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Microbial production of citric acid

Article in Brazilian Archives of Biology and Technology · December 1998

DOI: 10.1590/S1516-89131999000300001

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REVIEW
Microbial Production of Citric Acid
Luciana P. S. Vandenberghe1,2, Carlos R. Soccol1*, Ashok Pandey1 and Jean-Michel
Lebeault2
1
Laboratório de Processos Biotecnológicos, Departamento de Engenharia Química, Universidade Federal do
Paraná, CEP 81531-970 Curitiba-PR, Brazil; 2Laboratoire de Procédés Biotechnologiques, Génie Chimique,
Université de Technologie de Compiègne, 60205-Compiègne, Cedex, France

ABSTRACT

Citric acid is the most important organic acid produced in tonnage and is extensively used in food and
pharmaceutical industries. It is produced mainly by submerged fermentation using Aspergillus niger or Candida sp.
from different sources of carbohydrates, such as molasses and starch based media. However, other fermentation
techniques, e.g. solid state fermentation and surface fermentation, and alternative sources of carbon such as agro-
industrial residues have been intensively studied showing great perspective to its production. This paper reviews
recent developments on citric acid production by presenting a brief summary of the subject, describing micro-
organisms, production techniques, and substrates, etc.

Keywords: Citric acid, Aspergillus niger, submerged fermentation, solid state fermentation, substrates

INTRODUCTION was sold at high cost. This led extensive

attempts all over the world to find alternatives
Citric acid (C6H8O7, 2 – hydroxy – 1,2,3 – way for its production, which included chemical
propane tricarboxylic acid), a natural constituent and microbial techniques. In 1923, Wehmer
and common metabolite of plants and animals, is observed the presence of citric acid as a by-
the most versatile and widely used organic acid product of calcium oxalate produced by a culture
in the field of food (60%) and pharmaceuticals of Penicillium glaucum. Other investigations
(10%). It has got several other applications in showed the isolation of two varieties of fungi
various other fields. Currently, the global belonging to genus Citromyces (namely
production of citric acid is estimated to be Penicillium). However, industrial trials did not
around 736000 tones/year (Química e Derivados, succeed due to contamination problems and long
1997), and the entire production is carried out by duration of fermentation (Rohr et al., 1983). The
fermentation. In Brazil, almost the entire demand industrial process was first open by Currie, in
of citric acid is met through imports. There is 1917, who found that Aspergillus niger had the
constant increase (3.5-4%) each year in its capacity to accumulate significant amounts of
consumption, showing the need of finding new citric acid in sugar based medium. He also
alternatives for its manufacture. showed that high concentrations of sugar
favoured its production, which occurred under
Historical developments limitation of growth. In the thirties, some units
were implanted in England, in Soviet Union, and
Citric acid was first isolated by Karls Scheels in in Germany for the commercial production.
1874, in England, from the lemon juice imported However, the biochemical basis was only
from Italy. Italian manufacturers had monopoly cleared in the fifties with the discovery of the
for its production for almost 100 years, and it glycolytic pathway and the tricarboxylic acid
cycle (TCA). Consequently, an improved

*
Author for correspondence
process employing submerged fermentation was Applications of citric acid
developed in United States (Aboud-Zeid and
Ashy, 1984). Citric acid is mainly used in food industry
because of its pleasant acid taste an its high
Although methods were well developed to
synthesis citric acid using chemical means also, solubility in water. It is worldwide accepted as
better successes were achieved using microbial “GRAS” (generally recognized as safe),
fermentations, and over the period of time, this approved by the Joint FAO/WHO Expert
technique has become the method of ultimate Committee on Food Additives. The
choice for its commercial production, mainly pharmaceutical and cosmetic industries retain
due to economic advantage of biological
10% of its utilization and the remainder is used
production over chemical synthesis (Mattey,
1992). Much attention has been paid on research for various other purposes. Table 1 presents
to improve the microbial strains, and to maintain main applications of citric acid.
their production capacity.

