Process selection:

2.1 Types of processes:

Citric acid industrial scale production in past, surface or emerged production in
earlier years of twenty centuries dominated over traditional method of preparing
citric acid by extraction from various juices at the present time.

 Extraction from citrus fruits

 Synthesis
 Fermentation
The traditional method of preparing citric acid by extraction from the juice of
lemons, limes and pineapple wastes is still in practice in the developing world, but
its production is not significant, as it comprises less world production.
Citric acid can also be produced by purely chemical reactions. Citric acid was first
synthesized chemically in 1880 by Grimaux and Adam, using glycerol as a starting
material, but this method was not economically competitive enough compared to
other production routes such as fermentation. Therefore, microbial fermentation
became the choice for commercial citric acid fermentation as it was more
successful than chemical methods.

2.2 Selection of process:

Selection of process should include the following:

Fermentation:
This process utilize microorganisms to convert solid or liquid substrates into
various products. Commonly consumed fermented products include bread,
cheese, pickled vegetables, beer, wine, citric acid, glutamic acid etc.
Citric acid production by fermentation is the most economical and widely used
way of obtaining this product. More than 90% of the citric acid produced in the
world is obtained by fermentation.

Advantages:
1. Operations are simple and stable.
 2. The plant is generally less complicated and needs less sophisticated control
systems.
3. Technical skills required are less.
4. Energy consumption is less and frequent power failures do not critically
affect the functioning of the production plant.
Citric acid production by fermentation can be divided in three phases, which
include:
1. Preparation and inoculation of the raw material
2. Fermentation
3. Recovery of the product

Substrate:
 Molasses
 Sucrose
 Syrups
 Starch
 Hydrol
 Alkanes
 Oils and fats

Micro-organisms:
Aspergillus Niger is superior to other microorganisms for the commercial
synthesis of citric acid because of its better production yield. It is easy to handle,
can ferment various cheap raw materials and delivers high yields. As such, strains
of this microorganisms can be improved to create industrial strains for use in
commercial production. Mutagenesis and strain selection have been carried out
for such improvement.
In the history of citric acid fermentation, in the last hundred years, various strains
of genera fungi, yeast and bacteria were reported such as:
 Penicillium luterum,
 Penicillium restrictum,
 Paecilomyces divaricatum,
 Mucor piriformis,
  Trichoderma viride,
 Sacharomycopsis lipolitica,
 Arthrobacter paraffineius,
 Corynebacterium sp,
However, only mutants of Aspergillus and yeasts genus candida have almost
exclusively been utilized.
Apart from Aspergillus Niger the following species of Aspergillus have been
reported:
Aspergillus Niger, Aspergillus wentii, Aspergillus awamori, Aspergillus foetidus,
Aspergillus fenicis, Aspergillus fonsecalus, Aspergillus fumaricus, Aspergillus
luchensis, Aspergillus saitoi, and Aspergillus usumii.
From the genus candida the following have to be mentioned:
Candida lipolytica, candida tropicalis candida subtropicalix, candida fibriae,
candida intermedia, candida parapsilosis, candida zeylanoides, candida oleophila.
Mutants of Aspergillus Niger, Aspergillus wentii and on paraffine substrates
Candida lypolitica are used in industrial production.

Techniques:
 Batch techniques
 In fed batch process
 By continuous fermentation

Reactors:
Various kinds of bioreactors as:
 Stirred tank reactors
 Airlift reactors
 External loop reactors
 Magnetic drum contactors
 Reciprocated jet reactors
 Bio disc reactor
 Deep jet reactors
  Hollow fiber
 Fix bed reactor

2.3 Types of fermentation:

The industrial citric acid fermentation can be carried in three different ways:
1. Submerged fermentation
2. Surface fermentation
3. Solid state fermentation or koji process
 Solid- state Fermentation:
In solid state fermentation, the microorganisms grow on a moist solid with little
or no free water, although capillary water may be present. Example of this type of
fermentation are seen in mushroom cultivation, bread making and the processing
of cocoa, and in the manufacture of some traditional foods, misco (soy paste),
sake, soy sauce, temph (soybean cake) and gari (cassava) which are now
produced in large industrial operations.

