Critical Reviews in Microbiology

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Citric acid from Aspergillus niger: a comprehensive

overview

Bikash Chandra Behera

To cite this article: Bikash Chandra Behera (2020): Citric acid from Aspergillus�niger: a
comprehensive overview, Critical Reviews in Microbiology, DOI: 10.1080/1040841X.2020.1828815

To link to this article: https://doi.org/10.1080/1040841X.2020.1828815

Published online: 12 Oct 2020.

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CRITICAL REVIEWS IN MICROBIOLOGY
https://doi.org/10.1080/1040841X.2020.1828815

REVIEW ARTICLE

Citric acid from Aspergillus niger: a comprehensive overview

Bikash Chandra Behera
School of Biological sciences, National Institute of Science Education and Research, Bhubaneswar, Odisha, India

ABSTRACT ARTICLE HISTORY

Microbial citric acid has high economic importance and widely used in beverage, food, deter- Received 19 June 2020
gents, cosmetics and pharmaceutical industries. The filamentous fungus Aspergillus niger is a Revised 11 September 2020
work horse and important cell factory in industry for the production of citric acid. Although in- Accepted 18 September 2020
depth literatures and reviews have been published to explain the biochemistry, biotechnology Published online 8 October
2020
and genetic engineering study of citric acid production by Aspergillus niger separately but the
present review compiled, all the aspects with upto date brief summary of the subject describing KEYWORDS
microorganisms, substrates and their pre-treatment, screening, fermentation techniques, meta- Aspergillus niger; CRISPR/
bolic engineering, biochemistry, product recovery and numerous biotechnological application of Cas9 systems; fermentation;
citric acid for simple understanding of microbial citric acid production. The availability of genome immobilization; TCA cycle
sequence of this organism has facilitated numerous studies in gene function, gene regulation,
primary and secondary metabolism. An attempt has been also made to address the molecular
mechanisms and application of recent advanced techniques such as CRISPR/Cas9 systems in
enhancement of citric acid production.

Introduction tablets as a source of iron for body, ointments and cos-

Citric acid is a weak organic acid first isolated by
Scheele (1784), with systematic IUPAC name 2-
Hydroxypropane-1,2,3-tricarboxylic acid. Citric acid is
normally considered to be a tribasic due to its three car-
boxylic acid functional groups and hence has three pKa
value, that is, 3.13, 4.76 and 6.39. In the metabolism of
all aerobic organisms, it is an intermediate in the citric
acid cycle. Citric acid is a colourless white crystalline
powder which is practically odourless. It exists as
anhydrous form (C6H8O7, molecular weight 192.12) or
monohydrate form (C6H8O7.H2O, molecular weight
210.14). It is solid at room temperatures, melts at 153
C
and boiling point at 310
C (Kubicek 1998). It decom-
poses with loss of carbon dioxide above about 175
C.
Anhydrous citric acid is highly soluble in water, freely
soluble in ethanol and sparingly soluble in ether,
whereas monohydrate citric acid is soluble in water and
sparingly soluble in ether. It is the largest tonnage
organic acid produced by fermentation. Citric acid used
in food and beverage industry due to its antioxidant
properties to preserve the food or as an acidifier enhan-
ces the flavours and aromas of fruit juices, ice cream,
and marmalades. In the pharmaceutical industry, it is
used as an antioxidant to preserve vitamins, efferves-
cent, pH corrector, blood preservative, iron citrate
metic preparations (Max et al. 2010). In the chemical
industry, for softening and treatment of textiles, it is
used as a foaming agent. In metallurgy, certain metals
are utilised in the form of citrate. Because of less
eutrophic effect, citric acid is used in the detergent
industry as a phosphate substitute (Max et al. 2010).
Development of citric acid production increased greatly
since the last century due to biotechnology which pro-
vides proper knowledge of fermentation techniques
and product recovery. Biochemistry which provides
knowledge of different factor that affects synthesis and
blockage of citric acid production also enhanced citric
acid production and recovery. Besides biotechnology
and biochemistry, molecular regulatory mechanisms
and strategies helped a lot to enhance citric acid pro-
duction. In the past 60 years’, extensive reviews of lit-
erature along with more than thousands of reports
have been published in connection to citric acid pro-
duction (Prescott and Dunn 1959; Lockwood and
Schweiger 1967; Miall 1978; Dawson 1986;
Vandenberghe et al. 1999; Papagianni 2007; Max et al.
2010; Show et al. 2015; Tong et al. 2019). However,
these reviews have been published with respect to bio-
technology, biochemistry and metabolic engineering
separately for citric acid production. Several reports

