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In: Citric Acid ISBN 978-1-62100-353-3
Editor: Dominic A. Vargas eta al., pp. © 2011 Nova Science Publishers, Inc.

Chapter 4

CITRIC ACID: MICROBIAL PRODUCTION

AND APPLICATIONS IN FOOD AND
PHARMACEUTICAL INDUSTRIES

Manas R. Swain1, Ramesh C. Ray2 and

Jayanta K. Patra1
1
Department of Biotechnology,
College of Engineering and Technology, Bhubaneswar-751003, India
2
Central Tuber Crops Research Institute (Regional Centre),
Bhubaneswar-751019, India

ABSTRACT
Citric acid (2-hydroxy 2, 3-propanetricarboxylic acid) is a tri-
carboxylic acid and a universal intermediate product of plant and animal
metabolism. Citric acid is used as an acidifying agent and has wide
applications in food, beverage, pharmaceutical, cosmetics and other
industries for acidulation, anti-oxidation, flavour enhancement,
preservation, plasticizer, and as a synergistic agent. Among the organic
acids, citric acid is the most important in quantitative term with an
estimated annual production of about 1.4 million tonnes. The annual
growth of its demand/consumption rate is around 3.5-4.0 %. The food
and pharmaceutical industries consume about 70 and 12%, respectively of
the total citric acid produced, and the remaining 18 % is consumed by


Corresponding author: E- mail: rc_rayctcri@rediffmail.com
2 Manas R. Swain, Ramesh C. Ray and Jayanta K. Patra

other industries. Conventionally, it is produced by surface and submerged

fermentation processes using chemically defined media or molasses as
raw material by microorganisms such as Aspergillus niger, A.
accumulate, Yarrowia lipolytica, etc. However, recent research has
shown that citric acid can also be produced by solid state fermentation
using agricultural residues such as sugar cane and cassava bagasse, and
food producing solid residues such as grape and apple pomace. The
demand for this particular metabolite is increasing day by day which
requires more efficient fermentation process and genetically modified
microorganisms for higher yield and purity.

INTRODUCTION
Citric acid (2-hydroxy 2, 3-propanetricarboxylic acid) is the most
important organic acid produced in tonnage by fermentation. It is a tri-
carboxylic acid and a universal intermediate product of plant and animal
metabolism. Citric acid is a commodity chemical produced and consumed
throughout the World (Soccol et al., 2006). The acid was first isolated from
lemon juice in 1784 by Carl Scheele, a Swedish chemist and having molecular
weight of 210.14 Da. Citric acid is a universal intermediate product of
metabolism which are found in virtually all plants and animals in traceable
quantity. Wehmer (1893) first showed that a Penicillium glaucum on sugar
medium accumulated citric acid in culture medium that contained sugars and
inorganic salts.
Citric acid was first produced commercially in England from the imported
Italian lemons. Lemon juice remained the commercial source of citric acid
production until 1919 but when Currie (1917) discovered that some strains of
Aspergillus niger produced citric acid by growing abundantly in a nutrient
medium with a high concentration of sugar and mineral salts and an initial
medium pH of 2.5–3.5. This laid down the basis for industrial production of
citric acid by using A. niger.
Citric acid has wide applications in food, pharmaceutical, and chemical
industries as a major substrate for the production of a variety of products and
primarily as an acidulant. There have been increased interests in using natural
resources such as fruit sugars for the production of citric acid and to impart a
pleasant, tart flavour to foods and beverages. It also finds applications as a
function of additive detergents, pharmaceuticals, cosmetics and toiletries
(Kumar and Jain, 2008; Lazar et al., 2011). About 64 % of U.S. citric acid
usage in 2008 was for foods and beverages, 22 % for detergents and cleaning
 Citric Acid 3

products and 10 % for pharmaceutical and nutritional products. About 2 %

went into cosmetics and toiletries (Papagianni, 2007; Dhillon et al., 2011).
Although citric acid is one of the oldest industrial fermented products, the
demand is rapidly increasing day by day. Global production of citric acid in
2007 was over 1.6 million tonnes (Kumar and Jain, 2008).
Citric acid can be produced by fermentation technology using various
moulds, yeasts and bacteria. But, A. niger remains as a favourite mould strain
for industrial production of citric acid. Specific strains that are capable of
overproducing citric acid have been developed for various types of
fermentation processes. In this review an attempt has been made to elaborate
the biochemistry of citric acid fermentation, various microbial strains, as well
as various substrates used for citric acid production, bioprocess and product
recovery, and applications of citric acid in various fields, especially in food
and pharmaceutical industries (Berovic and Legisa, 2007).

