ASSIGNMENT
TOPIC: - Industrial Production of Acids ( Citric and Gluconic )
Submitted to: - Ms Ann Antony
Dept. Integrated Biology
Submitted by: - Jenna Marry
Submission Date: - 16/8/2024
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Contents
• Introduction
• Citric Acid
• Gluconic Acid
• Conclusion
• Reference
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INTRODUCTION
Citric, gluconic, itaconic and lactic acids are manufactured via large-scale bioprocesses.
Oxalic, fumaric and malic acids can be made through fungal bioprocesses, but the market
demand is small since competing chemical conversion routes are currently more economical.
The largest commercial quantities of fungal organic acids are citric acid and gluconic acid, both
of which are prepared by fermentation of glucose or sucrose by A. niger. Another Aspergillus
species, A. terreus, is used to make itaconic acid. A significant commercial source of lactic acid
at the time of writing this book is a bioprocess employing the Zygomycetes such as Rhizopus
oryzae. In soil, very often the availability of the sugar is not possible and the acid production
also will be less. When the strains are placed in the synthetic medium with enough sugar
concentration, the high uptake of sugar and high production of organic acids are formed. The
acidification of environment by producing citric acid is a main advantage for the soil fungi to
prevent the growth of the competitors. Many strains of A. niger can lower the pH of their
environment by oxidizing glucose outside the cell wall, converting it to gluconic acid via the
action of the enzyme glucose oxidase. The ability to catabolize gluconic acid is more unusual
than the catabolism of glucose, and gluconic acid is also an effective chelator and acidulant.
Other strains of A. niger produce citric acid intracellularly and export the acid, perhaps, as a
chelator and acidifier that can also be reabsorbed for use as a carbon source. The four
commercial organic acids (citric acid, lactic acid, gluconic acid and fumaric acid) produced by
fungi are employed in high-volume, low value applications.
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CITRIC ACID
Citric acid or 2-hydroxy-1, 2, 3-propanetricarboxylic acid was first isolated from lemon juice.
Although it occurs in rather high concentrations in citrus fruits, citric acid is ubiquitous in
nature as an intermediate in the citric acid (Krebs) cycle whereby carbohydrates are oxidized
to carbon dioxide. In the early days, citric acid was isolated from lemons (that contain 7-9%
citric acid ), and today about 99% of the worlds citric acid comes from microbial fermentation
Fig 1: Citric acid
Applications of citric acid
• Citric acid, due to its pleasant taste and palatability, is used as a flavouring agent in
foods and beverages e.g., jams, jellies , candies, desserts, frozen fruits, soft drinks, wine.
Besides brightening the colour, citric acid acts as an antioxidant and preserves the
flavors of foods.
• It is used in the chemical industry as an antifoam agent, and for the treatment of textiles.
In metal industry, pure metals are complexed with citrate and produced as metal citrates.
• In pharmaceutical industry, as trisodium citrate, it is used as blood preservative. Citric
acid is also used for preservation of ointments and cosmotic preparations. As iron
citrate, it serve as a good source of iron.
• Citric acid can be utilized as an agent for stabilization of fats, oils or ascorbic acid. lt
forms a complex with metal ions (iron, copper) and prevents metal catalysed reactions.
Citric acid is also used as a stabiliser of emulsions in the preparation of cheese.
• ln detergent/cleaning industry, citric acid has slowly replaced polyphosphates.
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Microbial strains for citric acid production
Many microorganisms can produce citric acid. The fungus Aspergillus niger is most commonly
used for industrial production of citric acid. The other organisms (although less important)
include A. clavatus, A. wentii, Penicillium luteum, Candida catenula, C. guilliermondii and
Corynebacterium sp.
Microbial biosynthesis of citric acid
Citric acid is a primary metabolic product (of primary metabolism) formed in the tricarboxylic
acid (Krebs) cycle. Glucose is the predominant carbon source for citric acid production. The
biosynthetic pathway for citric acid production involves glycolysis wherein glucose is
converted to two molecules of pyruvate. Pyruvate in turn forms acetyl CoA and oxaloacetate
which condense to finally give citrate.
Fig 2: Biosynthesis of citric acid
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Enzymatic regulation of citric acid production : During the synthesis of citric acid, there is
a tenfold increase in the activity of the enzyme citrate synthase while the activities of other
enzymes (aconitase, isocitrate dehydrogenase) that degrade citric acid are reduced. However,
recent evidence does not support the theory that reduction in the operation of tricarboxylic acid
(i.e. degradation of citric acid) contributes to accumulation of citric acid. Increased citric acid
is more likely due to enhanced biosynthesis rather than inhibited degradation. Further, there
are anaplerotic reactions that replenish the TCA cycle intermediates to keep the cycle
continuously in operation.