CONTACT Bikash Chandra Behera bikash@niser.ac.in

ß 2020 Informa UK Limited, trading as Taylor & Francis Group
2 B. C. BEHERA
have been also published regarding types of agro-
industrial product enhancing citric acid production sep-
arately but not combined yet to get the upto date
knowledge of the different types of agro-industrial sub-
strate used to reduce the cost of citric acid production.
Hence, in the present review an attempt has been
made to compile and up-to-date all the information for
citric acid production, associated with its biochemistry
biotechnology and metabolic engineering.
Microorganisms of choice for citric
acid production
In 1826, citric acid production started commercially in
England from Italian lemons. Chemical synthesis of cit-
ric acid was first invented by Grimoux and Adam
(1880), using glycerol as a starting material. Microbial
production of citric acid becomes industrially important
when Italian citrus exports stopped due to World War I.
As its commercial importance increased unexpectedly,
the production through chemical procedure was
replaced by other production routes such as fermenta-
tion. Therefore, microbial fermentation became eco-
nomically competitive enough and the choice for
commercial citric acid fermentation compared to chem-
ical synthesis. Wehmer (1893), discovered Penicillium
mould could produce citric acid from sugar. Currie
(1917) discovered that some strains of A. niger grew
abundantly in a nutrient medium that had initial pH of
2.5–3.5 and excreted large amounts of citric acid. This
finding of Currie laid down the mechanism for indus-
trial production of citric acid which is still the major
industrial route to citric acid used today. In addition to
fungi, some yeast that can able to produce citric acid
are Candida, Hansenula, Pichia, Debaromyces, Torula,
Torulopsis, Kloekera, Saccharomyces, Zygosaccharomyces
and Yarrowia. Bacteria that involved in citric acid pro-
duction are Arthrobacter paraffinens, Bacillus lichenifor-
mis and Corynebacterium spp. (Vandenberghe
et al. 1999)
Though a large number of micro-organisms includ-
ing bacteria, fungi and yeasts have been observed to
produce citric acid, but most of them are not able to
produce commercially acceptable yields. However, the
fungus A. niger has remained the organism of choice
for commercial production due several factors such as
its powerful polymer degrading enzyme systems to
hydrolyse many polymeric substrates, ability to grow
and ferment a wide variety of cheap agro industrial raw
materials with higher yields (Papagianni 2007). This
organism exhibits great robustness to extreme acid
environments, leading to outcompeting other rival
microorganisms and reducing contamination risk.
Substrates used for citric acid fermentation
In order to get optimal yield in fermentation conditions
to reduce cost, substrate is more important for product-
ivity and fermentation yield (Lesniak 1999). A substrate
of higher purity may result in increases in yield, or
reduction in fermentation time (Lesniak 1999). High-
quality molasses is often use for citric acid production
(Lesniak 1999). The ingredient of molasses which
favours citric acid production are sucrose, inverted
sugar, raffinose, kestose, arabinose, xylose, mannose,
glutaric, malonic, succinic, aconitic, malic and lactic
acid, oxalic, citric and tartaric acid (Lesniak 1999). These
can all react with calcium to form insoluble salts that
can influence the precipitation and recovery of the citric
acid crystals. It has been also reported that neutral, or
slightly alkaline molasses gave the best citric acid yields
(Lesniak 1999). Besides molasses, Aspergillus niger fer-
ment sucrose very well which refined from sugar beet
or cane. Syrups of beet or cane sugar, starch such as
potato, wheat and corn, Palm oil and hydrol obtained
as a by-product during crystalline glucose production
from starch can also be used as a substrate for citric
acid fermentation (Gutcho 1973; Kutermankiewicz et al.
1980; Lesniak et al. 1986; Pietkiewicz et al. 1996).
Agricultural residues produced from vast agricultural-
based industries are rich in bioactive compounds. These
residues can be used as an alternate source for the pro-
duction of citric acid fermentation which not only
reduce the production cost but also reduce the pollu-
tion load from the environment. However, such sub-
strates need pre-treatment prior to utilisation as a
substrate. The agricultural residues which are used in
citric acid production including coconut husk, brewery
wastes, coffee husk, rice bran, wheat bran, carrot waste,
cassava bagasses, decaying fruits, corn cob, orange
peel, kiwifruit peel, pineapple peel, banana peel, vege-
tables wastes, sugarcane baggase, tapioca, cheese
whey, rice straw, pomaces of grapes, and apples (Soccol
et al. 2006; Sawant et al. 2018; Dutta et al. 2019).
Biotechnology in production of citric acid
Techniques for qualitative screening of
microorganism for citric acid production
Screening of miocroorganism for citric acid production
is usually done by pour plate method on Czapek-Dox
agar medium supplemented with bromo-cresol purple.
Microbial strains with the widest yellow zone (Figure 1)