BIOCHEMISTRY OF CITRIC ACID FERMENTATION IN

A. NIGER
There have been many theories proposed to explain the phenomena of
citric acid accumulation by A. niger (Krzystek et al., 1996; Wolschek and
Kubicek, 1999), but so far no complete explanation is available. It can be said
that citric acid accumulates by an induced abnormality in the metabolism of
the mould during the operation of the tricarboxylic acid cycle (TCA)
postulated by Krebs in 1937, under its original name „„citric acid cycle‟‟
(Krebs and Johnson, 1937). The TCA cycle is a cyclic sequence of reactions of
almost universal occurrence in mitochondria in aerobic organisms. It is
catalyzed by multi-enzyme system, which accepts the acetyl group of acetyl-
Co enzyme A as fuel and dismembers it to yield CO2 and hydrogen atoms
(Jaklitsch et al., 1991). On each turn around within the TCA cycle, one
molecule of acetic acid (two carbon atoms) enters as acetyl-Co enzyme,
condenses with a molecule of the four-carbon compound oxalo-acetic acid to
form citric acid, the six-carbon compound. Citric acid is then degraded
through a reaction sequence that yields two molecules of CO2 and regenerates
the four-carbon oxalo-acetic acid. Another turn of the cycle may now start by
the reaction of the oxalo acetic acid with another molecule of acetyl-Co
enzyme A. Thus, in each turn of the cycle one molecule of acetic acid enters,
two molecules of ATP and CO2 are formed and a molecule of oxalo acetic acid
4 Manas R. Swain, Ramesh C. Ray and Jayanta K. Patra

is utilized to form citric acid, but is regenerated at the end of the cycle
(Papagianni et al., 1999; Prescott and Dunn, 2001) (Figure 1).

Figure 1. Schematic representation of the metabolic reactions involved in citric acid

production, the enzymes (italics), the known feedback loops (dashed lines) and their
locations within the cellular structure of Aspergillus niger (Papagianni, 2007).

Several unusual nutrient conditions are required in combination for

overproduction of citric acid i.e. excessive concentrations of carbon source,
hydrogen ions, dissolved oxygen, and sub-optimal concentrations of certain
 Citric Acid 5

trace metals and phosphate, which synergistically influence the yield of citric
acid (Kristiansen and Sinclair, 1978). Glycolysis pathway is inhibited by
accumulation of citric acid but in case of A. niger, citric acid overproduction
occurs by an active glycolytic pathway. Under particular nutrient conditions
citric acid inhibition is counteracted because of the accumulation of various
positive effects of the phosphofructokinase gene (pfk 1) (Arts et al., 1987).
According to Röhr and Kubicek (1981) and Habison et al. (1983), the protein
breakdown under manganese deficiency results in a high intracellular NH4+
concentration (the “ammonium pool”), that causes inhibition of the enzyme
phosphofructokinase, an essential enzyme in the conversion of glucose and
fructose to pyruvate. This leads to a flux through glycolysis and the formation
of citric acid. The high glucose and NH4+ concentrations strongly repress the
formation of 2-oxoglutarate dehydrogenase and thus inhibit the catabolism of
citric acid within the tricarboxylic acid cycle (Röhr and Kubicek, 1983).
Recent studies on the early stages of citric acid accumulation by A. niger
contradict the existence of an intracellular ammonium pool that has been
claimed to be responsible for inhibition of the enzyme phosphofructokinase
(Papagianni et al., 1999; Papagianni, 2007).

CITRIC ACID PRODUCING MICROORGANISMS

Several genera of microorganisms were isolated from natural habitats
mostly from soil which overproduce citric acid during metabolism. However,
for commercial production those native citric acid producing organisms are
genetically modified for higher organic acid yield and purity.

Microorganism from Natural Habitat

Citric acid fermentation was first observed as a fungal product by Wehmer

in 1893 by a culture of Penicillium glaucum on sugar medium, but reasonable
advance in citric acid production appeared when Zahorsky first patented a new
strain – A. niger (Berovic and Legisa, 2007). However, industrial trials did not
succeed due to contamination problems and long duration of fermentation. It
was the work of Thom and Currie (Thom and Currie, 1916; Berovic and
Legisa, 2007), which opened up the way for successful industrial production
of citric acid. In the history of citric acid fermentation, in the last hundred
years, various strains of fungi, yeast and bacteria were reported such as:
6 Manas R. Swain, Ramesh C. Ray and Jayanta K. Patra

Penicillium luterum, P. purpurogenum, P. restrictum, P. janthinellum, P.

citrinum, Paecilomyces divaricatum, Mucor piriformis, Trichoderma viride,
Sacharomycopsis lipolytica, Arthrobacter paraffineus, Corynebacterium sp.
and others (Sinha et al., 2001; Berovic and Legisa, 2007). Apart from A. niger,
the following species of Aspergillus have been reported: A. wentii, A.
awamori, A. foetidus, A. fenicis, A. fonsecalus, A. fumaricus, A. luchensis, A.
saitoi and A. usumii. From the genus, Candida the followings are to be
mentioned: Candida lipolytica, C. tropicalis, C. guilliermondii, C. intermedia,
C. parapsilosis, C. zeylanoides, C. fibriae, C. subtropicalis, C. oleophila.