Pyruvate carboxylase that converts pyruvate to oxaloacetate is also a key enzyme in citric acid
production.
Factors in the regulation of citric acid production
Carbohydrate source
A wide range of raw materials can be used for the supply of carbohydrates. These include
molasses (sugar cane or sugar beet), starch (from potatoes), date syrup, cotton wastes, banana
extract, sweet potato pulp, brewery waste and pineapple waste water. A high yield of citric acid
production occurs if the sugars that are rapidly metabolised are used e.g. sucrose, glucose,
maltose. At present, cane molasses and beet molasses are commonly used. lt is believed that a
high sugar concentration induces increased glucose uptake and consequently enhanced
citric acid production.
Trace metals
Certain trace elements (Fe, Cu, Zn, Mn, Mg, Co) are essential for the growth of A. niger. Some
of the trace metals particularly Mn 2+, Fe 3+ and Zn 2+ increase the yield of citric acid.
pH
The pH of the medium influences the yield of citric acid, and it is maximal when pH is below
2.5. At this pH, the production of oxalic acid and gluconic acid is suppressed. Further, at low
pH, transport of citric acid is much higher.
Dissolved O2
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The yield of citric acid production substantially increases when the dissolved O2 tension is
higher.
Nitrogen source
Ammonium salts, nitrates and urea are the nitrogen sources used in the media for citric acid
production. All the three compounds are equally good sources, as long as they do not adversely
effect the pH of the medium. Addition of ammonium ions stimulates citric acid production.
Production process for citric acid production
1. Surface process
2. Submerged process
Surface Processes
Solid surface fermentation
Surface processes using solid substrates are particularly carried out in less developed areas of
some Asian countries. The solid substrates such as wheat bran or pulp from sweet potato starch
are used, as culture media. The pH of the medium is adjusted to 4-5, and then sterilized. Now
the inoculum in the form of spores of A. niger is spread as layers (3-6 cm thickness) and
incubated at 28°C. The growth of the organisms can be accelerated by the addition of α-
amylase. Solid-state fermentation takes about 80 to 100 hours for maximal production of citric
acid. At the end of the process, citric acid can be extracted into hot water and isolated.
Liquid surface fermentation
Surface fermentation using liquid as nutrient medium is the oldest method for citric acid
production. lt is still in use due to a simple technology, low energy costs and h higher
reproducibility. Further, the interference of trace metals and dissolved O 2 tension are minimal.
The labour costs are however, higher since the manpower requirements are more for cleaning
the systems. About 20% of the citric acid in the world is produced by surface processes. The
nutrient supply for surface fermentation normally comes from beet molasses. The fermentation
is usually carried out in aluminium trays filled with sterile nutrient medium. The inoculum in
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the form of spores is sprayed over the medium. A sterile air is passed for supplying O 2 as well
as cooling. The temperature is maintained around 30°C during fermentation. As the spores
germinate (that occurs within 24 hours of inoculation), a layer of mycelium is formed over the
medium. The pH of the nutrient medium falls to less than 2, as the mycelium grows in size and
forms a thick layer on the surface of the nutrient solution. The fermentation is stopped after 7-
15 days. The mycelium and nutrient solution are separated. The mycelium is mechanically
pressed and thoroughly washed to obtain maximum amount of citric acid. The nutrient solution
is subjected to processing for the recovery of citric acid. The final yield of citric acid is in the
range of 0.7-0.9 of per gram of sugar.
Submerged Processes
Around 80% of the world's supply of citric acid is produced by submerged processes. This is
the most preferred method due to its high efficiency and easy automation. The disadvantages
of submerged fermentation are - adverse influence of trace metals and other impurities,
variations in O2 tension, and advanced control technology that requires highly trained
personnel. Two types of bioreactors are in use- stirred tanks and aerated towers. The vessels
of the bioreactors are made up of high-quality stainless steel. The sparging of air occurs from
the base of the fermenter.
The success and yield of citric acid production mainly depend on the structure of mycelium.
The mycelium with forked and bulbous hyphae and branches which aggregate into pellets is
ideal for citric acid formation. On the other hand, no citric acid production occurs if the
mycelium is loose and filamentous with Limited branches. An adequate supply of O2 (20-25 %
of saturation value) is required for good production of citric acid. The ideal aeration rate is in
the range of 0.2-1 vvm (volume/volume/minute). The submerged fermenters have the problem
of foam formation which may occupy about 1/3rd of the bioreactor. Antifoam agents (e.g. lard
oil) and mechanical antifoam devices are used to prevent foaming.
Production of citric acid from alkanes
Both yeasts and bacteria can be used for citric acid production from n-alkanes (C9-C23
hydrocarbons). The citric acid yield is better from hydrocarbons compared to sugars i.e. 145 %
of citric acid from paraffin. The most commonly used organism is Candida lipolytica. The
fermentation can be carried out in batch, semi-continuous or continuous modes. The pH should
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be kept above 5. The major limitations of citric acid production from alkanes are-very low
solubility of alkanes and increased production of unwanted isocitric acid.