Figure 1. Halo zone produced by A. niger on Czapek-Dox
agar-BPB medium indicating citric acid production (Dutta et
al. 2019).
are considered as positive (Dutta et al. 2019). Though
the actual mechanism of the screening procedure is not
understood properly but it is known that the structure
of bromo cresol purple (BCP) which is an indicator dye
changes with pH. When microorganism produce citric
acid in the medium it diffuses through the agar
medium, react with the dye and changes the purple
colour of the dye to yellow (Ali 2004).
Improvement of strains for citric acid production
Improvement of strain is a technique used to increase
citric acid production. Strain improvement through
para-sexual cycle, was first described by Pontecorvo
et al. (1953). Diploids displayed higher citric acid yields
compared to their parent haploids was reported by Das
and Roy (1978). Genetic manipulation of A. niger with
respect to citric acid production through protoplast
fusion was described by Kirimura et al. (1988a). Other
two methods such as single spore technique (SST) and
passage method (PM) are well-known alternative meth-
ods for selection of improved strains (Soccol et al.
2006). Improvement of strain can be also achieved by
modifying metabolism of the microorganism by induc-
ing mutations in them through physical or chemicals
mutagens (Swain et al. 2012). Among physical muta-
gens, the most common mutagens used are gamma-
radiation and UV-radiation (Pelechova et al. 1990).
Among chemical mutagens, diethyl sulphonate (DES),
N-Methyl-N-Nitroso-guanidine, ethidium bromide aziri-
dine, N-nitroso-N-methyl urea or ethyl methane-sul-
phonate are very common (Musilkova et al. 1982).
CRITICAL REVIEWS IN MICROBIOLOGY 3
Screening and identification of mutated improved
strain can be carried out based on enzymatic reaction
on plate media through enzyme diffusion zone analysis
(Prasad et al. 2014). In most cases, mutations are very
harmful which may lead to either increase, decrease or
abolish the activity of mutant. Due to several molecular
tools, such as homologous recombination (HR), RNA
interference (RNAi), CRISPR/CAS9 gene editing tools etc.
genetic engineering of fungal strains became more suc-
cessful for the development of improved strains.
Pre-fermentation treatments of substrate
Concentration of trace metals has profound effect on
citric acid production and various techniques have
been used to reduce trace metals in fermentation
media (Kristiansen et al. 1999). Hence, substrate used in
citric acid fermentation required pre-treatment for the
removal of trace metals. It has been found that cane
molasses used in fermentation contains calcium, mag-
nesium, manganese, iron and zinc, which have a retard-
ing effect on the accumulation of citric acid. Therefore,
chemical pre-treatment such as potassium ferrocyanide
treatment has been extensively used which can able to
precipitate iron and zinc very effectively. The chemical
is either added to the fermentation medium prior to
inoculation. On the other hand, development of fungal
strains that can able to tolerate excess trace metals and
produce high levels of citric acid are preferred to use
(Vandenberghe et al. 1999).
Fermentation technologies used for citric
acid production
Industrial citric acid fermentation can be carried out in
three different ways such as surface, submerged and
solid state fermentation. However, each of these fer-
mentation methods require media preparation, inocula-
tion, fermentation and recovery of the citric acid.
Submerged fermentation
Become the method of choice, submerged fermenta-
tion adopted as most widely used fermentation tech-
nique for higher yields of citric acid in the world today
(Show et al. 2015). In submerged fermentation, micro-
organism grows throughout the broth medium inside
the nutrient substratum and decomposed the carbon
source anaerobically (Reddy 2002; Swain et al. 2012).
Fermentation carried out in an aseptic bioreactor con-
sists of a stirred vessel with an air supply system, incor-
porating a heat exchange facility, such as snake tubes
4 B. C. BEHERA
and water jacket. Spargers and impeller blades are used
to maintain the culture conditions. The air flow is intro-
duced into the vessel at high speed, and the agitation
equipment mixes and breaks the air bubbles, to
increase oxygen levels. The heat exchange system is
used to control the medium temperature at 28 to 30
C
by supplying warm water or cool water depending on
the fermentation requirement. Normally, citric fermen-
tation is concluded in 5 to 12 days, depending on the
process conditions. (Soccol et al. 2006). The advantages
of submerged fermentation include a better control of
the fermentation process and maximum use of a wide
range of substrates (Max et al. 2010). It provides higher
yields, lower capital, maintenance, labour costs and
contamination risks make it more suitable for citric acid
production (Rohr et al. 1983). In contrast disadvantage
of submerged fermentation is the formation of foam,
which can be avoided using antifoam agents such as
animal or vegetable fats and chambers with volume of
up to one-third of the total fermenter volume.
Surface fermentation
Surface fermentation or Liquid surface culture tech-
nique is the beginning of citric acid fermentation pro-
cess characterized by low yield, lower energy
consumption and is also foam free. (Moyer 1953).
However, it is labour intensive and their maintenance
cost is higher, compared to submerged processes, due
to the heavy labour required for cleaning pipes, trays
and reactor walls. (Drysdale and McKay 1995). This pro-
cess is also sensitive to changes in composition of the
media (Benghazi et al. 2014).
Surface fermentation performed in fermentation
chambers, using number of trays arranged in shelves
and made from materials such as stain less steel
(Bauweleers et al. 2014). The trays are shallow pans with
capacity of 0.4 to 1.2 where the fungal mycelium devel-
ops on the surface of the medium. Cane molasses, beet
molasses, wheat bran, potato starch and glucose syrup
of 15 to 20% (w/v), are used as a raw material for surface
fermentation. Surface fermentation is a stationary batch
fermentation process which is completed in 8 to 12 days.
After sterilisation, the nutrient medium contained in
trays (Manzoni 2006), is inoculated with spore suspen-
sion, and incubated at 28-30
C (Marzona 1996) for 24 h.
After incubation, germinated spores form a continuous
mycelium on the surface and pH drops from 6.0–6.5 to
1.5–2.0. High amount of heat is generated during fer-
mentation, so high aeration required to control the tem-
perature, to supply air to the microorganism and to
supplant the CO2 generated which would inhibit the
production of citric acid in concentrations higher than
10%. Since the chamber needs to be effectively venti-
lated, the fermentation chambers are provided with an
effective air circulation, which passes over the medium
surface through a bacteriological filter in order to control
humidity and temperature by evaporative cooling
(Soccol et al. 2006). The chambers should be always 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, other Aspergilli, yeasts and lactic bacteria. After
completion of the fermentation citric acid is recovered
by separating and washing the mycelial mats with boil-
ing water and eventually pressed to remove the impreg-
nated citric acid (Max et al. 2010).
Solid-state fermentation
Solid-state fermentation, popularly known as Koji process,
was first initiated in Japan where fruit wastes and rice
bran are abundantly available (Vandenberghe et al. 2000).
Solid-state fermentation is carried out by growing micro-
organisms in a low-water containing insoluble material
that acts both as physical support and as a source of
nutrients instead of free flowing liquid (Pandey 1992). The
process is completed within 4–5 days (Drysdale and
McKay 1995). The substrate used is solid and moistened
to about 70% moisture before fermentation. The opti-
mum pH required for this process is 4.5–6.0, and tempera-
ture between 28–30
C. The main advantage of solid-state
fermentation is higher yield and easy utilisation of cheap
and widely available agro-industrial substrates without
any pre-treatment as the system is less sensitive to the
presence of trace elements compared to submerged fer-
mentation (Berovic and Legisa 2007). This process has
low-energy requirements, low risk of contamination, less
efforts in downstream processing, less effluent generation
and simple operation as compared to submerged fermen-
tation. It does not require complex equipment and can
operate under less water and less operating costs. On the
other hand, this method has some disadvantages also
such as difficulties to scale up, low amenability of the pro-
cess to standardisation, difficult control of process param-
eters and problems with heat buildup. It cannot utilize
available nutrients completely owing to poor heat and
oxygen transfer in the substrate (Kapilan 2015).
Citric acid production using
immobilisation technique
Some of the problems raised in the downstream proc-
essing of citric acid produced by submerged cultivation,