Genetically Modified Microorganisms

Most of the native producers, however, are not able to produce

commercially acceptable yields due to the fact that citric acid is a metabolite
of energy metabolism and its accumulation rises in appreciable amounts only
under conditions of drastic imbalances. Hence, these microbes are genetically
engineered to yield in much higher capacity (Papaginni, 2007).
Mutants of A. niger, A. wentii and C. lipolytica are used in industrial
production (Berovic and Legisa, 2007). The main advantages of using these
mutant organisms are: (i) ease of handling; (ii) ability to ferment a wide
variety of cheap raw materials; and (iii) higher yields. The improvement of
citric acid producing strains has been carried out by employing mutations in
parental strains using mutagens (Vandenberghe et al., 1999; Pandey, 2001;
Haq et al., 2001; Soccol et al., 2006). Among mutagens, -radiation, UV
radiation and chemical mutagens are frequently used (Soccol et al., 2006). A
list of microorganisms (native and mutants), capable of producing citric acid is
given in Table 1.

BIOPROCESS OF CITRIC ACID PRODUCTION

Raw Materials

Agricultural residues (i.e., cassava and sugarcane bagasse) and fruit and
vegetable processing wastes (i.e., apple and grape pomace) have been
employed as substrates for commercial submerged citric acid production
(Table 2) (Mourya and Jauhri, 2000; Vandenberghe, 2000; Soccol et al.,
 Citric Acid 7

2006), although citric acid is mostly produced from starch or sucrose- based
medium using submerged fermentation (Soccol et al., 2006).
Molasses is preferably used as the source of sugar for microbial
production of citric acid due to its relatively low cost and high sugar content
(40–55 %) in the form of sucrose, glucose and fructose (Grewal and Kalra,
1995). Since it is a by-product of sugar refining, the quality of molasses varies
considerably, and not all types are suitable for citric acid production. The
molasses composition depends on various factors like the variety of beet and
cane, methods of cultivation, conditions of storage and handling (transport,
temperature variations), etc (Soccol et al., 2006).

Table 1. Microorganisms capable of producing citric acid

Fungi Yeasts Bacteria

Native Strain
Aspergillus niger Candida tropicalis Arthrobacter paraffinens
A. aculeatus C. oleophila Bacillus licheniformis
A. carbonarius C. guilliermondii Corynebacterium spp.
A. awamori C. citroformans
A. foetidus C. intermedia
A. fonsecaeus C. parapsilosis
A. phoenicis C. zeylanoides
A. fumaricus C. fibriae
A. luchensis C. subtropicalix
A. saitoi Candida lipolytica
A. usumii Hansenula anamola
A. wentii Yarrowia lipolytica
Penicillium janthinellum
Penicillium luterum
P. purpurogenum
P. restrictum
P. citrinum
Paecilomyces divaricatum
Mucor piriformis
Trichoderma viride
Sacharomycopsis lipolitica
Mutant strain
Aspergillus niger C192
A. niger GCB-75
A. niger YW-112
Aspergillus niger CBX-209
8 Manas R. Swain, Ramesh C. Ray and Jayanta K. Patra

Table 2. Agricultural and food processing wastes as raw materials used

for citric acid fermentation

Raw material Yield (%)

Apple pomace 88 a
Bagasse hydrolysates 60 a
Beet molasses 68.7 a
Brewery waste 78.5 a
Brewery waste 42-58 a
Cane molasses 100
Carob pod 60
Carob sugar 40-60 a
Carrot waste 36
Cassava bagasse 67
Cellulose hydrolysate 44
Coffee husk 32-46a
Coconut oil 99.6 c
Corn starch 62
Corncob 50
Date syrup 60 a
Date syrup 50
Deoiled rice bran 22a
Grape pomace 60 a
Grape pomace 88
Hydrolysate starch 75
Kiwi fruit peel 60 a
Mandarin orange waste 55-65 b
Molasses (sugarcane bagasse) 64.5
Okara (soy residue) 53
Olive oil 119 c
Palm oil 155 c
Pineapple waste 74
Pineapple waste water 50-60 b
Rapeseed oil 115 c
Soybean oil 63
Spent grain liquor 63 a
Tubers ofAsphodelus aestivus 24-26a
Turnip whey (enriched with molasses) 27-46 a
Whey permeate 19-33 a
Yam bean starch 74 a
a - based on sugar consumed.
b - based on total sugars.
c- based on oils and fatty acids.
 Citric Acid 9