Recovery of Citric Acid
The steps for the recovery of citric acid either from surface process or submerged process are
comparable. The recovery starts with the filtration of the culture broth and washing of
mycelium (which may contain about 10% of citric acid produced). Oxalic acid is an unwanted
byproduct and it can be removed by precipitation by adding lime at pH < 3. The culture broth
is then subjected to pH 7.2 and temperature 70-90°C for precipitating citric acid. For further
purification, citric acid is dissolved in sulfuric acid (calcium sulfate precipitate separates). The
final steps for citric acid recovery are - treatment with activated charcoal, cation and anion-
exchangers and crystallization. Citric acid monohydrate formed below 36°C is the main
commercial product. Above 40°C, citric acid crystallizes in an anhydrous form. The degree of
purity of citric acid produced depends on the purpose for which it is required. For instance,
pure forms of citric acid are needed for use in food preparations, while for industrial use it can
be crude form.
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Fig 3:- Flow chart for industrial production of citric acid by surface or submerged processes
GLUCONIC ACID
Gluconic acid can be produced by several bacteria and fungi. Glucose, on a simple direct
dehydrogenation, forms D-gluconolactone which is then converted to gluconic acid.
Applications of gluconic acid
l. Gluconic acid is used in the manufacture of metals, stainless steel and leather, as it can
remove the calcareous and rust deposits.
2. lt is used as an additive to foods and beverages.
3. Gluconic acid has pharmaceutical applications - calcium and iron therapy.
4. Sodium gluconate is used as a sequestering agent in many detergents.
5. Gluconate is used for desizing polyester or polyamide fabrics.
6. lt is utilized in the manufacture of highly resistant (to frost and cracking) concrete.
Microbial production of gluconic acid
Gluconic acid can be produced by a wide variety of prokaryotic and eukaryotic
microorganisms.
Bacterial species of the genera - Gluconobacter, Acetobacter, Pseudomonas, Vibrio.
Fungal species of the genera - Aspergillus, Penicillium, Gliocladium.
Principle of production: In bacteria, intracellular glucose is converted to extracellular
gluconic acid. A membrane bound enzyme, glucose dehydrogenase utilizes pyrroloquinoline
quinone (PQQ) as coenzyme and converts glucose to δ-D-gluconolactone which undergoes
hydrolysis (spontaneous or enzymatic) to form gluconic acid.
As regards fungal production, glucose is oxidized by the extracellular enzyme glucose oxidase
to form δ-D-gluconolactone, which subsequently gets converted to gluconic acid by lactonase.
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Glucose oxidase is an inducible enzyme that can be induced by high concentrations of glucose,
and at pH above 4. lt is believed that H2O2, produced by glucose oxidase acts as an antagonist
against other microorganisms (antimicrobial activity) in the surroundings.
Production process for gluconic acid
Submerged processes, by employing either A. niger or G. suboxidans, are used for producing
gluconic acid. The culture medium contains glucose at a concentration of 12-15% (usually
obtained from corn). The fermentation is carried out at pH 4.5-6.5, and at temperature 28-30°
C for a period of about 24 hours. Increasing the supply of O2 enhances gluconic acid yield.
Biotechnologists exploit the fermentation process of gluconic acid for the production of the
enzyme glucose oxidase, besides producing calcium gluconate and sodium gluconate.
Chemical synthesis of gluconic acid: By employing the immobilized enzyme glucose
oxidase, gluconic acid can also be produced.
Conclusion
Citric acid has high economic potential owing to its numerous applications. It is
mostly produced by microbial fermentation using Aspergillus niger. In view of
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surges in demand and growing markets, there is always a need for the discovery
and development of better production techniques and solutions to improve
production yields and the efficiency of product recovery. To support the enormous
scale of production, it is necessary and important for the production process to be
environmentally friendly by utilizing readily available and inexpensive agro-
industrial waste products, while maintaining high production yields. This article
reviews the biochemistry of citric acid formation, choices of citric-acid producing
microorganisms and raw materials, fermentation strategies, the effects of various
fermentation conditions, citric acid recovery options and the numerous
applications of citric acid, based on information drawn from the literature over
the past 10 years. Gluconic acid is produced by microbial fermentation. The
filamentous fungus A. niger is the primary microorganism used for the production
of gluconic acid, with glucose as a main carbon source.
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Reference
• Kalaichelvan P T et al; Bioprocess Technology. MJP Publishers, 2007
• U. Satyanarayana; Biotechnology. Books and Allied (p) LTD,2005
• S.S. Purohit; Biotechnology fundamentals and applications. Agrobios
(India), 2001
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