Table 1. Applications of citric acid

Industry Applications
Beverages Provides tartness and complements fruits and berries flavors. Increases the
effectiveness of antimicrobial preservatives. Used in pH adjustment to provide
uniform acidity.
Jellies, Jams Provides tartness. pH adjustment.
and Preserves
Candy Provides tartness. Minimizes sucrose inversion. Produces dark color in hard
candies. Acts as acidulant.
Frozen fruit Lowers pH to inactivate oxidative enzymes. Protects ascorbic acid by
inactivating trace metals
Dairy products As emulsifier in ice creams and processed cheese; acidifying agent in many
cheese products and as an antioxidant.
Fats and oils Synergist for other antioxidants, as sequestrant.
Pharmaceuticals As effervescent in powders and tablets in combination with bicarbonates.
Provides rapid dissolution of active ingredients. Acidulant in mild astringent
formulation. Anticoagulant.
Cosmetics and toiletries pH adjustment, antioxidant as a metallic–ion chelator, buffering agent.
Industrial applications Sequestrant of metal ions, neutralizant, buffer agent
Metal cleaning Removes metal oxides from surface of ferrous and nonferrous metals, for
preperational and operational cleaning of iron and copper oxides
Others In electroplating, copper plating, metal cleaning, leather tanning, printing inks,
bottle washing compounds, floor cement, textiles, photographic reagents,
concrete, plaster, refractories and moulds, adhesives, paper, polymers,
tobacco, waste treatment, etc.

MICRO-ORGANISMS USED FOR metabolism and its accumulation rises in

CITRIC ACIC PRODUCTION appreciable amounts only under conditions of
drastic imbalances. Kubicek and Rohr (1986)
A large number of micro-organisms including reviewed the strains reported to produce citric
bacteria, fungi and yeasts have been employed to acid. Table 2 shows the micro-organisms used to
produce citric acid. Most of them, however, are produce citric acid. Among these, only A. niger
not able to produce commercially acceptable and certain yeasts such as Saccharomycopsis sp.
yields. This fact could be explained by the fact are employed for commercial production.
that citric acid is a metabolite of energy However, the fungus A. niger has remained the
organism of choice for commercial production. technique” and the “passage method” have been
The main advantages of using this micro- used for selecting citric acid producing micro-
organism are: (a) its ease of handling, (b) its organisms The single-spore technique has the
ability to ferment a variety of cheap raw disadvantage that mineral acid or organic acids
materials, and (c) high yields. (gluconic acid, oxalic acid) simulate the
presence of citric acid. Rohr et al. (1979)
Strains selection and improvement improved this method by incorporating a specific
stain for citric acid (para-di-methylamino
The two principal methods of selecting benzaldehyde), instead of using the indicator.
populations, namely, “the single-spore

Table 2. Micro-organisms employed for citric acid production

Micro-organisms References

Fungi
Aspergillus niger Hang & Woodams 1984, 1985, 1987, Roukas 1991, Garg & Hang
1995, Lu et al. 1997, Pintado et al. 1998, Vandenberghe et al.,
1999b, c
A. aculeatus El Dein & Emaish, 1979
A. awamori Grewal & Kalra, 1995
A. carbonarius El Dein & Emaish, 1979
A. wentii Karow & Waksman, 1947
A. foetidus Chen, 1994; Tran et al., 1998
Penicillium janthinelum Grewal & Kalra, 1995

Yeasts
Saccahromicopsis Ikeno et al., 1975; Maddox et al., 1985; Kautola et al., 1992
lipolytica Wojtatowicz et al., 1993; Rane & Sims, 1993
Candida tropicalis Kapelli et al., 1978
C. oleophila Ishi et al., 1972
C. guilliermondii Miall & Parker, 1975; Gutierrez et al., 1993
C. parapsilosis Omar & Rehm, 1980
C. citroformans Uchio et al., 1975
Hansenula anamola Oh et al., 1973

Bacteria
Bacillus licheniformis Sardinas, 1972
Arthrobacter paraffinens Kroya Fermentation Industry, 1970
Corynebacterium sp. Fukuda et al., 1970