Both beet and cane molasses are suitable for citric acid production,
however, beet molasses is preferred to sugarcane due to its lower content of
trace metals, supplying better production yields than cane molasses, but there
are considerable yield variations within each type (Berovic and Legisa, 2007).
In the case of cane molasses, generally it contains some metals (iron,
calcium, magnesium, manganese, zinc), which retard citric acid synthesis and
it requires some pretreatment for the reduction of them. Palmyra jaggery,
sugar syrup from the palmyra palm is a novel substrate for increasing the yield
of citric acid production (Ambati and Ayyanna, 2001). The addition of phytate
(an important plant constituent) at the beginning of incubation of beet
molasses results in about 3-fold increase in citric acid accumulation (Lu et al.,
1998).
A variety of agro-industrial residues and by-products such as cassava
bagasse, coffee husk, wheat bran, apple pomace, pineapple waste, kiwi fruit
peel, grape pomace, citrus waste, etc. has also been investigated with solid-
state fermentation techniques for their potential to be used as substrates for
citric acid production (Vandenberghe et al., 1999; Vandenberghe, 2000 ).
Bagasse is the most suitable carrier, as it does not show agglomeration after
moistening with medium, resulting in better heat and mass transfer during
fermentation and higher product yield (Kumar et al., 2008).
Syrups of beet or cane sugar can also be used as basic substrate for the
submerged citric acid fermentation. The great advantage with this substrate is
its purity; however, the quality of the syrups deteriorates rapidly during
storage. Because of this they can only be used during the sugar campaign
season and only if the citric acid plant is not too far from the sugar factory
because of the large transport costs (Soccol et al., 2006). Good citric acid
yields have been also obtained using dextrose syrup, obtained by enzymatic
hydrolysis of starch. This method is now employed also in industrial scale
(Soccol et al., 2006).
Yarrowia clade (Aciculoconidium aculeatum, Candida hispaniensis and
Candida bentonensis) were screened for citric acid production with pure
glycerol as the carbon source. The cultures were grown under nitrogen-limited
conditions. The highest yielding strain, Y. lipolytica NRRL YB-423, produced
21.6 g/l citric acid from 40 g/l glycerol (54% yield). Further work on medium
optimization with this strain showed that the optimum C/N ratio for the rate of
citric acid production was 172 while the best combination of rate and yield
was obtained at a C/N ratio of 343. The citric acid to isocitric acid ratios
produced reached an optimum at C/N ratios of 343–686 (Levinson et al.,
2007). Crolla and Kennedy (2004) reported n-paraffin as the carbon source for
10 Manas R. Swain, Ramesh C. Ray and Jayanta K. Patra

production of citric acid using Candida lipolytica in fermentor agitation and

fed-batch mode of operation.

Fermentation Methods

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, which has its own 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, and (3) recovery of the product. The industrial citric acid
fermentation can be carried in three different ways: (1) submerged
fermentation, (2) surface fermentation and (3) solid-state fermentation or Koji
process (Vandenberghe et al., 2004). All of these methods require raw material
and inoculum preparation.

Submerged Fermentation

Submerged Fermentation is the process of choice for industrial operations

because of the very well known engineering aspects such as fermentation
modeling, 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 (Ray and Ward, 2006).
The submerged technique is widely used for citric acid production. It is
estimated that about 80 % of world production is obtained by submerged
fermentation (Vandenberghe, 2000; Soccol and Vandenberghe, 2003). The
fermentation process employed in large scale requires more sophisticated
installations and rigorous control. On the other hand, it presents several
advantages such as (1) higher productivity and yield , (2) lower labour costs,
and (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 11