especially in a continuous process (Eikmeier and Rehm
1984; Mittal et al. 1993). Hence, in the past few years to
increase the rate of microbial production some new
technique has been developed. It has been observed
that successful application of living biocatalysts such as
immobilised micro-organisms enhanced production
rates than free microorganisms in the bioreactor. In cit-
ric acid production, the immobilisation of A. niger
depends on the type of the support material and pro-
cess conditions. Citric acid production by immobilised
A. niger has been performed on a laboratory scale with
the use of calcium alginate gel (Eikmeier and Rehm
1984), polyacrylamide gel (Mittal et al. 1993), polyureth-
ane foam and cryopolymerized acrylamide (Wang
et al. 2000).
Techniques used for recovery of citric acid
After the end of the citric acid fermentation, the prod-
uct obtained contains, citric acid and various undesir-
able by-products such as mycelium, other organic acids,
mineral salts, proteins and varying amounts of other
impurities. Hence, it is necessary to recover the pure cit-
ric acid only from the fermentation medium which is
depends on raw materials and techniques used for pro-
duction (Grewal and Kalra 1995). At first mycelia are
separated from fermentation broth by filtration or cen-
trifugation as it contains about 15% of the citric acid
formed during fermentation. In surface fermentation,
the product obtained is drained off the trays and boiled
water is used to recover the remaining amount of citric
acid from the mycelia. Whereas in submerged fermen-
tation, separation of citric acid from fermented prod-
ucts are more difficult. After fermentation the mycelium
containing broth is heated 70
C for 15 min, and then
filtered by rotating vacuum drum or belt discharge fil-
ters or in centrifuges (Noyes 1969). Waste mycelia may
also be pressed in filter presses and washed additional
citric acid that it contains (Max et al. 2010). It has been
observed that oxalic acid is formed during the fermen-
tation which can be removed by increasing the pH up
to 3.0 with calcium hydroxide at 72-75˚C (Sawant et al.
2018). Subsequently, calcium oxalate is formed. Calcium
oxalate is further precipitated and eliminated by centri-
fugation or filtration process. Whereas the citric acid
remained in the original solution in the form of calcium
salt (calcium citrate) can be further extracted by various
techniques such as precipitation, solvent extraction,
adsorption, electro-dialysis, ultra-filtration and
liquid membranes.
Citric acid can be recovered from the insoluble cal-
cium citrate in the original solution through
CRITICAL REVIEWS IN MICROBIOLOGY 5
precipitation by adding an equivalent amount of cal-
cium oxide at 90
C and pH nearly equal to 7.0 (Soccol
et al. 2006). The acid is transformed into tri-calcium cit-
rate tetrahydrate, which is then filtered off and subse-
quently treated with 70% sulphuric acid to form citric
acid and insoluble calcium sulphate (gypsum). After fil-
tering off the gypsum a solution of 25-30 per cent of
citric acid is obtained. To remove residual impurities,
the filtrate is treated with activated carbon or can be
purified in ion-exchange columns. Further it is concen-
trated by evaporation in a vacuum evaporator at 40
C
and crystals of citric acid monohydrate can be formed
in a vacuum crystallizer at a temperature of 20– 25
C
or anhydrous citric acid can be formed at crystallisation
temperatures above 36.5
C (Kubicek 1986; Grewal and
Kalra 1995). For one tonne of citric acid, 579 kg of cal-
cium hydroxide, 765 kg of sulphuric acid and 18 m3 of
water are consumed and approximately one tonne of
waste calcium sulphate is produced (Glusz and
Ledakowicz 1999). Wastes formed in this process
include calcium sulphate (gypsum), and microorganism
residues containing amino acids, sugar, colloid, pigment
and inorganic matter, are supplied to cement factories
and dried as forage for use in feed factories, respect-
ively (Glusz and Ledakowicz 1999).
Purification and crystallization of citric acid from fer-
mentation broth can be alternatively performed by
solvent extraction method in which insoluble or spar-
ingly soluble solvent such as n-octyl alcohol, tridodecyl-
amine and isoalkane (Soccol et al. 2006), Alanine 336 in
heptane or xylene (Sirman et al. 1990), mixture of buty-
lacetate and N, N-disubstituted alkylamide (Yi et al.
1987), aliphatic alcohols, ketones, ethers or esters
(Kasprzycka et al. 1989), organophosphorus com-
pounds, such as tri-n-butylphosphate (TBP) (Pagel and
Schwab 1950), alkylsulphoxides, (Grinstead 1976) and
water-insoluble amines or a mixture of two or more of
such amines are used (Prochazka et al. 1994). The
advantage of the solvent extraction method is to pre-
vent the use of lime and H2SO4 thus production of gyp-
sum can be avoided (Grewal and Kalra 1995). In this
process, citric acid is recovered from the aqueous solu-
tion by washing off the extract with water, subse-
quently crystallised and concentrated. The
concentrated citric acid solution dissolved in acetone is
passed with compressed CO2. The anti-solvent effects
of CO2 remove the residual impurities and food grade
citric acid is obtained by simple decolourization and
crystallisation (Shishikura et al. 1992).
In ion-exchange separation technique, two static
beds used which are connected with appropriate
valve. Feed mixture is passed through one adsorbent
6 B. C. BEHERA