The most employed technique to improve citric using a suitable selection technique on model
acid producing strains has been by inducing medium with non-specific carbon sources, a
mutations in parental strains using mutagens. strain yielding high amounts of citric acid from
Among physical mutagens, γ-radiation (Bonatelli unusual substrates can be obtained from the
and Azevedo, 1983 ; Gunde-Cimerman, 1986 ; mutants produced.
Islam et al., 1986) and UV-radiation (Pelechova
et al., 1990) have often used. To obtain hyper- Another approach for strain improvement has
producer strains, frequently UV treatment could been the para-sexual cycle, as first described by
be combined with some chemical mutagens, e.g. Pontecorvo et al. (1953). According to Das and
aziridine, N-nitroso-N-methylurea or ethyl Roy (1978), diploids displayed higher citric acid
methane-sulfonate (Musilkova et al., 1983). By yields compared to their parent haploids, but
tended to be less stable (Bonatelli and Azevedo, magnesium, manganese, iron and zinc, which
1983). Protoplast fusion appeared to be a have a retarding effect on the synthesis of citric
promising tool to extend the range of genetic acid. Consequently, some pre-treatment is
manipulation of A. niger with respect to citric required for the removal/reduction of trace
acid production. Kirimura et al. (1988a) studied
metals. Despite that, cane molasses posses
protoplast fusion of production strains. They
were able to obtain fusants with acid production difficulties in achieving good fermentation
capacities exceeding those of the parent strains yields.
in solid state fermentation, but not in submerged
fermentation. Some other aspects of strain Various other agro-industrial residues such as
improvement could be the resistance to apple pomace, cassava bagasse, coffee husk,
detrimental constituents of fermentation raw wheat straw, pineapple waste, sugar beet cosset,
materials, capability of utilizing raw materials kiwi fruit peel, etc. have been investigated with
(starch, cellulose, pectin containing and other solid state fermentation techniques for their
waste materials). However, there is no single potential to be used as substrates for citric acid
effective technique to achieve hyper-producing production (Pandey and Soccol, 1998, Pandey et
mutants and much remains to be done in this al. 1999, Vandenberghe et al., 1999a,b, c). In
area. fact, these residues are very well adapted to
solid-state cultures due to their cellulosic and
starchy nature. However, despite the fact that
PRODUCTION TECHNIQUES AND these solid residues provide rich nutrients to the
RAW MATERIALS micro-organisms, and are good substrates for
growth and activity of micro-organisms, much
Although citric acid is mostly produced from remains to be done for developing commercially
starch or sucrose based media using liquid feasible process utilizing these residues (Pandey
fermentation, a variety of raw materials such as 1992, 1994, Pandey and Soccol 1998).
molasses, several starchy materials and
hydrocarbons have also been employed. Rohr et Liquid fermentation
al. (1983) classified raw materials used for citric
acid production in to two groups: (i) with a low Submerged fermentation: The submerged
ash content from which the cations could be fermentation (SmF) process is the commonly
removed by standard procedures (e.g. cane or employed technique for citric acid production. It
beet sugar, dextrose syrups and crystallized is estimated that about 80% of world production
dextrose); (ii) raw materials with a high ash is obtained by SmF. Several advantages such as
content and high amounts of other non sugar higher yields and productivity and lower labour
substances (e.g. cane and beet molasses, crude costs are the main reasons for this. Two types of
unfiltered starch hydro-lysates). fermenters, conventional stirred fermenters and
tower fermenters are employed, although the
latter is preferred due to the advantages it offers
Several attempts have been made to produce
on price, size and operation (Rohr et al., 1983).
citric acid using molasses, which is preferred due Preferentially, fermenters are made of high-
its low cost and high sugar content (40-55%). grade steel and require provision of aeration
The composition of molasses depends on various system, which can maintain a high dissolved
factors, e.g. the kind of beet and cane, methods oxygen level. Fermenters for citric acid
of cultivation of crops and fertilizers and production do not have to be built as pressure
pesticides applied during cultivation, conditions vessels since sterilization is performed by simply
of storage and handling (e.g. transport, steaming without applying pressure. Cooling can
temperature variations), production procedures, be done by an external water film over the entire
outside wall of the fermenter.
etc. Both, cane and beet molasses are suitable for
citric acid production. However, beet molasses is In SmF, different kinds of media are employed
preferred due to its lower content of trace metals. such as sugar and starch based media (Table 3).
Generally, cane molasses contains calcium, Molasses and other raw materials demand pre-
treatment, addition of nutrients and sterilization. Surface fermentation: The first individual
Inoculation is performed either by adding a process for citric acid production was the liquid
suspension of spores, or of pre-cultivated surface culture (LSC), which was introduced in
mycelia. When spores are used, a surfactant is 1919 by Société des Produits Organiques in
added in order to disperse them in the medium. Belgium, and in 1923 by Chas Pfizer & Co. in
For pre-cultivated mycelia, an inoculum size of US. After that, other methods of fermentation,
10% of fresh medium is generally required. such as submerged fermentation were developed.
Normally, submerged fermentation is concluded Although this technique is more sophisticated,
in 5 to 10 days depending on the process surface method required less effort in operation
conditions. It can be carried out in batch, and installation and energy cost (Grewal and
continuous or fed batch systems, although the Kalra, 1995).
batch mode more frequently used.

