AN INVESTIGATORY PROJECT IN PARTIAL

FULFILLMENT OF AISSCE, 2023-24

CITRIC ACID FERMENTATION AND

PRODUCTION

SUBMITTED BY:
NAME: Aastha Patel

GUIDED BY:- MS.LIPSITA MOHAPATRA

AISSCE PGT BIOLOGY
ROLL NO………………….
 CERTIFICATE

This is to certify that the project entailed" Project Title "is

done by Aastha Patel a bonafide student of ODM Public School,
Bhubaneswar, bearing AISSCE Roll No………………..,
investing along project is in accordance with the norms of 2024
and the project is submitted to CBSE for partial fulfillment of
AISSCE, 2024.

The project has been carried out under my supervision and

guidance. I certify that this project contain all the required
information needed along with the meeting standards and
demands of CBSE rules and guidelines.
 ACKNOWLEDGEMENT

I have taken efforts in the project. However it would

not have been possible without the kind support and help of
many individual. I would like to thank my Principal Dr. S.
Minaketan and vice principal Dr. Rajesh Kumar Padhy and
school for providing facilities required to do my project. I
am highly indebted to my biology teacher Ms. Lipsita
Mohapatra for her invaluable guidance which has sustained
my efforts in all the stages of this project work. I am also
thankful to my parents for their continuous support and
encouragement.

My thanks and appreciation also goes to my fellow

classmates and friends in developing this project and to
people who have willingly helped me out with their abilities.

Name: Aastha Patel

AISSCE ROLL-
 INDEX

S.No. Praticulars Page No.

1- Introduction
2- MICRO-ORGANISMS USED FOR
CITRIC ACIC PRODUCTION
3- PRODUCTION TECHNIQUES AND RAW
MATERIALS
3.1- SUBMERGED FERMENTION
3.2- SURFACE FERMENTATION
3.3- SOLID- STATE FERMENTATION
4- FACTORS AFFECTING CITRIC ACID
PRODUCTION
5- CIRIC ACID MARKET SNAPSHOT AND
MARKET SIZE
6- CONCLUSION
CITRIC ACID PRODUCTIONFERMENTATION PROCESS
Aastha Patel

1. INTRODUCTION:

Citric acid fermentation was first observed as a fungal product by

Wehmer in 1893 by a culture of Penicillium glaucum on sugar medium.
After a few years, he isolated two new fungal strains with the ability to
accumulate citric acid, which were designated Citromyces (Penicillium).
However, industrial trials did not succeed due to contamination problems
and long duration of fermentation. It was the work of Currie which opened
up the way for successful industrial production of citric acid. In 1916, he
found that numerous strains of Aspergillus niger produced significant
amounts of citric acid. The most important finding was that Aspergillus
niger grew well at pH values around 2.5–3.5 and high concentrations of
sugars favour citric acid production.
The first citric acid fermentations were carried out in surface cultures. In
the 1930s, some units were implanted in England, in Soviet Union, and in
Germany for the commercial production. In general, citric acid is
commercially produced by submerged microbial fermentation of molasses;
the fermentation process using Aspergillus niger is still the main source of
citric acid worldwide. Although methods were well developed to synthesise
citric acid using chemical means, better successes were achieved using
microbial fermentations, and over the period of time, this technique has
become the method of ultimate choice for its commercial production over
chemical synthesis. Despite that, the introduction of submerged fermentation
presented several problems, including the choice of productive strains with
low sensitivity to trace elements. It was necessary to consider raw material
much more carefully. Several works were dedicated to the optimization of
the conditions for the utilization of cheap material like sugar cane molasses,
beet molasses, starch and hydrolysate starch. Various processes for treating
and purifying molasses were developed, especially for the removal of trace
metals. Moreover, it was found that a small excess of copper ions was
beneficial to achieve high yields of citric acid.

