SELECTION OF MICRO-ORGANISMS.

INTRODUCTION
Submerged Fermentation is the technique of biological conversion of complex
substrates into simple compounds by various microorganisms such as bacteria,
yeast and fungi. They also release several additional compounds apart from the
usual products of fermentation, such as carbon dioxide and alcohol. These
additional compounds are called secondary metabolites.
Several types of microorganisms such as bacteria, fungi, and yeast have been
reported for the bio-production of CA (Citric Acid), as shown in Table 2.

Yeast
During the last 30 years the interest of researchers has been attracted by the use of
yeasts as citric acid producers. A number of different strains, mostly belonging to
the Candida (Yarrowia) genus have been used for citric acid production, mainly in
conventional batch processes, but also in continuous culture, and with immobilized
cells. The yeast species which were reported to produce citric acid are; Candida
(Yarrowia) lipolytica, Candida guilliermondii and Candida oleophila.
Among the yeast species, Yarrowia lipolytica is known as a potential producer of
citric acid and has been developed as a microbial cell factory for citric acid
production in recent years. Candida (Yarrowia) lipolytica can be obtained from
several sources, including
commercial sources: Companies that operate in industrial biology may supply
strains of Candida (Yarrowia) lipolytica for use in research and industrial
applications. These strains can be purchased from specialist commercial suppliers.
Tissue and microbial banks:
Some tissue and microbial banks maintain collections of microbial strains, and
you can obtain Candida (Yarrowia) lipolytica from these banks. Some of these
popular banks include the American Type Culture Collection (ATCC) and the
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).
Industrial companies:
Industrial food or biological manufacturing companies may maintain special
strains of Candida (Yarrowia) lipolytica that have been optimized for enhanced
performance in industrial applications.

The main advantages of using yeasts:

Yeasts are characterized by greater resistance to high substrate concentrations than
fungi, with comparable conversion rates and have greater tolerance to metal ions
that allows the use of less refined substrates. Using yeasts also gives a better
process control due to their unicellular nature. It was reported that citric acid
production by yeast could be in the future an alternative to A. Niger one, especially
if the yeast biomass became an additive to animal food and not a by-product.

The major disadvantage of using yeasts is the simultaneous production of citric

and iso-citric acids. It is reported that the ratio of citric:isocitric acid can vary
between 1:1 to 20:1 according to the yeast strain, carbon source and micronutrient
concentration. Selection of a yeast strain with high citric acid production and
giving high citric acid:isocitric acid ratios has been reported as the principal step of
a citric acid production process. Table 2 shows citric acid production by several
yeast strains at different conditions.

From about 1965 methods using yeasts were developed, first from
carbohydrate sources, then from n-alkanes. At this time hydrocarbons
were relatively cheap and plants were built to use this method. The
economics have altered since then and plants that have been built to
utilize both yeast technologies have apparently switched back to
carbohydrate feedstocks.
The industrial production of citric acid from n-alkanes is not now
economic, although a plant was built, and operated, around 1970 at
Saline, Reggio Calabria, Italy (Liquichimica). This process was based on
a low aconitase mutant of C. lipolytica in a batch process with stirred,
aerated tank reactors of 400 m3, operating on a 72-hour cycle. The
conversion from alkanes was reported to exceed 130 per cent (by
weight). The theoretical yield is 250 per cent, but part of the alkanes was
converted to biomass and carbon dioxide. The yeast was removed by
centrifugation and the purification was traditional. The medium used
was based on the process developed for the yeast strain that had a
substrate concentration of 10 per cent n-decane, although n-alkanes from
9 to 20 carbons could be used. The availability and cost of Libyan n-
alkanes, which lead to the development of this and other plants,
including the dual substrate plants, has changed over the last three
decades. One unique feature of the n-alkane process is the insolubility of
the substrate. The efficiency and scalability of yeast in citric acid
production are limited due to the natural metabolic pathways of yeast,
which are not optimized for citric acid synthesis.
Bacteria
Bacteria are not commonly used for large-scale industrial citric acid production.
Citric acid production has historically been associated with fungi, particularly
Aspergillus Niger. Certain bacterial strains have the capability to produce organic
acids, including citric acid. Some examples include strains of Acetobacter, bacillus
licheniformis and arthrobacter paraffinens. Bacteria typically have different
optimal fermentation conditions compared to fungi. Factors such as temperature,
pH, and nutrient requirements need to be carefully controlled to maximize citric
acid production. Bacteria usually have an optimal temperature range for growth
and metabolism, in general, mesophilic bacteria prefer moderate temperatures
however, these factors depend on type of bacteria used and industrial process.
Bacteria might face challenges in competing with fungi in terms of citric acid yield
and efficiency. Fungi, especially Aspergillus Niger, have evolved natural
capabilities for citric acid production, making them well-suited for industrial-scale
processes.
Fungi
The most used microorganism in CA industrial production is the fungus
Aspergillus Niger. This is related to A. Niger's ability to generate large amounts of
CA using low-cost raw materials, such as molasses and corn flour liquefied.
Furthermore, A. Niger is easy to handle and does not produce significant amounts
of by-products, such as iso-citric acid, observed in fermentation using Y.lipolytica
yeast. Despite that, several bacteria, such as Arthrobacter paraffinens, Bacillus
licheniformis, and Corynebacterium and yeast, such as Candida tropicalis and
Yarrowia lipolytica, can also produce CA.
This table shows fungi and yeast through yield, raw material and scale.