Table 3. Raw materials employed in submerged fermentation for citric acid production
Raw material Strain Citric acid Yield, % References
Brewery wastes A. niger ATTC 9142 19 g/L 78.5 Roukas & Kotzekidou, 1986
Beet molasses A.niger ATTC 9142 109 g/L - Ogawa & Fazeli, 1976
Yarrow lipolytica 54 g/L 68.7a Kautola et al., 1992
A101
Cane molasses A. niger T 55 - 65 Kundu et al, 1984
Wood Hemicellulose A. niger IMI- 41874 27 g/L 45a Maddox et al., 1985
S. lipolytica IFO 1658 9 g/L 41 Maddox et al., 1985
Date syrup A. niger ATTC 9142 - 50 Roukas & Kotzekidou, 1997
Corn starch A. niger IM-155 - 62 Nguyen et al., 1992
Starch hydrolysate Y. lipolytica DS-1 - - Shah et al., 1993
Y. lipolytica A-101 - 75 Wojtatowicz et al., 1993
Rapeseed oil Y. lipolytica A-101 - 57 Wojtatowicz et al., 1993
Soybean oil Y. lipolytica A-101 - 63 Wojtatowicz et al., 1993
Coconut oil C.lipolytica N-5704 - 99.6b Ikeno et al., 1975
Palm oil C.lipolytica N-5704 - 155b Ikeno et al., 1975
Olive oil C.lipolytica N-5704 - 119b Ikeno et al., 1975
Soybean oil C.lipolytica N-5704 - 115b Ikeno et al., 1975
Glycerol C.lipolytica N-5704 - 58.8b Ikeno et al., 1975
n-Paraffin C.lipolytica N-5704 - 161b Ikeno et al., 1975
a
based on sugar consumed; b based on oils and fatty acids

In the classical process for citric acid manu- generally used as sources of carbon. When
facture, the culture solution is held in shallow applied, molasses is diluted to 15-20% and is
trays (capacity of 50-100 L) and the fungus treated with hexacyanoferrate (HFC).
develops as a mycelial mat on the surface of the
medium. The trays are made of high purity Solid-state fermentation
aluminium or special grade steel and are
mounted one over another in stable racks. The Solid-state fermentation (SSF) has been termed
fermentation chambers are provided with an as an alternative method to produce citric acid
effective air circulation in order to control from agro-industrial residues (Pandey 1991,
temperature and humidity. Fermentation 1992, 1994, Soccol 1994, Pandey and Soccol
chambers are always in aseptic conditions, 1998). Citric acid production by SSF (the Koji
which might be conserved principally during the process) was first developed in Japan and is as
first two days when spores germinate. Frequent the simplest method for its production. SSF can
contamination are mainly caused by Penicilia, be carried out using several raw materials (Table
other Aspergilli, yeast and lactic bacteria (Rohr 4). Generally, the substrate is moistened to about
et al, 1983; Morgant, 1988). Refined or crude 70% moisture depending on the substrate
sucrose, cane syrup or beet molasses are absorption capacity. The initial pH is normally
adjusted to 4.5-6.0 and the temperature of used for citric acid fermentation in SSF.
incubation can vary from 28 to 30°C. The most Vandenberghe et al. (1999a,b) used Erlen-meyer
commonly organism is A. niger. However there flasks and glass columns for the production of
also have been reports with yeasts (Maddox and citric acid from gelatinized cassava bagasse.
Kingston, 1983; Tisnadjaja et al., 1996). One of
Higher yields were obtained in flasks without
the important advantages of SSF process is that
the presence of trace elements may not affect any aeration, and very little sporulation was
citric acid production so harmfully as it does in observed. The same yields were found in column
SmF. Consequently, substrate pre-treatment is reactors only with variable aeration. This
not required. showed great perspective to use SSF process for
citric acid production in simple tray type
Different types of fermenters such as conical fermenters.
flasks, glass incubators and trays, etc. have been