2. MICRO-ORGANISMS USED FOR CITRIC ACIC PRODUCTION:

A large number of micro-organisms including bacteria, fungi and yeasts
have been employed to produce citric acid. Most of them, however, are not able
to produce commercially acceptable
yields. This fact could be explained by the fact that citric acid is a metabolite of
energy metabolism and its accumulation rises in appreciable amounts only
under conditions of drastic imbalances. The strains reported to produce citric
acid. Table 2 shows the micro-organisms used to produce citric acid. Among
these, only A. niger and certain yeasts such as Saccharomycopsis sp. are
employed for commercial production. However, the fungus
A. niger has remained the organism of choice for commercial production. The
main advantages of using this microorganism are: (a) its ease of handling, (b)
its ability to ferment a variety of cheap raw materials, and (c) high yields.
Table 2: Micro-organisms employed for citric acid production
Micro-organisms
Fungi
Aspergillus niger

A. aculeatus
A. awamori
A. carbonarius
A. wentii Karow & Waksman, 1947
A. foetidus
Penicillium janthinelum
Yeasts
Candida tropicalis
C. oleophila
C. guilliermondii
C. parapsilosis
C. citroformans
Hansenula anamola
Bacteria
Bacillus

3. PRODUCTION TECHNIQUES AND RAW MATERIALS:

Although citric acid is mostly produced from starch or sucrose based media
using liquid fermentation, a variety of raw materials such as molasses, several
starchy materials and hydrocarbons have also been employed. Classified raw
materials used for citric acid production in to two groups:
I. Raw materials with a low ash content from which the cations could be
removed by standard procedures (e.g. cane or beet sugar, dextrose syrups
and crystallized dextrose);
II. Raw materials with a high ash content and high amounts of other non-
sugar substances (e.g. cane and beet molasses, crude unfiltered starch
hydro-lysates).
Several attempts have been made to produce citric acid using molasses, which is
preferred due its low cost and high sugar content (40-55%). The composition of
molasses depends on various factors, e.g. the kind of beet and cane, methods of
cultivation of crops and fertilizers and pesticides applied during cultivation,
conditions of storage and handling (e.g. transport, temperature variations),
production procedures, etc. Both, cane and beet molasses are suitable for citric
acid production.
 Various other agro-industrial residues such as apple pomace, cassava
bagasse, coffee husk, wheat straw, pineapple waste, sugar beet cosset, kiwi fruit
peel, etc. have been investigated with solid state fermentation techniques for
their potential to be used as substrates for citric acid production.

3.1 Submerged Fermentation:

The submerged fermentation (SmF) process is the commonly employed
technique for citric acid production. It is estimated that about 80% of world
production is obtained by SmF. Several advantages such as higher yields and
productivity and lower labour costs are the main reasons for this. Two types of
fermenters, conventional stirred fermenters and tower fermenters are employed,
although the latter is preferred due to the advantages it offers on price, size and
operation. Preferentially, fermenters are made of high grade steel and require
provision of aeration system, which can maintain a high dissolved oxygen level.
Fermenters for citric acid production do not have to be built as pressure vessels
since sterilization is performed by simply steaming without applying pressure.
Cooling can be done by an external water film over the entire outside wall of the
fermenter.
Molasses and other raw materials demand pre-treatment, addition of nutrients
and sterilization. Inoculation is performed either by adding a suspension of
spores, or of pre-cultivated mycelia. When spores are used, a surfactant is added
in order to disperse them in the medium. For pre-cultivated mycelia, an
inoculum size of 10% of fresh medium is generally required. Normally,
submerged fermentation is concluded in 5 to 10 days depending on the process
conditions. It can be carried out in batch, continuous or fed batch systems,
although the batch mode more frequently used. [5]

Table 3: Raw materials and micro-organisms employed in submerged

fermentation for citric acid production
Raw material Strain Citric acid Yield, %
Brewery wastes A. niger ATTC 9142 19 g/L 78.5

Beet molasses A.niger ATTC 9142 109 g/L -

Yarrow lipolytica 54 g/L
A101
Cane molasses A. niger T 55 - 65
Wood A. niger IMI- 41874 27 g/L 45
Hemicellulose S. lipolytica IFO 9 g/L 41
1658
Date syrup A. niger ATTC 9142 - 50

Corn starch A. niger IM-155 - 62

 Starch hydrolysate Y. lipolytica DS-1 - -
Y. lipolytica A-101 - 75
Rapeseed oil Y. lipolytica A-101 - 57
Soybean oil Y. lipolytica A-101 - 63
Coconut oil C.lipolytica N-5704 -
Palm oil C.lipolytica N-5704 - 155b
Olive oil C.lipolytica N-5704 - 119
Soybean oil C.lipolytica N-5704 - 115
Glycerol C.lipolytica N-5704 - 58.8
n-Paraffin C.lipolytica N-5704 - 161