bed while the desorbent material is passed through
another. In such a system, the adsorption and desorp-
tion operations are continuously taking place, which
allows both continuous production of an extract and a
raffinate stream and the continual use of feed and
desorbent streams (Edlauer et al. 1990). Two catego-
ries of resins such as macro-porous adsorption and ion
exchange are commonly employed in this method.
The polymeric adsorbent to be used should be neutral,
non-ionogenic, micro-reticular and water-insoluble
styrene-based polymers. Weakly basic anionic-
exchange resins used to achieve better selectivity and
higher capacity of the adsorbent which is impregnated
with tertiary amine or pyridine or strongly basic anion
exchange resins. The major advantage of ion-
exchange separation techniques is quick recovery,
high capacity, specificity and low regeneration con-
sumption, not production of any calcium sulphate co-
product. In contrast disadvantage of this process is it
require a large amount of desorbent causing consider-
able dilution of the resultant citric acid solution and
formation of waste liquor in large quantities
(Kristiansen and Sinclair 1979).
Citric acid recovery is also carried out by another
method called electrodialysis separation in which elec-
trically charged membranes and electrical potential dif-
ference are used to separate ionic species from
aqueous solutions. Separation of ionic species from
clarified fermentation broths is more economical
through this process (Grewal and Kalra 1995). The dis-
advantage of electrodialysis technique is its costs which
are found to be 50% greater than precipitation process
(Amenaghawon and Aisien 2012).
Nowadays, determination and quantification of citric
acid are carried out in laboratory using spectrophotom-
etry, gas chromatography and high-performance liquid
chromatography (Jham et al. 2002), high-resolution
nuclear magnetic resonance spectroscopy (del Campo
et al. 2006) in addition to the traditional method of
titration (Williams 1984).
Biotechnological application of citric acid
Due to its non-toxicity citric acid is widely used in various
industries. It is widely accepted as “GRAS” (generally rec-
ognised as safe) and approved by the joint FAO/WHO

Table 1. Biotechnological application of citric acid.

Industry Application Uses References
Food and beverage industry Carbonated beverages Used to provide taste and tartness as well as Fukui and Tanaka 1980
stimulates and fruit flavours
Confectionery industry Used as flowing agent Buchard and Merrit 1979
Frozen food products Preventing the deterioration of colour and Soccol et al. 2006
flavour in frozen fruit. By inactivating trace
metals prevents ascorbic acid
Jam and jellies Taste and pH adjustment Crueger and Crueger (1984)
Fats, oils and fat containing foods Used as an antioxidant synergism Buchard and Merrit 1979
Ice cream and cheese Used as an emulsifying agent Buchard and Merrit 1979
Caramel and candies Used in preventing sucrose crystallisation Buchard and Merrit 1979
Protection of foodstuffs Esters of citric acid, employed as non-toxic Buchard and Merrit 1979
plasticisers in plastic films used to
protect foodstuffs
Wines and ciders Prevents browning, turbidity of wines Soccol et al. 2006
and ciders,
Candies Act as an acidulant Soccol et al. 2006
Pharmaceutical industry Pharmaceutics Combined with carbonates and bicarbonates Vandenberghe et al. 1999; Gul and
produce effervescent effect in antacid and Monga 2014
soluble aspirin, anticoagulant, prevent
kidney stones,
Detergent industry Chelating agent Used for softening water Ciriminna et al. 2017
Metal industry Cleaning and chelating agent Solutions of citric used in the cleaning of Smith and Munton 1941; Krummel
power station boilers to remove buildup and Gault 1976; Verhoff 2016
of limescale
Citrates used in platting of copper, nickel,
chromium, lead etc.
Chemical industry Pectin extraction Pectin from apple pomace Canteri-Schemin et al. 2005
Cross-linker Used in crosslinking of materials Ciriminna et al. 2017
Environmental Soil bioremediation Citric acid with rhamnolipid biosurfactants Wan et al. 2015
used in soil remediation through biobased
chemical agents
Biomedical Disinfectant Citric acid in combination with alcohol is an Ionidis et al. 2016; Ciriminna
excellent and safe disinfectant against et al. 2017
several viral and bacterial pathogen
Other Other Printing ink, leather tanning, concrete plaster, Soccol et al. 2006
paper polymer, soldering, used as dip for
oily fish, cosmetic formulations for pH
adjustment etc.