Table 4. Raw materials employed in solid state fermentation for citric acid production.
Raw material Strain Citric acid Yield(%) Reference
Apple pomace A.niger NRRL2001 766 g/kga Hang & Woodams, 1984
NRRL 2270 816 g/kga
NRRL 599 771 g/kga
NRRL 328 798 g/kga
NRRL 567 883 g/kga
Grape pomace A.niger NRRL2001 413 g/kga 88 Hang & Woodams, 1985
NRRL 2270 511 g/kga
NRRL 599 498 g/kga
NRRL 328 523 g/kga
NRRL 567 600 g/kga
Kiwifruit peel A .niger NRRL 567 100 g/kga Hang & Woodams, 1987
Cellulose hydrolysate and A. niger 29 g/kg 44 Mannomani & Sreekantiah, 1987
Sugar cane
Orange waste A. niger 46 g/kg Aravantinos-Zafiris et al., 1994
Beet molasses A.niger ATCC 9142 35 g/L Roukas, 1991
(Ca-alginate gel)
Saccharose (Sugar cane A. niger CFTRI 30 174 g/kgb Shankaranand & Lonsane, 1993
bagasse)
Coffee husk A. niger CFTRI 30 150 g/kgb Shankaranand & Lonsane, 1994
Carrot waste A.niger NRRL 2270 29 g/kga 36 Garg & Hang, 1995
Okara (soy residue) A. niger 96 g/kga Khare et al., 1995
Pineapple waste A.niger ATCC 1015 132 g/kgb Lima et al., 1995
A.niger ACM 4942 194 g/kgb 74 Tran et al., 1998
Glucose A.niger CBS733.88 21.24 g/L Pallares et al., 1996
(Sugar cane bagasse)
Kumara A. niger Yang no 2 103 g/kgb Lu et al., 1997
(starch containing)
Mussel processing A. niger 300 g/kg Pintado et al., 1998
wastes (polyurethane foams)
Cassava bagasse A. niger LPB-21 347 g/kgb 67 Vandenberghe et al., 1999c