 Liquid surface fermentation:

The surface fermentation methodology consist of inoculating with spore of
Aspergillus Niger, shallow aluminum or stainless pans containing sugar solution
along with source of assailable nitrogen, phosphate, magnesium and various trace
minerals. These pans are stacked vertically in a chamber. Growth of the mold
occurs on the solution surface forming a rubbery, con-volute mycelia mass. Air is
passed over the surface and controls temperature by evaporating cooling.

 Submerged Fermentation:
In this process microorganism is grown dispersed through a liquid medium. The
fermentation vessel usually consists of a sterilized vessel of desired volume
equipped with a mechanical agitator and a source of introducing sterile air.
Submerged fermentation are used for pickling vegetables, producing yoghurt,
brewing beer and producing wine and soy sauce.
Each fermentation may be subdivided into:
 Oxygen-requiring aerobic processes
 Anaerobic process
Aerobic process are those, which require continuous supply of oxygen. Examples
of aerobic fermentation include:
 Submerged culture
 Citric acid by Aspergillus Niger
 Solid state fermentation
Anaerobic processes are those which are carried out in absence of oxygen supply.
Fermentation meat products such as bologna sausage, dry sausage, pepperoni
and salami are produced by solid-state anaerobic fermentation utilizing acid
forming bacteria, particularly lactobacillus, Pediococcus and Micrococcus species.
A submerged culture anaerobic Fermentation occurs in yoghurt making.

2.4 Submerged Fermentation:

It is the process of choice for industrial operations because of the very well-
known engineering aspects such as fermentation modelling, bioreactor design
and process control. It is the process in which the growth and anaerobic/ partially
anaerobic decomposition of the carbohydrates by microorganisms in liquid
medium occur with plenty availability of free water.
The submerged technique is widely used for citric acid production. It is estimated
that about 80% of world production is obtained by submerged fermentation. The
fermentation process employed in large scale require more sophisticated
installations and rigorous control.

2.4.1 Advantages:
On the other hand, it presents several such as:
1. Higher productivity and yield
2. Lower labor costs
3. Lower contamination risk.
Submerged fermentation can be carried out in batch, fed batch or continuous
systems, although the batch mode is more citric acid frequently used. Normally,
citric acid fermentation is concluded in 5 to 12 days, depending on the process
conditions.
Disadvantages:
The disadvantages are the higher energy costs and more sophisticated control
which requires more highly trained personal.

2.4.2 Main factors:

Three main factors especially important or highly yielding citric acid production in
submerged processes are:
1. Quality of the stainless steel or the construction of the bioreactor
2. Mycelium structure
3. Oxygen fermenters

2.4.3 Technique:
Batch techniques in stirred tank or air lift bioreactors are in general use. Very
promising results were obtained in fed-batch process and by continuous
fermentation where various kinds of bioreactors as stirred tank reactors, airlift
reactors, external loop reactors, magnetic drum contactors, reciprocated jet
reactors, bio disc reactor, deep jet, hollow fiber or by use of fix bed reactor.

2.4.4 Types of fermenters:

There are two types of fermenter
1. Conventional stirred fermenters
2. Tower fermenters

