

Lactose

Lactobacillus helveticus Both lactose and galactose

Lactobacillus amylophylus and

L.amylovirus

Starch

Lactobacillus lactis Glucose, sucrose and galactose

Lactobacillus pentosus Sulfite waste liquor

PRODUCTION OF LACTIC ACID The organisms responsible for the production of lactic acid includes

Bacteria : Lactobacillus helveticus, L. salivarus. L. brevis. L viridescens. L.

plantarurn and Pediococcus damnosus.

Fungi : Candida krusei, Saccharomyces cerevisiae, Rhizopus sp,

It requires only a simple medium and produces L (+) lactic acid. It also

requires vigorous aeration.

Rhizopus sp : Utilize glucose aerobically to produce lactic acid.

Rhzopus species such as R. oryzae and R. arrhizus have amylolytic

enzyme activity, which enables them to convert starch directly to

L (+)-lactic acid.

In fungal fermentation, the low production rate, below 3 g/(Lh), 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 by-products, such as fumaric acid and ethanol.

NUTRITION REQUIREMENTS FOR PRODUCTION OF ACETIC ACID

LAB requires : complex nutritional requirements (Due to their limited ability to

synthesize their own growth factors such as B vitamins and amino acids).

There are several growth-stimulation factors that have a considerable effect on the

production rate of lactic acid.

The mixture of amino acids, peptides, and amino acid amides usually stimulates

the growth of LAB.

Fatty acids also influence LAB growth, and phosphates are the most important salt in lactic acid fermentation.

Ammonium ions cannot serve as the sole nitrogen source, but they seem to have

some influence on the metabolism of certain amino acids.

Since minerals do not seem to be essential to LAB growth, the amount found in

commercial complex media is usually sufficient.

Temperature and pH are also important factors influencing LAB growth and lactic

acid production.

RAW MATERIALS Cheap, Low levels of contaminants, Rapid production rate, High yield, Little or no

by-product formation, ability to be ferment with little or no pre-treatment, and

year-round availability.

When refined materials are used : The costs for Purification should be cheap.

Starchy and Cellulosic materials [because they are cheap, abundant, and

renewable], whey, and molasses, have been used for lactic acid production.

STARCHY MATERIALS : Sweet sorghum, Wheat, Corn, cassava, potato, rice, rye

and barley.

These materials have to be hydrolyzed into fermentable sugars before

fermentation, because they consist mainly of a(1,4)- and a(1,6)-linked

glucose.

This hydrolysis can be carried out simultaneously with fermentation.

CELLULOSIC MATERIALS : These materials consist mainly of B(1,4)-glucan, and

often contain xylan, arabinan, galactan, and lignin that have previously

attempted to produce lactic acid from pure cellulose through simultaneous

saccharification and fermentation (SSF).

Some industrial waste products, such as whey and molasses, are of interest for

common substrates for lactic acid production.

FERMENTATION APPROACHES TO LACTIC ACID

PRODUCTION : Batch, fed-batch, repeated batch, and continuous fermentations are the most frequently used

methods for lactic acid production.

Higher lactic acid concentrations : Obtained in batch and fed-batch cultures

Higher productivity : May be achieved by using of continuous cultures.

Fermentation generally carried out in the Bioreactor which is suitable for the production of large quantity products.

Bio process having the processes of upstream and downstream processes.

Upstream process : Includes the R&D development of the strains that will be used in the fermentation. After the development of proper strain, thi initial culture ans the secondary culture were made in the flask.

Simultaneously the bio reactor was cleaned and avoided of microbial contamination. Aseptically the nutrient media will be prepared and from the flask culture, the inoculum will be introduced into the reactor.

At the end of the fermentation, the crude raw product will be collected and preserved aseptically to get the final products.

Downstream processes : The crude product will be undergone for the purification and extraction of the compound that we need.

Extraction can be performed using the lysis method in the case of products persists in side of the cell. Otherwise, the centrifugation method only will be used to get the final products.

These above processes are termed as Downstream processes.

PROBLEMS OF LACTIC ACID BACTERIA IN INDUSTRIES

While using the LAB in the laboratories, there are several chances of

getting the contaminations and other problems.

Attack by bacteriophage

Inhibition by penicillin and other antibiotics

Undesirable strains.

Acid produced by the lactic starters introduce elasticity in the

curd, a property desirable in the final qualities of cheese.

PRODUCTION OF LA AS BY PRODUCTS

During the production of Kaffir Beer and Other Traditional Sorghum

Beers Lactic acid is produced as the by products.

The final product is the result of alcohol produced mainly by

S.cerevisiae: the lactic acid in the beverage is produced by several

Lactobacilli.

In some palm wine, microbial malo-lactic fermentation occurs.

In this fermentation, malic acid is first converted to pyruvic acid

and then to lactic acid.

The most important contaminants in distilling industries are lactic

acid which affects the flavor of the product.

USES OF LATIC ACIDS INDUSTRY : It is used in the baking industry, plastics(Polymers of lactic acids are

biodegradable thermoplastics), food industry as emulsifiers

Lactic acid is used as acidulant/ flavoring/ pH buffering agent or inhibitor of

bacterial spoilage in a wide variety of processed foods.

It is a very good preservative and pickling agent.

Addition of lactic acid aqueous solution to the packaging of poultry and fish

increases their shelf life.

IN MEDICINE : It is sometimes used to introduce calcium in to the body in the

form of calcium lactate, in diseases of calcium deficiency.

PHARMACEUTICAL AND COSMETIC : Lactic acid has many applications and

formulations in topical ointments, lotions, anti acne solutions, humectants,

parenteral solutions and dialysis applications, for anti carries agent

They are high boiling, non-toxic and degradable components.

Poly L-lactic acid with low degree of polymerization can help in controlled

release or degradable mulch films for large-scale agricultural applications.

It is non-volatile, odorless and is classified as GRAS (generally regarded as safe)

by the FDA.

As salts

The sodium and potassium salts of acetic and lactic acid are widely used in

foods, and they have a long history of use.

For example, sodium diacetate (CH 2 COONa·CH 3 COOH·xH 2 O) is used

widely in the baking industry to prevent moldiness of bread and cakes.

REFERENCES

Acetic Acid Production M

Cheryan, University of

Illinois, Urbana, IL, USA 2nd

edition, volume 1, pp. 13–17,

Industrial Pharmaceutical Biotechnology.

Heinrich Klefenz, 2002 Wiley-VCH Verlag GmbH. ISBNs: 3-527-

29995-5 (Hardcover); 3-

527-60012-4 (Electronic)

Modern Industrial

Microbiology and

Biotechnology.Nduka Okafor,

SCIENCE PUBLISHERS, 2007

Biotechnological Production of

Lactic Acid and Its Recent

Applications. Y.-J. WEE et al,

Food Technol. Biotechnol. 44

(2) 163–172 (2006)

L (+) lactic acid fermentation

and its product polymerization. Niju Narayanan et al., Electronic

Journal of Biotechnology ISSN: 0717-3458 Vol.7 No.2, Issue

of August 15, 2004

Lactic Acid Fermentation.

Lary et al., Carcass

Disposal: A Comprehensive

Review –Chapter 5,

August 2004.

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Ed, 2005.

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