expert committee on food additives. Some up-to-date
and recent application of citric acid are discussed in this
section as well as presented in Table 1.
Food and beverage industry
Because of its pleasant taste, high water solubility, che-
lating and buffering properties, citric acid is widely
used as a safe acidulant in the food, sugar, confection-
ery and beverages industry. During carbonated bever-
ages preparation citric acid is used to provide taste as
well as complement fruit and berry flavours (Fukui and
Tanaka 1980). In confectionery industry, it is used as
flowing agent (Buchard and Merrit 1979). The chelating
and pH adjusting properties of citric acid applied in the
food industry to enhance the stability of frozen food
products by preventing the deterioration of colour and
flavour in frozen fruit. It also helps to prolong the shell-
fire of frozen fish and shellfish. Crueger and Crueger
(1984) reported that for taste and pH adjustment in jam
and jellies, citric acid is widely used. It is used in wine
industry to prevent turbidity of wines and ciders
(Soccol et al. 2006). Used in candies to provide dark col-
our and tartness. Citric acid is used as an antioxidant
synergism in fats, oils and fat containing foods
(Buchard and Merrit 1979). Citric acid is used in sher-
bets as a flavour adjunct (Buchard and Merrit 1979).
Citric acid can be added to ice cream as an emulsifying
agent, added to caramel for preventing sucrose crystal-
lisation and in recipes in place of fresh lemon juice. It is
also used in culinary applications, as an alternative to
vinegar or lemon juice, where a pure acid is needed.
Citric acid can be used in food colouring to balance the
pH level of a normally basic dye. Citric acid can com-
plex with heavy metals such as iron and copper hence
used as a stabiliser of oils and fats where it greatly
reduces oxidation catalysed by these metals. It is also
employed as an aid to emulsification of cheese. In add-
ition, esters of citric acid, such as triethyl, tributyl and
acetyltributyl are employed as non-toxic plasticisers in
plastic films used to protect foodstuffs (Buchard and
Merrit 1979).
Pharmaceutical industry
Due to their sequestering action, such as stabilisation of
ascorbic acid and good buffering capacity, citric acid
and its salts are widely used in the pharmaceutical
industries as oral pharmaceutical liquids, elixirs and sus-
pensions to buffer and maintain stability of active ingre-
dients of the pharmaceutical product (Moledina et al.
1977). Citric acid is often used as the anion in
CRITICAL REVIEWS IN MICROBIOLOGY 7
pharmaceutical preparations which make use of basic
substances as the active agent. It combined with carbo-
nates and bicarbonates to produce the effervescent
effect in antacid and soluble aspirin. Trisodium citrate is
widely used as a blood preservative, where it prevents
clotting by complexing calcium (Vandenberghe et al.
1999; Ciriminna et al. 2017). In combination with
sodium citrate, acetic acid is used to prevent kidney
stones (Gul and Monga 2014). Consuming fruit juice
prevents stone formation not only because it increases
urine volume but also because it is high in potassium
and citric acid which prevents stone formation by bind-
ing with urinary calcium and thereby reducing the
supersaturation of urine (Gul and Monga 2014)
Although other iron salts are preferred for treatment of
anaemia but ferric ammonium citrate is still used.
Detergent industry
Citric acid is used is for softening water which makes it
useful in household detergents and dishwashing
cleaners or soap (Ciriminna et al. 2017). By chelating
the metals ions like Ca2þ and Mg2þ ions in hard water,
it helps to produce foam and work better without need
for water softening. Contrarily to phosphate builders, it
does not contribute to the eutrophication of aquatic
systems. It can also be used in shampoo for washing
colour and wax from the hair.
Cleaning and chelating agent
Due to its excellent metal chelating properties it binds
to metals and makes them soluble. Hence, solutions of
citric are used in the cleaning of power station boilers
and similar installations to remove and discourage the
buildup of limescale (Verhoff 2016). Citric acid-based
metal cleaning formulations efficiently remove metal
oxidation products from the surface of ferrous and non-
ferrous metals. Citric acid has also found uses in electro-
plating (Ates et al. 2002). Citrates are required in plat-
ting of copper (Krummel and Gault 1976), nickel,
chromium, lead (Smith and Munton 1941) and various
heavy metals (Buchard and Merrit 1979). Citric acid is
the active ingredient in some bathroom and kitchen
cleaning solutions. A solution with a six percent con-
centration of citric acid will remove hard water stains
from glass without scrubbing.
Extracting agent
In 2005, Brazilian researchers first showed that citric
