Production of lactic acid and acidic acid

2. PRODUCTION OF
LACTIC ACID AND
ACETIC ACID

4. 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.

5. ACETIC ACID

6. 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

7. 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

8. 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.

9. 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.

10.  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.

11.  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 these
compounds 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 for
yeast extract.

13. INDUSTRIAL PRODUCTION OF ACETIC ACID
 LET-ALONE METHOD
 SURFACE FERMENTATION
 SUBMERGED FERMENTATION
 BATCH FERMENTION

14. 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.

15. 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

17. 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.

19. 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).

20. USES OF ACETIC ACID

21. LACTIC
ACID

22. 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.

23. 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

24. 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.

25. 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.

28. 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.

29. 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

30. 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.

34. 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.

36. 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.

38. 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.

39. 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.

40. 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.

41. 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.

42. 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.

43. REFERENCES
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Cheryan,
University of
Illinois, Urbana,
IL, USA 2nd
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1, pp. 13–17,
Industrial
Pharmaceutical
Biotechnology.
Heinrich Klefenz,
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ISBNs: 3-527-
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(Electronic)
Modern
Industrial
Microbiology
and
Biotechnology.
Nduka Okafor,
SCIENCE
PUBLISHERS, 2007
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Production of
Lactic Acid and
Its Recent
Applications. Y.-
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Food Technol.
Biotechnol. 44
(2) 163–172
(2006)
L (+) lactic acid
fermentation
and its product
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