a
based on sugar consumed; b based on dry matter

FACTORS AFFECTING CITRIC ACID carbohydrates has been found essential for good
PRODUCTION production of citric acid. Hossain et al. (1984)
showed that sucrose was the most favourable
Medium and its components carbon source followed by glucose, fructose and
galactose. Galactose contributed to a very low
Carbon source: Citric acid accumulation is growth of fungi and did not favour citric acid
strongly affected by the nature of the carbon accumulation.Other sources of carbon such as
source. The presence of easily metabolized
sorbose, ethanol, cellulose, manitol, lactic, malic biomass production. Urea has a tampon effect,
and α-acetoglutaric acid, allow a limited growth which assures pH control (Raimbault, 1980).
and low production. Starch, pentoses (xyloses The concentration of nitrogen source required
and arabinoses), sorbitol and pyruvic acid slow for citric acid fermentation is 0.1 to 0.4 N /liter.
down growth, though the production is minimal A high nitrogen concentration increases fungal
(Yokoya, 1992). growth and the consumption of sugars, but
decreases the amount of citric acid produced
According to Kovats (1960), initial sugar (Hang et al., 1977).
concentration was critical for citric acid
production and other organic acids produced by Phosphorous source: Presence of phosphate in
A. niger. Xu et al. (1989) reported that A. niger the medium has a great effect on the yield of
strains needed an initial sugar concentration of citric acid. Potassium dihydrogen phosphate has
10-14% as optimal; no citric acid was produced been reported to be the most suitable
at sugar concentration of less than 2.5%. phosphorous source. Shu and Johnson (1948)
Honecker et al. (1989) showed that immobilized reported that phosphorous at concentration of 0.5
cells of A. niger needed lower concentrations of to 5.0 g/L was required by the fungus in a
sucrose than free cells culture, in order to obtain chemically defined medium for maximum
high yields (200 g of citric acid/L for free cells production of citric acid. Phosphate is known to
culture, and 120 g/L for immobilized cells). be essential for the growth and metabolism of A.
Maddox et al. (1985) reported the influence of niger (Shankaranand and Lonsane, 1994). Low
different sources of carbon on citric acid levels of phosphate favour citric acid production,
production by A. niger and Saccharomycopsis however, the presence of excess of phosphate
lipolytica. Glucose, maltose, galactose, xylose was shown to lead to the formation of certain
and arabinose were tested. Fermentation was sugar acids, a decrease in the fixation of CO2,
carried out in 8 and 4 days, respectively, at 30°C and the stimulation of growth. Phosphates acts at
and 180 rpm. Better results were found for A. the level of enzyme activity and not at the level
niger with 0.45 g of citric acid/ g of glucose of gene expression (Kubicek et al., 1979). It is
corresponding to 27 g/L. S. lipolytica produced interesting to note that different strains require
0.41 g/g of glucose or 9 g/L which was not so distinct nitrogen and phosphorous concentrations
bad. in the medium. In fact, nitrogen and
phosphorous limitation is a crucial factor in
As presented previously, several raw materials citric acid production as there is an interaction
can be employed successfully for citric acid between them. Consequently, the study of their
production. There are some critical factors combined effect is necessary (Pintado et al.,
(costs, need of pretreatment), which should be 1993; Chen, 1994). Pintado et al. (1998) reported
considered for substrate determination. One how the culturing modality conditions the
another aspect is the presence of trace elements, behavior of the micro-organisms referring to the
which can act as inhibitors or stimulants. tendencies of production as a function of the
Consequently, sometimes it is necessary to levels of N and P. The author used as first order
conduce a pre-treatment, e.g.; precipitation of an empirical model based on rotatable design to
trace metals of molasses by potassium study the effect of both nutrients. As expected,
ferrocyanide. for the two studied strains, a similar behavior
was noticed, showing an improvement towards
Nitrogen source: Citric acid production is low levels of N and P in submerged culture, and
directly influenced by the nitrogen source. toward high levels in solid state culture, and with
Physiologically, ammonium salts are preferred, superior productions for the last one.
e.g. urea, ammonium sulfate, ammonium Shankaranand and Lonsane (1994) affirmed that
chlorure, peptone, malt extract, etc. Nitrogen the specificity of solid state culture is largely due
consumption leads to pH decrease, which is very to a lower diffusion rate of nutrients and
important point in citric acid fermentation (Rohr metabolites, which occurs in low water activity
et al., 1983, Kubicek and Rohr, 1986). However, conditions. Consequently, strains with large
it is necessary to maintain pH values in the first requirements of N and P seems to be disfavored,
day of fermentation prior to a certain quantity
due to the restriction of accessibility to the glucose and other crude carbohydrate.