acid can be successfully used in place of toxic mineral
8 B. C. BEHERA
acids to recover pectin from apple pomace (Canteri-
Schemin et al. 2005). Pectin extraction yield with citric
acid showed the highest average value (13.75%).
Although nitric acid sometimes showed the highest
yield, the associated variability was very large, as the
harmful effluents generated
Cross-linker
For crosslinking of materials, use of citric acid is widely
accepted (Ciriminna et al. 2017). As a crosslinker it has
several use such as in ultrafine protein fibres for bio-
medical applications (Reddy and Reddy, 2015), polyols
for making biodegradable films like bio-plastic suitable
for eco-friendly packaging (Seligra et al. 2016), hydroxy-
apatite to make bioceramic composites for orthopaedic
tissue engineering (Sun et al. 2014).
Environmental remediation
As already discussed, citric acid has excellent metal che-
lating properties, hence widely used to clean nuclear
sites contaminated with radio-nuclides (Kantar and
Honeyman 2006), and bioremediation of soils contami-
nated with heavy metals (Ates et al. 2002). It has been
reported that citric acid in combination with rhamnoli-
pid biosurfactants affords very excellent results in soil
environmental remediation through bio-based chemical
agents. This combination is not only environmentally
compatible, but also promotes soil ecological restor-
ation after remediation (Wan et al. 2015). Citric acid
also enhances the soil desorption of hydrophobic
organic compounds from soils (Gao et al. 2010).
Disinfectant
Common hand disinfectant, 70% ethanol are widely
used as a disinfectant to get protection from patho-
genic microorganism. It has been observed that appli-
cation of 70% ethanol solution, for one minute fails to
deactivate the non-enveloped poliovirus (Ionidis et al.
2016). Citric acid in combination with alcohol is an
excellent and safe disinfectant against several viral and
bacterial pathogens (Ionidis et al. 2016; Ciriminna et al.
2017). When it added to norovirus-like particles, citrate
precisely binds at the binding pocket on the histo-
blood group antigens of the virus that involved in
attaching to host ligands, hence, preventing the trans-
mission of these viruses (Koromyslova et al. 2015). A
commercial paper tissue, with citric acid (7.51%) and
sodium lauryl sulphate (2.02%), have been prepared in
which sodium lauryl sulphate kills the enveloped
viruses by disrupting the lipid envelope of the viruses,
whereas citric acid disrupts rhinoviruses, which do not
have a lipid envelope. Another important recent
advance is the production of perorganic acids from the
combination of citric acid, lactic acid and hydrogen per-
oxide (Gurtler et al. 2016) which is very effective disin-
fectant against the pathogens. The powerful oxidising
agents perorganic acid can quickly penetrate the lipid
bilayer membrane providing rapid inactivation of
pathogenic such as Salmonella, Listeria monocytogenes
and Escherichia coli. This product is also very safe and
after its use the constituent ingredients break down
into water, oxygen, and organic acids. Hence, no toxic
compounds are released to the environment. (U.S. Food
and Drug Administration 2015). Citric acid alone as a
buffer (within pH 4.0) can make the bacterial mem-
branes more vulnerable to leakage, and thus can inhibit
bacterial growth.
Though, a combination of ethanol (70%), urea (1%)
and citric acid (1.5%) had a strong inactivation effect on
poliovirus but insufficient for adenovirus and polyoma-
virus. Ionidis et al. (2016) found that a combination of
70% ethanol with 2% urea and 2% citric acid could suf-
ficiently deactivate all enveloped viruses such as polyo-
mavirus, norovirus and adenovirus vaccinia virus
on surfaces.
Other uses
As a component of printing plate emulsions in various
bleaches, fixers and stabilisers, citric acid and its esters
are used in photography (France Patient 1937), in oil
well treatment and cements (Buchard and Merrit 1979),
in textile industry (U.S. Patient 1949), in paper industry
and tobacco industry (Hushedeck 1965). It is used as an
ingredient in cosmetic formulations for pH adjustment,
and in antioxidant systems as a metallic-ion chelator
(Wells et al. 1972). Citric acid is an excellent soldering
flux, either dry or as a concentrated solution in water
(Nissan et al. 1995). It should be removed after solder-
ing, especially with fine wires, as it is mildly corrosive. It
dissolves and rinses quickly in hot water. A mixture of
citric acid and ascorbic acid is used as a dip for oily fish
to prevent surface tissue from becoming brownish and
gummy, a condition known as rusting.
Biochemistry of citric acid production and
accumulation
From the technology viewpoint, understanding of the
biochemistry of citric acid production is very meaning-
ful for the investigations of the kinetics, and reactor