2.4.5 Bio-chemistry of citric acid:

Citric acid is a normal metabolite and an intermediate in cellular oxidative
metabolism. It is formed in the mitochondrion after condensation of acetate
with oxaloacetate. The six-carbon acid is then successively degraded to a
series of four-carbon acids, effectively accomplishing the oxidation of
acetate in the cell. Thus, citric acid is metabolized to carbon dioxide and
water, and has only a transient effect on systemic acid-base status; it works
as a temporary buffer component. Oxidation is virtually complete, and less
than 5% of citrates are excreted in urine unchanged Citric acid is commonly
consumed as part of a normal diet. Man’s total daily consumption of citric
acid from natural sources and from food additive sources may exceed
500 mg/kg of body weight. Citric acid is well absorbed from the
gastrointestinal tract, and reacts with the enzyme citrates to yield
oxaloacetic acid and acetic acid. The total circulating citric acid in the
serum of a man is approximately 1 mg/kg of body weight. Normal daily
excretion in the urine of humans is 0.2-1.0 g.
Orally ingested citric acid is absorbed, and is generally regarded as a
nontoxic material when used as an excipient. However, excessive or
frequent consumption of citric acid has been associated with erosion of the
teeth.
Citric acid (5 percent in the diet) did not depress food intake, but caused a
loss in body weight gain and survival time in mice, with a slightly greater
influence on mature animals. The effects on body weight gain and survival
time may have resulted from the chelating ability of citric acid, which could
impair absorption of calcium and iron. The use of large volumes of blood
anti-coagulated with citrates has been shown to decrease plasma levels of
calcium and magnesium.
Citric acid and citrates also enhance intestinal aluminum absorption in renal
patients that may lead to increased and harmful serum aluminum levels.
Although aluminum is an abundant terrestrial element, it is toxic to tissues,
including the brain. It has therefore been suggested that patients with renal
failure taking aluminum compounds to control phosphate absorption should
not be prescribed citric acid and or citrate-containing products.
Krebs cycle:
The Krebs cycle is a part of cellular respiration. Named after Hans Krebs it is a
series of chemical reactions used by all aerobic organisms to generate energy The
Krebs cycle comes after the link reaction and
provides the hydrogen and electrons needed for electron transport chain. Its take
place inside mitochondria.
It is also known as Tricarboxylic Acid (TCA) cycle .In prokaryotic cells, the citric
acid cycle occurs in the cytoplasm; in eukaryotic cells, the citric acid cycle takes
place in the matrix of the mitochondria. The cycle was first elucidated by scientist
“Sir Hans Adolf Krebs” (1900 to 1981) He shared the Noble prize for physiology
and medicine in 1953 with Fritz Albert Lipmann, the father of ATP cycle.
The process oxidizes glucose derivatives, fatty acids and amino acids to carbon
dioxide through a series of enzyme controlled steps. The purpose of the Krebs
cycle is to collect high energy electrons from these fuels by oxidizing them, which
are transported by activated carriers NADH and FADH2 to the electron transport
chain. The Krebs cycle is also the source for the precursors of many other
molecules, and is therefore an amphibolic pathway (meaning it is both anabolic
and catabolic). To review the biochemistry of citric acid formation immense task.
Shortly, it can be said that the overproduction of citric acid requires a unique
combination of unusual nutritional conditions (excess of carbon source, hydrogen
ions and dissolved oxygen, and suboptimal concentrations of certain trace metals
and phosphate), which influence the fermentation performance.
A deficiency of manganese, or phosphate and nitrogen limitation inhibits the A.
Niger anabolism, and the resulting degradation of proteins leads to increased
ammonium ion concentration. This increase is able to counterbalance the
inhibition exerted by citric acid on phosphofructokinase being a positive end-
effector high concentrations of ammonium and glucose also repress the synthesis
of ketoglutarate dehydrogenase, inhibiting the citric acid catabolism via the Krebs
cycle, leading to its accumulation of citric acid is the result of high speed flow of
income and the reduction in outflow velocity.
Other smaller issues are of interest to other enzymes: invertase, hexokinase,
glucose oxidase, phosphofructokinase, other enzymes of the pentose phosphate
pathway, pyruvate kinase and citrate synthase.
An important aspect concerns the need that the Krebs cycle can be completed to
support the continuous production of citric acid. To address the lack of cycle
intermediates consequent to the metabolic dysfunction responsible for the
accumulation of citric acid, pyruvic acid produced from glucose is not only
decarboxylate to acetyl CoA by the pyruvate dehydrogenase complex, but it is
also partially carboxylate to oxaloacetic acid during the iodophase, by the action
of pyruvate carboxylase acetyl CoA combine with oxaloacetic produce citric acid.
2.5. Process Description:
Process description should include the following:

Pre-treatment of Molasses:
A pre-treatment with precipitating agents or ion exchange resins is necessary to
remove cations and then promote the metabolic function responsible for citric
acid accumulation. Alternatively, molasses are often treated with potassium
hexacyanoferrate to precipitate heavy metals.