frequently used. Normally, citric acid fermentation is concluded in 5 to 12

days, depending on the process conditions (Soccol et al., 2006).
The following main factors were found to affect citric acid fermentation:
(1) type and concentration of the carbon source, (2) nitrogen and phosphate
limitation, (3) pH, (4) aeration, (5) concentration of trace elements, and (6) the
physiology of the producer organism. Certain nutrients needed to be in excess
(i.e. sugar, protons and oxygen), others had to be limiting (i.e. nitrogen,
phosphate) and some otherwise common feed components had to remain
below defined limits (i.e. trace metals, especially manganese).
Some of the recent works in submerged fermentation for production of
citric acid are discussed below.
Haq et al. (2003) demonstrated production of citric acid from raw starch
by A. niger. Shake flask and semi solid culture methods were compared using
A. niger GCB-47 (parental strain) and GCMC-7 (mutant strain). When
cultivated in shaking culture with 150 g/l soluble starch as a carbon source, the
mutant strain GCMC-7 produced 69.5 g/l citric acid, which was, 1.48-fold
greater than the parental strain GCB-47. From a practical viewpoint, direct
production of citric acid from corn and potato starch was examined using
semi-solid culture. On the basis of a comparison of kinetic parameters namely
the volumetric substrate uptake rate (Qs), the specific substrate uptake rate
(qs), the volumetric productivity, theoretical yield and specific product
formation rate, it was observed that the mutant strain was a faster growing
organism. The mutant strain GCMC-7 produced 71.4 and 92.9 g/l citric acid,
approximately 1.44 and 1.12 times as much as the parental strain GCB-47,
from 200 g/l corn and potato starch, respectively. The findings suggest that
GCMC-7 possesses enhanced ability for sugar metabolism and citric acid
production. Dhillon et al. (2011) evaluated the potential of different agro-
industrial wastes for hyper production of citric acid through submerged
fermentation by A. niger NRRL 567 and NRRL 2001. Apple pomace ultra-
filtration sludge gave highest citric acid production rate of 9.0±0.3 g/l and
8.9±0.3 g/l of substrate by A. niger NRRL 567 and NRRL 2001 by submerged
fermentation, respectively.
Biosensor-controlled substrate feeding was used in a citric acid production
process with the yeast strain Y. lipolytica H222 with glucose as the carbon
source. The application of an online glucose biosensor measurement facilitated
the performance of long-time repeated fed-batch process with automated
bioprocess control. Ten cycles of repeated fed-batch fermentation were carried
out in order to validate both the stability of the microorganism for citric acid
production and the robustness of the glucose biosensor in a long-time
12 Manas R. Swain, Ramesh C. Ray and Jayanta K. Patra

experiment. In the course of this fermentation with a duration of 553 h, a slight

loss of productivity from 1.4 g/(l × h) to 1.1 g/(l × h) and of selectivity for
citric acid from 91% to 88% was observed. The glucose biosensor provided
6,227 measurements without any loss of activity (Moeller et al., 2011). An
external-loop airlift bioreactor (11.5 l), with a ratio of height to diameter of the
riser of 2.9, was used for the production of citric acid from the mash of dried
sweet potato with its dregs by A. niger. The effects of air flow rate, liquid
volume and sparger hole diameter on citric acid production were investigated.
A comparison of citric acid fermentation was made between the external-loop
airlift bioreactor and the mechanically stirred tank bioreactor. After a total
fermentation time of 65 h, an average of 10.6 mg: ml citric acid concentration
was obtained in the external-loop airlift bioreactor under an air flow rate of 1.3
vvm, liquid volume of 8.5 l as compared to 9.6 mg : ml citric acid
concentration in the 10 l mechanically stirred tank bioreactor under optimized
operation conditions: agitation rate 200 rpm, air flow rate 1.0 vvm and liquid
volume 6.5 l. It was demonstrated conclusively that the external-loop airlift
bioreactor could be used to produce citric acid economically using A. niger
from the mash of dried sweet potato with its dregs (Yuguo et al., 1999).
The yeast Y. lipolytica Wratislavia AWG7, an acetate (acet−) and
morphological (fil−) mutant, was cultured in a nitrogen- and phosphorus-
limited medium at the dilutionrate of 0.009–0.031 /h in the chemostat. Under
steady-state conditions, the increase in the dilution rate was paralleled by the
decrease in citric acid concentration (from 86.5 to 51.2 g/ l), as well as by the
increase in the volumetric rate (from 0.78 to 1.59 g/ l/ h) and specific rate
(from 0.05 to 0.18 g/ g/ h) of citric acid production. The yield of the
production process varied from 0.59 to 0.67 g/g. In a 550-h continuous culture
of the yeast test, at a dilution rate of 0.01 /h, in a medium with enhanced
concentrations of carbon, nitrogen and phosphorus sources, the concentration
of citric acid, the concentration of biomass and the volumetric rate of citric
acid production were 97.8 g /ll, 22.2 g/ l and 0.98 g/ l/h, respectively.
The yield of the process decreased to 0.49 g /g. Owing to the low content
of iso-citric acid and polyols, the fermentation process was characterized by a
high purity. This study has produced the following finding: the double mutant
Y. lipolytica AWG7 was an effective citric acid producer, with the ability to
preserve its properties unchanged during the long run of the continuous
chemostat process. This is a valued technological feature of such mutants
(Rywinska et al., 2011).
 Citric Acid 13