3.1 - Surface Fermentation:

The first individual process for citric acid production was the liquid surface
culture (LSC), which was introduced in 1919 US. After that, other methods of
fermentation, such as submerged fermentation were developed. Although this
technique is more sophisticated, surface method required less effort in operation
and installation and energy cost. In the classical process for citric acid
manufacture, the culture solution is held in shallow trays (capacity of 50-100 L)
and the fungus develops as a mycelial mat on the surface of the medium. The
trays are made of high purity aluminium or special grade steel and are mounted
one over another in stable racks. The fermentation chambers are provided with
an effective air circulation in order to control temperature and humidity.
Fermentation chambers are always in aseptic conditions, which might be
conserved principally during the first two days when spores germinate. Frequent
contamination is mainly caused by Penicillin, other Aspergilli, yeast and lactic
bacteria. Refined or crude sucrose, cane syrup or beet molasses are generally
used as sources of carbon. When applied, molasses is diluted to 15-20% and is
treated with hex cyanoferrate (HFC). [6]
3.2- Solid-state Fermentation:
Solid-state fermentation (SSF) has been termed as an alternative method to
produce citric acid from agro- industrial residues. Citric acid production by SSF
(Koji process) was first developed in Japan and is as the simplest method for its
production. SSF can be carried out using several raw materials. Generally, the
substrate is moistened to about 70% moisture depending on the substrate
absorption capacity. The initial pH is normally adjusted to 4.5-6.0 and the
temperature of incubation can vary from 28 to 30°C. The most commonly
organism is Aspergillus niger. However there also have been reports with yeasts.
One of the important advantages of SSF process is that the presence of trace
elements may not affect citric acid production as harmfully as it does in SmF.
Consequently, substrate pre-treatment is not required. [7]
Different types of fermenters such as conical flasks, glass incubators and
trays, etc. have been used for citric acid fermentation in SSF. Used Erlenmeyer
flasks and glass columns for the production of citric acid from gelatinized
cassava bagasse. Higher yields were obtained in flasks without any aeration,
and very little sporulation was observed. The same yields were found in column
reactors only with variable aeration. This showed great perspective to use SSF
process for citric acid production in simple tray type fermenters.
Table 4: Raw materials and micro-organisms employed in solid state
fermentation for citric acid production.
Raw material Strain Citric acid Yield(%)
Apple pomace A.niger NRRL2001 766 g/kg
NRRL 2270 816 g/kg
NRRL 599 771 g/kg
NRRL 328 798 g/kg
NRRL 567 883 g/kg
Grape pomace A.niger NRRL2001 413 g/kg
NRRL 2270 511 g/kg 88
NRRL 599 498 g/kg
NRRL 328 523 g/kg
NRRL 567 600 g/kg
Kiwifruit peel A .niger NRRL 567 100 g/kg
Cellulose A. niger 29 g/kg 44
hydrolysate and
Sugar cane
Orange waste A. niger 46 g/kg

Beet molasses A.niger ATCC 9142 35 g/kg

(Ca-alginate gel)
Saccharose (Sugar A. niger CFTRI 30 174 g/kg
cane
bagasse)
Coffee husk A. niger CFTRI 30 150 g/kg

Okara (soy A. niger 96 g/kg

residue)
Pineapple waste A.niger ATCC 1015 132 g/kg 74
A.niger ACM 4942 194 g/kg
 Glucose A.niger CBS733.88 21.24 g/kg
(Sugar cane
bagasse)
Mussel processing A. niger 300 g/kg
wastes
(polyurethane
foams)
Cassava bagasse A. niger LPB-21 347 g/kg 67

4. FACTORS AFFECTING CITRIC ACID PRODUCTION:

4.1 - Medium and its components:

Carbon source:
Citric acid accumulation is strongly affected by the nature of the carbon source.
The presence of easily metabolized carbohydrates has been found essential for
good production of citric acid. That sucrose was the most favourable carbon
source followed by glucose, fructose and galactose. Galactose contributed to a
very low growth of fungi and did not favour citric acid accumulation. Other
sources of carbon such as sorbose, ethanol, cellulose, manitol, lactic, malic and
acetoglutaric acid, allow a limited growth and low production. Starch, pentoses
(xyloses and arabinoses), sorbitol and pyruvic acid slow down growth, though
the production is minimal.
According initial sugar concentration was critical for citric acid production and
other organic acids produced by Aspergillus niger. That Aspergillus niger
strains needed an initial sugar concentration of 10-14% as optimal; no citric acid
was produced at sugar concentration of less than 2.5%. That immobilized cells
of Aspergillus niger needed lower concentrations of sucrose than free cells
culture, in order to obtain high yields (200 g of citric acid/L for free cells
culture, and 120 g/L for immobilized cells). The influence of different sources
of carbon on citric acid production by Aspergillus niger and Saccharomycopsis
lipolytica. Glucose, maltose, galactose, xylose and arabinose were tested.
Fermentation was carried out in 8 and 4 days, respectively, at 30°C and 180
rpm. Better results were found for Aspergillus niger with 0.45 g of citric acid/ g
of glucose corresponding to 27 g/L. Saccharomycopsis lipolytica produced 0.41
g/g of glucose or 9 g/L which was not so bad.
Nitrogen source:
Citric acid production is directly influenced by the nitrogen source.
Physiologically, ammonium salts are preferred, e.g. urea, ammonium sulfate,
ammonium chlorure, peptone, malt extract, etc. Nitrogen consumption leads to
pH decrease, which is very important point in citric acid fermentation.
However, it is necessary to maintain pH values in the first day of fermentation
prior to a certain quantity biomass production. Urea has a tampon effect, which
assures pH control. The concentration of nitrogen source required for citric acid
fermentation is 0.1 to 0.4 N/liter. A high nitrogen concentration increases fungal
growth and the consumption of sugars, but decreases the amount of citric acid
produced. [9]
Phosphorous source:-
Presence of phosphate in the medium has a great effect on the yield of citric
acid. Potassium dihydrogen phosphate has been reported to be the most
suitable phosphorous source. The phosphorous at concentration of
0.5 to 5.0 g/L was required by the fungus in a chemically defined medium for
maximum production of citric acid. Phosphate is known to be essential for the
growth and metabolism of Aspergillus niger. Low levels of phosphate favour
citric acid production, however, the presence of excess of phosphate was shown
to lead to the formation of certain sugar acids, a decrease in the fixation of CO2,
and the stimulation of growth. Phosphates acts at the level of enzyme activity
and not at the level of gene expression. It is interesting to note that different
strains require distinct nitrogen and phosphorous concentrations in the medium.
In fact, nitrogen and phosphorous limitation is a crucial factor in citric acid
production as there is an interaction between them. Consequently, the study of
their combined effect is necessary. The culturing modality conditions the
behaviour of the micro-organisms referring to the tendencies of production as a
function of the levels of N and P. The author used as first order an empirical
model based on rotatable design to study the effect of both nutrients. As
expected, for the two studied strains, a similar behaviour was noticed, showing
an improvement towards low levels of N and P in submerged culture, and
toward high levels in solid state culture, and with superior productions for the
last one. The specificity of solid state culture is largely due to a lower diffusion
rate of nutrients and metabolites, which occurs in low water activity conditions.
Consequently, a strain with large requirements of N and P seems to be
disfavoured, due to the restriction of accessibility to the nutrients in the
medium. [10]
Trace elements:
Trace element nutrition is probably the main factor influencing the yield of
citric acid. A number of divalent metals such as zinc, manganese, iron, copper
and magnesium have been found to affect citric acid production by Aspergillus
niger. However, it is crucial to take into account the interdependence of
medium constituents in SmF and, probably, in SSF. Zinc favoured the
production of citric acid if added with KH2PO4. On the other hand, the presence
of manganese ions and iron and zinc (in high concentrations) could cause the
reduction of citric acid yields only in phosphate free medium. There were few
differences in the response of Aspergillus niger to metal ions and minerals in
SSF and in SmF systems. SSF systems were able to overcome the adverse
effects of the high concentrations of these components in the medium. As a
consequence of this, the addition of chelating agents such as potassium
ferrocyanide to the medium proved to be of no use.
Copper was found to complement the ability of iron at optimum level, to
enhance the biosynthesis of citric acid. Manganese deficiency resulted in the
repression of the anaerobic and TCA cycle enzymes with the exception of
citrate synthetase. This led to overflow of citric acid as product of glycolysis. A
low level of manganese (ppm) was capable to reduce the yield of citric acid by
10%. Citric acid accumulation decreased by the addition of iron, which also had
some effect on mycelial growth. The presence of different copper
concentrations in the pellet formation medium was very important in order to
enhance a suitable structure, related to cellular physiology, for citric acid
production. The optimal initial CuSO4.5H2O concentration was 78 mg/L.
Magnesium is required both for growth as well as for citric acid production.
Optimal concentration of magnesium sulfate was found in the range of 0.02-
0.025%. [11]
Lower alcohols:
Addition of lower alcohols enhances citric acid production from commercial
glucose and other crude carbohydrate. Appropriate alcohols are methanol,
ethanol, isopropanol or methyl acetate. The optimal amount of methanol/ethanol
depends upon the strain and the composition of the medium, generally optimum
range being 1-3%.
The addition of ethanol resulted in two-fold increase in citrate synthetase
activity and 75% decrease in aconitase activity. Whereas the activities of other
TCA cycle enzymes increased slightly. They also found that coconut oil
influenced citric acid production in a sucrose medium when added at 3% (v/w).
Alcohols have been shown to principally act on membrane permeability in
micro-organisms by affecting phospholipid composition on the cytoplasmatic
membrane. The alcohols stimulate citric acid production by affecting growth
and sporulation through the action not only on the cell permeability but also the
spatial organization of the membrane, or changes in lipid composition of the
cell wall. [12]
Process
parameters:

pH:
The pH of a culture may change in response to microbial metabolic activities.
The most obvious reason is the secretion of organic acids such as citric, acetic
or lactic acids, which will cause the pH to decrease. Changes in pH kinetics
depend highly also on the micro-organism. With Aspergillus sp., Penicillium sp.
and Rhizopus sp., pH can drop very quickly until less than 3.0. For other groups
of fungi such as Trichoderma, Sporotrichum, Pleurotus sp., pH is more stable
(between 4 and 5). Generally, a pH below 2.0 is required for optimum
production of citric acid. A low initial pH has the advantage of checking
contamination and inhibiting oxalic acid formation. A pH of 2.2 was reported to
be optimum for the growth of the mould as well as for the production of citric
acid whereas; a higher pH i.e. 5.4 and 6.0-6.5 has been found optimum for citric
acid production in molasses medium.
Aeration:
Aeration has been shown to have a determinant effect on citric acid
fermentation. Increased aeration rates led to enhanced yields and reduced
fermentation time. The influence of dissolved oxygen concentration on citric
acid formation has been examined. It is important to maintain the oxygen
concentration above 25% saturation and interruptions in oxygen supply may be
quite harmful. The high demand of oxygen is fulfilled by constructing
appropriate aeration devices, which is also dependent on the viscosity of the
fermentation broth. This is an additional reason why small compact pellets are
the preferred mycelial forms of Aspergillus niger during fermentation. When the
organism turns into filamentous developments, e.g. due to metal contamination,
the dissolved oxygen tension rapidly falls to less than 50% of its previous value,
even if the dry weight has not increased by more than 5%. Aeration is
performed during the whole fermentation with the same intensity through the
medium at a rate of 0.5 to 1.5 vvm. However, because of economic reasons, it's
usually preferred to start with a low aeration rate (0.1 to 0.4 vvm). High aeration
rates lead to high amounts of foam, especially during the growth phase.
Therefore, the addition of antifoaming agents and the construction of
mechanical “deformers” are required to tackle this problem. [14]
5. CIRIC ACID MARKET SNAPSHOT AND MARKET SIZE –
6. CONCLUSIONS:
Since the beginning of this century, citric acid production has been intensively
studied and great alternatives to this process have been found to follow its great
demand. The use of alternative raw materials to produce citric acid by SmF,
LSC, and SSF seems to be a suitable possibility. However, it is necessary to
adapt the right type of raw material to the right technique e.g. cassava bagasse
employed as substrate in SSF, or cellulose hydrolysate used in SmF. The need
of some pre-treatment of raw materials may enhance the fermentation
efficiency. One area, which needs attention is the development of continuous
culture techniques which have been attempted but only at the laboratory scale.
Another area is the strain improvement with improved substrate utilization
efficiency.