nutrients in the medium. Appropriate alcohols are methanol, ethanol, iso-
propanol or methyl acetate. The optimal amount
Trace elements: Trace element nutrition is of methanol/ethanol depends upon the strain and
probably the main factor influencing the yield of the composition of the medium, generally
citric acid. A number of divalent metals such as optimum range being 1-3%. The effect of
zinc, manganese, iron, copper and magnesium methanol or ethanol have been extensively
have been found to affect citric acid production studied by many authors (Hamissa, 1978;
by A. niger. However, it is crucial to take into Mannomani and Sreekantiah, 1988; Georgieva et
account the interdependence of medium al., 1992; Dasgupta et al., 1994).
constituents in SmF and, probably, in SSF. Zinc
favoured the production of citric acid if added Mannomani and Sreekantiah (1987) reported
with KH2PO4. On the other hand, the presence of that addition of ethanol resulted in two-fold
manganese ions and iron and zinc (in high increase in citrate synthetase activity and 75%
concentrations) could cause the reduction of decrease in aconitase activity. Whereas the
citric acid yields only in phosphate free medium. activities of other TCA cycle enzymes increased
Shankaranand and Lonsane (1994) noticed that slightly. They also found that coconut oil
there were few differences in the response of A. influenced citric acid production in a sucrose
niger to metal ions and minerals in SSF and in medium when added at 3% (v/w). Alcohols
SmF systems. SSF systems were able to have been shown to principally act on membrane
overcome the adverse effects of the high permeability in micro-organisms by affecting
concentrations of these components in the phospholipid composition on the cytoplasmatic
medium. As a consequence of this, the addition membrane (Orthofer et al., 1979). However
of chelating agents such as potassium Meixner et al. (1985) argued against a role of
ferrocyanide to the medium proved to be of no membrane permeability in citric acid
use. accumulation. Ingram and Buttke (1984) found
that alcohols stimulate citric acid production by
Copper was found to complement the ability of affecting growth and sporulation through the
iron at optimum level, to enhance the action not only on the cell permeability but also
biosynthesis of citric acid. Manganese deficiency the spatial organization of the membrane, or
resulted in the repression of the anaerobic and changes in lipid composition of the cell wall.
TCA cycle enzymes with the exception of citrate
synthetase. This led to overflow of citric acid as Miscellaneous: Some compounds which are
an end product of glycolysis (Kubicek and Rohr, inhibitors of metabolism such as calcium
1978). A low level of manganese (ppm) was fluoride, sodium fluoride and potassium fluoride
capable to reduce the yield of citric acid by 10%. have been found to accelerate the citric acid
Citric acid accumulation decreased by the production, while, potassium ferrocyanide has
addition of iron, which also had some effect on been found to decrease the yield. There are many
mycelial growth. Benuzzi and Segovia (1996) compounds, which act in many ways to favour
reported that the presence of different copper citric acid accumulation. Some of them are
concentrations in the pellet formation medium capable to impair the action of metal ions and
was very important in order to enhance a suitable other toxic compounds influence growth during
structure, related to cellular physiology, for citric the initial phase. Some of these are: 4-Methyl-
acid production. The optimal initial CuSO4.5H2O umbelliferone, 3-hydroxi-2-naphtoic, benzoic
concentration was 78 mg/L. acid, 2-naphtoic acid, iron cyanide, quaternary
ammonium compounds, amine oximes, starch,
Magnesium is required both for growth as well EDTA, vermiculite, etc.
as for citric acid production. Optimal
concentration of magnesium sulfate was found in Process parameters
the range of 0.02-0.025% (Kapoor et al., 1983).
pH: The pH of a culture may change in response
Lower alcohols: Addition of lower alcohols to microbial metabolic activities. The most
enhances citric acid production from commercial obvious reason is the secretion of organic acids
such as citric, acetic or lactic acids, which will phase. Therefore, the addition of antifoaming
cause the pH to decrease. Changes in pH kinetics agents and the construction of mechanical
depend highly also on the micro-organism. With “defoamers” are required to tackle this problem.
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, PRODUCT RECOVERY
Sporotrichum, Pleurotus sp., pH is more stable
(between 4 and 5). Besides, the nature of the The recovery of citric acid from liquid
substrate also influences pH kinetics fermentation is generally accomplished by three
(Raimbault et al., 1997). basic procedures, precipitation, extraction, and
adsorption and absorption (mainly using ion
Generally, a pH below 2.0 is required for exchange resins). Citric acid extraction has been
optimum production of citric acid. A low initial described by the Food and Drug Administration
pH has the advantage of checking contamination (1975) of the United States and by Colin