Surface Fermentation

Originally surface culture was the method employed for large scale
manufacture of microbial citric acid, employing mostly filamentous fungi. It
was introduced in 1919 by Societe des Produits Organique in Belgium and in
1923 by Chas Pfizer and Co. in the United States. Surface fermentation is still
used in industries of small and medium scale because it requires less effort in
operation, installation and energy cost. The process is carried out in
fermentation chambers where a great number of trays are arranged in shelves.
The culture solution is held in shallow trays with capacity of 0.4 to 1.2 m3 and
the fungus develops as a mycelial mat on the surface of the medium. The trays
are made of high purity aluminum, special grade steel or polyethylene,
however steel trays supply better yields of citric acid (Grewal and Kalra, 1995;
Soccol et al., 2006). The fermentation chambers are provided with an effective
air circulation, which passes over the surface in order to control humidity and
temperature by evaporative cooling. This air is filtered through a
bacteriological filter and the chambers should always be in aseptic conditions
and must be conserved principally during the first two days when spores
germinate. The most common contaminations are mainly caused by penicillia,
aspergilli, yeasts and lactic bacteria.
During fermentation, which is completed in 8 to 12 days (Pandey et al.,
2001), high amount of heat is generated, so high aeration rates are needed in
order to control the temperature and to supply air to the microorganism. After
fermentation, the tray contents are separated into crude fermentation fluid and
mycelial mats which are washed to remove the impregnated citric acid (Soccol
et al., 2006).

Solid-State Fermentation

Solid-state fermentation is characterized by the development of

microorganisms in a low-water activity environment on an insoluble material
that acts both as physical support and source of nutrients (Castilho et al.,
2009). It has a series of advantages over submerged fermentation including (1)
lower cost, (2) improved product characteristics, (3) higher product yield, (4)
easiest product recovery, and (5) reduced energy requirement (Ray et al.,
2008). Solid-state fermentation, also known by Koji process, was first
developed in Japan where abundant raw materials such as fruit wastes and
mainly rice bran are available. It is the simplest method for citric acid
14 Manas R. Swain, Ramesh C. Ray and Jayanta K. Patra

production and it has been an alternative method for using agro-industrial

residues (Vandenberghe, 2000). The great advantage of solid-state
fermentation processes is the extremely cheap raw material used as main
substrate. For citric acid production, the substrate is solid and it is moistened
to about 70 % moisture, depending on the substrate absorption capacity. The
initial pH of the material is normally adjusted to 4.5–6.0 and the temperature
of incubation is about 28–30 °C, depending on the microorganism used
(Vandenberghe, 2000). The solid culture process is completed within 96 h
under optimal conditions (Kubicek and Röhr, 1986). The most common
organism used in solid-state fermentation for citric acid production is A. niger.
However, there have also been reports with yeasts such as Saccharomyces
cereviseiae, Zygosaccharomyces bailli (Nielsen and Arneborg, 2011). The
strains with large requirements of nitrogen and phosphorus are not ideal
microorganisms for solid culture due to lower diffusion rate of nutrients and
metabolites occurring at lower water activity in solid- state process. The
presence of trace elements may not affect citric acid production so harmfully
as it does in submerged fermentation, thus, substrate pretreatment is not
required. This is one of the important advantages of the solid culture (Aghdam
and Taherzadeh, 2008; Karthikeyan and Sivakumar, 2010).Few recent studies
on solid state fermentation for production of citric acids are discussed in the
following paragraph.
Apple pomace with 66.0±1.9 g/kg of dry substrate proved to be an
excellent substrate for citric acid production by A. niger NRRL 567 at 72 h of
incubation. A. niger NRRL 2001 resulted in slightly lower citric acid
concentration of 61.0±1.9 g/kg of dry substrate at the same incubation time.
Addition of 3% (v/w) ethanol and 4% (v/w) methanol to apple pomace gave
significantly higher citric acid values of 127.9±4.3 g/kg and 115.8±3.8 g/kg of
dry substrate by A. niger NRRL 567. Higher citric acid values of 18.2±0.4 g/l
and 13.9±0.4 g/l of apple pomace ultrafiltration sludge-1 were attained after
addition of 3% (v/v) ethanol and 4% (v/v) methanol, respectively by A. niger
NRRL 567. Apple pomace solid waste and apple pomace ultrafiltration
sludge-1 thus proved to be an excellent source for citric acid production, of the
different substrates chosen (Dhillon et al., 2011).
Karthikeyan and Sivakumara (2010) showed peels of banana (Musa
acuminata) as an inexpensive substrate for the production of citric acid using
A. niger. Various crucial parameters that affect citric acid production such as
moisture content, temperature, pH, inoculum level and incubation time were
quantified. Moisture (70%), 28 °C temperature, an initial pH 3, 108 spores/ml
as inoculum and 72 h incubation was found to be suitable for maximum citric
 Citric Acid 15