The production of Aspergillus Niger involves several procedures, from

isolation and refinement to fermentation and genetic improvement.
Aspergillus Niger need a medium to grow and multiplies in order to
settle this point a medium was prepared of the following composition:
Distilled water 1,000 cc. Cane sugar 30-gram Ammonium dihydrogen
phosphate 2-gram Potassium chloride 0.2-gram Magnesium sulfate 0.2-
gram. Aspergillus Niger spored just as abundantly as on a medium of the
same composition to which iron salts were added.
Using Aspergillus Niger in citric acid production is preferred for several
reasons:
 High Yield:
o Aspergillus Niger is known for its ability to produce citric
acid in high yields. The fungus has naturally evolved
efficient metabolic pathways for citric acid synthesis, making
it a robust and productive choice for industrial-scale
fermentation processes.

 Optimized Metabolic Pathways:

o The metabolic pathways of Aspergillus Niger are well-suited
for the production of citric acid. The fungus efficiently
converts sugars into citric acid through the tricarboxylic acid
(TCA) cycle, contributing to its high yield and productivity.

 Adaptability to Industrial Conditions:

o Aspergillus Niger is adaptable to a wide range of
fermentation conditions, including temperature, pH, and
nutrient availability. This adaptability is crucial for
maintaining optimal growth and citric acid production in
large-scale industrial bioreactors.

 Safety and Regulatory Approval:

o Aspergillus Niger has a long history of safe use in various
industrial processes, including food production. Regulatory
bodies often have established safety assessments for the use
of this fungus, facilitating its regulatory approval in industrial
applications.

 Economic Viability:
 o The established knowledge base and infrastructure for using
Aspergillus Niger in citric acid production contribute to its
economic viability. The fungus has been extensively studied
and optimized, making it a cost-effective choice for large-
scale production.

 Research and Industrial Standard:

o The use of Aspergillus Niger in citric acid production has
been a standard practice for decades. Research efforts have
focused on optimizing strains and fermentation conditions,
further solidifying its position as the preferred microorganism
for this purpose.

 Efficiency and Process Expertise:

o Aspergillus Niger has demonstrated efficiency in utilizing
various carbon sources for citric acid production. Its well-
characterized physiology and growth characteristics
contribute to the overall expertise in the fermentation
process.

In conclusion, the selection of Aspergillus Niger for citric acid

production is based on its historical success, high yield, adaptability to
industrial conditions, safety profile, and economic viability. While other
microorganisms may have unique qualities, Aspergillus Niger remains
the predominant and well-established choice in the industry.

References
 Citric Acid Biotechnology by BJØRN KRISTIANSEN,
MICHAEL MATTEY and JOAN LINDEN.
 HE CITRIC ACID FERMENTATION OF ASPERGILLUS
NIGER. * BY JAMES N. CURRIE.
 Review article from society of science and nature (SFSN).
 Citric Acid Production- Microbes, Methods, Steps, Factors
(microbenotes.com).
 CITRIC ACID PRODUCTION FERMENTATION PROCESS
Tushar Patel1, Hiral Pandya2 1 Student, Chemical Engineering
Department, L.D. college of Engineering, Gujarat, India 2
Assistant Professor, Chemical Engineering Department, L.D.
college of Engineering, Gujarat, India.
 Citric acid production by yeasts: Fermentation conditions, process
optimization and strain improvement
Seda Karasu Yalcin1, M. Tijen Bozdemir2 and Z. Yesim Ozbas3*
1Department of Food Engineering, Faculty of Engineering and
Architecture, Abant Izzet Baysal University, Golkoy
14280, Bolu, Turkey.
2 Department of Chemical Engineering, Faculty of Engineering,
Hacettepe University, Beytepe 06532, Ankara, Turkey.
3Department of Food Engineering, Faculty of Engineering,
Hacettepe University, Beytepe 06532, Ankara, Turkey.
*Corresponding author: e-mail: yesim@hacettepe.edu.tr, Phone:
+90 312 297 71 12, Fax: +90 312 299 21 23.