and inhibiting oxalic acid formation. A pH of 2.2 (1960,1962). Citric acid extracted by this method
was reported to be optimum for the growth of has been recommended suitable for use in food
the mould as well as for the production of citric and drugs. Precipitation is the classical method
acid (Srivastava and De, 1980) whereas, a higher and it is performed by the addition of calcium
pH i.e. 5.4 and 6.0-6.5 has been found optimum oxide hydrate (milk of lime) to form the slightly
for citric acid production in molasses medium soluble tri-calcium citrate tetrahydrate. The
(Roukosu and Anenih, 1980). precipitated tri-calcium citrate is removed by
filtration and washed several times with water. It
Aeration: Aeration has been shown to have a is then treated with sulphuric acid forming
determinant effect on citric acid fermentation calcium sulphate, which is filtered off. Mother
(Rohr et al., 1983; Dawson et al., 1986). liquor containing citric acid is treated with active
Increased aeration rates led to enhanced yields carbon and passed through cation and anion
and reduced fermentation time (Grewal and exchangers. Several anion-exchange resins are
Kalra, 1995). commercially available. Finally, the liquor is
concentrated in vacuum crystallizers at 20-25°C,
The influence of dissolved oxygen concentration forming citric acid monohydrate. Crystalization
on citric acid formation has been examined. It is at temperatures higher to this is used to prepare
important to maintain the oxygen concentration anhydrous citric acid.
above 25% saturation and interruptions in
oxygen supply may be quite harmful (Kubicek et
al., 1980). The high demand of oxygen is CONCLUSIONS AND PERSPECTIVES
fulfilled by constructing appropriate aeration
devices, which is also dependent on the viscosity Since the beginning of this century, citric acid
of the fermentation broth. This is an additional production has been intensively studied and
reason why small compact pellets are the great alternatives to this process have been found
preferred mycelial forms of A. niger during to follow its great demand. The use of alternative
fermentation (Kubicek and Rohr, 1986). When raw materials to produce citric acid by SmF,
the organism turns into filamentous LSC, and SSF seems to be a suitable possibility.
developments, e.g. due to metal contamination, However, it is necessary to adapt the right type
the dissolved oxygen tension rapidly falls to less of raw material to the right technique e.g.
than 50% of its previous value, even if the dry cassava bagasse employed as substrate in SSF,
weight has not increased by more than 5%. or cellulose hydrolysate used in SmF. The need
Aeration is performed during the whole of some pre-treatment of raw materials may
fermentation with the same intensity through the enhance the fermentation efficiency. One area,
medium at a rate of 0.5 to 1.5 vvm. However, which needs attention is the development of
because of economic reasons, it's usually continuous culture techniques which have been
preferred to start with a low aeration rate (0.1 to attempted but only at the laboratory scale.
0.4 vvm). High aeration rates lead to high
amounts of foam, especially during the growth
Another area is the strain improvement with by Aspergillus foetidus. Process Biochem.,
improved substrate utilization efficiency. 29, 399-405
Colin, P. Extraction of citric acid from aqueous
solution. (1960) Fr. Pat., 1,211,066.
RESUMO Colin, P. and Mention, M. Extraction of organic
acids made by fermentation. (1962) Fr. Pat.,
O ácido cítrico é o ácido mais produzido em 1,300,250
termos de tonagem e é extensivamente utilizado Das, A. and Roy, P. (1978) Improved production
pelas indústrias alimentícia e farmacêutica. É of citric acid by diploid strain of Aspergillus
produzido principalmente por fermentação niger. Can. J. Micobiol., 24, 622-625
submersa utilizando o fungo Aspergillus niger e Dasgupta, J., Nasim, S., Khan, A. W. and Vora,
leveduras do gênero Candida sp. à partir de V.C. (1994) Production of citric acid in
diferentes fontes de carbono, como a glicose e molasses medium: effect of addition of lower
meios à base de amido. No entanto, outras alcohols during fermentation. J. Microbiol.
técnicas de fermentação, e.g. fermentação no Biotechnol., 9, 123-125
estado sólido e em superfície, e fontes Dawson, M. W., Maddox, I. S. and Brooks, J.D.
alternativas de carbono tem sido intensamente (1986) Effect of interruption of oxygen
estudadas mostrando grande perspectivas para o supply on citric acid production by
processo. O presente trabalho apresenta um Aspergillus niger. Enzyme Microb. Technol.,
resumo dos últimos avanços sobre a produção do 8, 37-40
ácido cítrico, descrevendo de maneira sucinta os El Dein, S. M. N. and Emaish, G. M. I. (1979)
trabalhos mais recentes, descrevendo Effect of various conditions on production of
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substratos empregados, etc. potassium ferrocyanide by A. aculeatus and
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production by Aspergillus niger in solid-state
fermentation on coffee husk. III Internatl Sem

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