acid production by A. niger using banana peel as a substrate. Solid state

fermentation was carried out to compare efficiency of acid, alkaline and urea
pretreatment of sugarcane bagasse for production of citric acid using
Aspergillus niger ATCC 9142. Plackett–Burman statistical design was used to
evaluate significance of variables. Pretreatment of bagasse by urea was known
as the most influential treatment to increase citric acid production (137.6 g/kg
of dry sugarcane bagasse and citric acid yield of 96% based on sugar
consumed). Finally, up- scaling was achieved to a 20 l solid state fermentor in
which humidity was constant in gas phase and urea-treated sugarcane bagasse.
The produced acid concentration and yield in fermentor was 82.38 g/kg of dry
substrate and 26.45 g/kg day, respectively (Khosravi-Darani and Zoghi, 2008).
A statistical design and one-factor-at-a-time method was employed to
optimize the media constituents for the improvement of citric acid production
from oil palm empty fruit bunches through solid state bioconversion using A.
niger IBO-103MNB. The results obtained from the Plackett–Burman design
indicated that the co-substrate (sucrose), stimulator (methanol) and minerals
(Zn, Cu, Mn and Mg) were found to be the major factors for further
optimization. Based on the one-factor-at-a-time method, the selected medium
constituents and inoculum concentration were optimized by the central
composite design under the response surface methodology. The statistical
analysis showed that the optimum media containing 6.4% (w/w) of sucrose,
9% (v/w) of minerals and 15.5% (v/w) of inoculum gave the maximum
production of citric acid (337.94 g/kg of dry EFB). The analysis showed that
sucrose (p < 0.0011) and mineral solution (p < 0.0061) was more significant
compared to inoculum concentration (p < 0.0127) for the citric acid production
(Bari et al., 2009).

APPLICATIONS OF CITRIC ACID IN FOOD AND

PHARMACEUTICAL INDUSTRIES
Citric acid is a versatile and innocuous alimentary additive. It is accepted
worldwide as GRAS (generally recognized as safe), approved by the Joint
FAO/WHO Expert Committee on Food Additives (Vandenberghe, 2000;
Papagianii, 2007; Berovic and Legisa, 2007). The food and pharmaceutical
industries utilize 60% of citric acid extensively because of its general
recognition of safety, pleasant acid taste, high water solubility and chelating
16 Manas R. Swain, Ramesh C. Ray and Jayanta K. Patra

and buffering properties. Citric acid is used in cosmetics and toiletries as

buffer, and as chelating agent.

In Food Industries
The various uses of citric acid (Table 3) in food and beverage industries
are discussed below.

Table 3. Applications of citric acid in food and pharmaceutical industries

Industry
Food Industry
Animal fats and oils
Candies
Cosmetics and toiletries
Dairy products
Fruits and vegetable juices
Gelatin desserts
Jellies and jams
Soft drinks and syrups
Frozen fruits
Wines and ciders
Pharmaceuticals
Uses
Synergist for other antioxidants, as sequestrant.
Prevent crystallization of sucrose, produce dark
colour in hard candies, inversion of sucrose.
For pH adjustment, antioxidant and buffering
agent
As emulsifier in ice creams and processed
cheese, acidifying agent in many cheese products
and as an antioxidant.
Acts as stabilizer in commercially prepared
juices of fruits and vegetables.
Adjusts pH to the desired levels and helps in
setting of gelatin desserts.
gelling agent, provides the desired degree of
tartness, tang and flavour.
As acidulant in carbonated and sucrose based
beverages, stimulates natural fruit flavour,
incorporates tartness.
Neutralizes the residual lye, lowers pH to
inactivate oxidative enzymes, protects ascorbic
acid by inactivating trace metals.
Prevents turbidity of wines and ciders, prevents
browning in some white wines, adjusts pH,
inhibits oxidation.
As effervescent in powders and tablets in
combination with bicarbonates, solubilization
action for cathartics, antioxidant in vitamin
preparations, acidulant in mild astringent
formulations, anticoagulant.
 Citric Acid 17