Mash Preparation:
After filtration molasses sent to the dilution tank from the storage tank and dilute
the molasses by using hot water. The purpose of dilution of molasses is to reduce
the concentration don not support it, then sent it to the section called
sterilization.

Sterilization:
In this section, we sterilized this mesh at above 120C with high pressure steam to
separate unwanted micro-organisms, then this mesh is sent to the cooling
section.

Cooling section:
In this section, by using cold water, we reduced the temperature of mesh at 32C,
then pumped into the fermentation tank.

Fermentation:
In the fermentation process, by adding a specific enzyme known as Aspergilla’s
Niger in fermentation tank. For adjust the PH of material by adding HCL, H2SO4
and MgSO4. The fermentation process complete in ten days. Add some nutrient
in the fermentation tank for micro-organism nutrient. A cooling system is placed
with the Fermentation and we get the beer and mycelium from the bottom of
fermentation tank and sent into the next section.

Separation of mycelium:
From this solution, we separate mycelium from the process called filtration. The
remaining material is known as broth. This broth is sent into the precipitation
tank to adding Ca (OH) 2.

Liming of broth:
In this section of liming of broth, we add calcium hydroxide Ca (OH) 2 into the
material and then agitate it by mechanical stirrer, and control the temperature at
80C to 90C. After sometime Calcium citrate is formed. We separate the mixture of
calcium citrate through vacuum filter and enter it into the next section.

Acidulation of calcium citrate:

In this section, acidulate the calcium citrate with the dilute H2SO4. This process
can be done with the help of mechanical stirrer. Calcium citrate react with H2SO4
and produced calcium sulphate and citric acid. Calcium sulphate as by product.
This solution is passed through filter to separate the calcium sulphate and citric
acid, then sent into the section.

Concentration of citric acid:

In this section, we passed the citric acid from triple effect Evaporator and
concentrate it. The material is heat up by using stream heating coil and then into
the bleaching section.

Bleaching section:
In this section, using the granular CO2 to bleach the concentrated citric acid and it
becomes colorless. This colorless citric acid is sent into the crystallization section.
Crystallization section:
In this section, bleached citric acid is sent to the crystallizer. In crystallizer, citric
acid crystals are formed. The citric acid crystals and remaining solution sent into
the centrifuge section.

Drying section:
In drying section, water is removed by evaporation and we get the dry crystals of
citric acid. And then sent into storage.

Storage:
In this section, the dry crystals of citric acid are packed and stored.
2.6. Block flow diagram of process:
2.7. Process flow diagram:

[Link] conditions:
An industrial scale stirred bioreactor fermenter of 175 L capacity having working
volume of 115 L was use for cultivation process and nutritional analysis. The strain
CGBT7 Aspergilla’s Niger was found to enhance citric acid production.
 Volume of fermenter: 175 L
 Working volume: 115 L
 Initial PH value of molasses solution =6.0
 Incubation temperature =30C
 Sterilization temperature =120C
 Cooling temperature =32C
 Raw molasses sugar mainly sucrose (Substrate):150g/L
 Fermentation period=6 day
 Time of fillings=1 days
 Time for empty fermenter=1 day
 Time of sterilization and cleaning=1 day
 Total process time=9 days
 Agitation rate=75rpm
 Ammonium nitrate (Nitrogen source) =0.2%=2g/L
 The dry cell mass, [Link] (biomass):1.85g/L
 Nutrient required=Cu, Mg, Mn, Fe, Zn