Food Preservation
Citric acid helps to prolong the shelf- life of frozen fish and shellfish.
Citric acid also inhibits colour and flavour decorations of the frozen fruit
(Buchard and Merrit, 1979). In general, lower pH value exerts a protective
effect on fruit juice pigment. Strawberry fountain syrup in particular is colour
stabilized by citric acid. The natural flavours of grape and other fruit juices are
greatly enhanced by the tartness which citric acid gives. Citric acid is used for
two purposes in the processing of fruits for frozen packs. First, since lye
peeling operations are common, it is important after thorough water washing
to neutralize the residual lye by dipping fruits or vegetables in 1 - 2% citric
acid solutions. Since residual alkali destroys natural ascorbic acid, it is
extremely important that the last traces be neutralized. The citric acid further
stabilizes ascorbic acid by lowering the pH to inactivate the oxidative enzymes
present such as catalase and peroxidase and ascorbic acid oxidase. In addition,
the citric acid retards destruction of ascorbic acid by binding harmful metal
contaminants. Citric acid in addition to D-erythorbic acid (D-araboascorbic
acid) or sodium-D-erythorbate, when added to products such as peaches,
apricots, plums, pears and cherries protects the fruit from undesirable colour
and flavour changes due to oxidation. Citric acid complexes contain metals
which accelerate oxidation. The net result is that discolouration is retarded and
flavours and natural vitamins are protected.
In the manufacture of Gelatin desserts, careful control of pH is important,
as the setting qualities of the gelatin are a function of pH. Citric acid not only
permits pH adjustment to the optimum (3.0 to 3.5) but adds flavour and
refreshing properties that account for its popularity and wide use. The
solubility and non toxic qualities of citric acid are also important in this
application (Roukas and Kotzekidou, 1987).

Carbonated Beverage and Syrups

Since citric acid occurs naturally in fruits, it is the preferred acidulant for
carbonated and still beverages. Citric acid adds refreshing properties to the
drink, often duplicating natural fruit products. It acts as a preservative in
syrups and the finished beverage and aids in obtaining the desired bouquet by
modifying the sweet flavours. It sequesters harmful metals which cause haze
and accelerates deterioration of colour and flavour. The amount of citric acid
added to soft drinks depends upon the flavour and the particular end uses
intended. Some syrup flavours, such as grape or orange, contain as little as 0.5
OZ of citric acid per gallon of syrup, whereas the citric content of certain
mixers may be as high as 4 OZ per gallon. Sufficient citric acid should be
18 Manas R. Swain, Ramesh C. Ray and Jayanta K. Patra

added to give a final pH of 2.5 to 4.5. Anhydrous citric acid is generally used
as a 50% solution prepared by dissolving 2 kg of anhydrous citric acid in
enough water to make 5 litres of final solution (Berovic and Legisa, 2007).

Fruit Jam and Jelly

Citric acid serves to adjust the pH of jellies, jams or preserve mixtures to
the optimum range, where pectin can act most effectively. Certain foods such
as guava, mangos, blackberries, cherries, sweet peaches, sweet plums etc.
would not naturally contain sufficient acids to give the proper pH. The use of
citric acid also gives the desired degree of flavour, which are important to
jellies. After the juice-pectin- sugar mixture is cooked and concentrated, citric
acid is added. Many processors use juice concentrates as a starting point and
add citric acid and other ingredients directly to the concentrates (Stops et al.,
2006).

Soft Drink Tablets

Tablets containing citric acid, an alkaline salt, a sweetening agent and
various flavours have been placed in the market, which effervesce when
dissolved in water. Carbonation of the water solution is caused by the release
of CO2 when citric acid reacts with metal bicarbonate (Khosravi-Darani and
Zoghi, 2008).

In Pharmaceutical Industries

Citric acid is a standard ingredient in cosmetic formulations for pH

adjustment, and in antioxidant systems as a metallic-ion chelator. The
detergent-building properties of citrate enable it to be used as a rapidly
biodegradable environmentally acceptable phosphate substitute in non-
phosphate detergent powders (Berovic and Legisa, 2007).
The citrate ion is rapidly and almost completely oxidized in the human
metabolism, less than 1% being excreted unchanged in the urine. Intravenous
injection shortens the coagulation time of the blood, but in vitro the citrate ion
acts as an anticoagulant. The free acid is also employed in pharmaceuticals
preparations as an acidulant and to enhance the flavour of syrups, solutions
and elixirs. The free citric acid may also be employed as an acidulant in mild
astringent preparations.
 Citric Acid 19

CONCLUSION
With a growing demand, the production of citric acid is expected to
increase. Its production has now reached 1.4 million tonnes per year and
continues to increase more each year. The main reason for constant increase is
the large number of applications that can be found for citric acid, mainly in the
food and pharmaceutical industries. Traditional processes, such as the
submerged fermentation using the fungus Aspergillus niger, dominate the
global production. However, different techniques of production such as
continuous fermentation and solid-state fermentation are continuously being
studied showing new perspectives for the production of citric acid.

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