Figure 2. Production of citric acid by A. niger (PFK ¼ phosphofructokinase, PC ¼ pyruvate carboxylase, ACO ¼ aconitase. (Slight
modification to Soccol et al. 2006).
designs. In the last few decades’ biochemical basis of
knowledge about the circumstances, under which citric
acid is accumulated was published by several research
groups (Papagianni 2007; Max et al. 2010; Show et al.
2015). Research investigation revealed that a number of
biochemical factors including carbon source concentra-
tion, dissolved oxygen, hydrogen ions, and suboptimal
concentrations of phosphate and trace metals were
found to be responsible for citric acid overflow but var-
ied amongst individual strain (Kristiansen and
Sinclair 1979).
Citric acid biosynthesis started with uptake of sugar
which undergoes glycolysis to produce two molecule of
pyruvate (Figure 2). Pyruvate molecules produced by
glycolysis are actively transported across the inner mito-
chondrial membrane, and into the matrix. Here, they
can be oxidized and combined with coenzyme A to
form CO2, Acetyl-CoA and NADH. Acetyl-co-A formed is
combines with the oxaloacetate to produce citrate. On
the other hand, it is also possible that pyruvate pro-
duced during glycolysis can be carboxylated by pyru-
vate carboxylase (PC) to form oxaloacetate (a TCA cycle
intermediate) to fills up the amount of oxaloacetate in
the citric acid cycle to metabolise Acetyl-CoA when the
CRITICAL REVIEWS IN MICROBIOLOGY 9
tissue’s energy needs are suddenly increased by activ-
ity. Citric acid thus synthesised due to combination of
Acetyl-co-A and oxaloacetate is then transformed
through a reaction sequence that yields two molecules
of CO2 and regenerates the four-carbon oxaloacetate
again. Another turn of the cycle may now start by the
reaction of the oxaloacetate with another molecule of
Acetyl-Co enzyme A. Thus in each turn of the cycle one
molecule of Acetyl-CoA enters, two molecules of ATP
and CO2 are formed and a molecule of oxaloacetate is
utilised to form citrate (Prescott and Dunn 1959).
Accumulation of citric acid by Aspergillus niger was
at first proposed by Ramakrishnan et al. (1955). They
proposed that citric acid accumulation was only
occurred when enzymes such as aconitase, isocitrate
dehydrogenase and succinic dehydrogenase were
severely inhibited during the TCA-cycle. In contrary,
during citric acid fermentation and throughout the
fermentation period, presence of these enzymes in
very less amounts was reported by several workers
(Kubicek and Rohr 1977). These enzymes play very
important role for the production of several intermedi-
ates, required for biomass formation (Ahmed et al.
1972). Therefore, citric acid accumulation may more
10 B. C. BEHERA
likely be the result of enhanced (deregulated) biosyn-
thesis rather than inhibited degradation (Max
et al. 2010).
Kubicek (1986), explained that the accumulation of
citric acid is associated to tricarboxylate transporter
activity which competes with aconitase for citric acid.
When affinity for citric acid is greater than that of aconi-
tase, this enzyme ejects citric acid out of the mitochon-
dria without inhibition of enzymes of the cycle.
According to Martin and Wilson (1951) and Cleland and
Johnson (1954) pentose phosphate and glycolytic path-
ways act as a channel by Aspergillus niger to metabolise
glucose and accumulation of citric acid. Mischak et al.
(1985) explained that citrate synthase is an enzyme respon-
sible for reversible catalysis between acetyl coenzyme A
(acetyl CoA) and oxaloacetate favours citrate production.
Some of the enzymes of A. niger plays very crucial role
during citric acid formation. Outside the cell sucrose is first
converted into glucose and fructose by a membrane-
bound enzyme invertase, and transported into the cell
(Rubio and Maldonado 1995) via glucose transporters.
Hexokinase is another key enzyme which is present inside
the cell and converted the transported glucose into glu-
cose-6-phosphate and initiates the process of glycolysis. A.
niger can also oxidise glucose using enzyme glucose oxi-
dase which produced early in fermentation and converts
glucose into gluconic acid (Mischak et al. 1985). However,
it is not clear whether gluconic acid is used or not by A.
niger to produce citric acid later in the fermentation.
In addition to the TCA cycle enzymes, high flux
through glycolysis also responsible for accumulation of
citric acid by A. niger (Rohr et al. 1992). It has been
reported that deficiency of manganese, or phosphate and
nitrogen limitation, inhibits anabolism of the fungus and
results in degradation of proteins leads to increased intra-
cellular NH4þ concentration (Ro €hr and Kubicek 1981;
Habison et al. 1983). This high concentration of intracellu-
lar NH4þ inhibits of the enzyme phosphofructokinase
(PFK), an essential enzyme with magnesium as a cofactor,
changes fructose 6-phosphate into fructose 1,6-bisphos-
phate in glycolysis (Figure 2). Inhibition of phosphofructo-
kinase (PFK) leads to a flux through glycolysis. According
to Ro €hr and Kubicek (1981), high concentrations of glu-
cose and ammonium pool repress the synthesis of a-keto-
glutarate dehydrogenase, an important enzyme required
to regulate TCA cycle and thus inhibited the citric acid
cycle movement, resulting in the accumulation of citric
acid. The NADPþ, dependent enzyme isocitrate dehydro-
genase are found both in mitochondria and cytoplasm of
A. niger, require Mg2þ or Mn2þ for activation, and their
inhibition by a-ketoglutarate and citrate favours citric acid
accumulation (Ratledge and Kristiansen 2001).
Kubicek-Pranz et al. (1990) reported that high con-
centrations of ATP, citrate and manganese inhibit PFK1,
but it can be activated by the products of the PFK2
reaction, such as Zn2þ, Mg2þ, NH4þ and fructose-2,6-
bisphosphate.
A mutant strain of A. niger with citrate insensitive
PFK2 enzyme was found to be more tolerant of manga-
nese when accumulating citric acid and showing that
the inhibitory effect of Mn2þ on PFK1 is antagonised by
fructose-2:6-bisphosphate (Schreferl et al. 1986). Citrate
accumulation also occurs when a certain amount of
NH4þ ions is taken up at the start of the batch fermen-
tation. In steady-stage fermentation, PFK enzyme has
no immense effect on the flux through glycolysis
(Torres 1994b). Mattey (1992) found that the combin-
ation of the levels of inhibitors and activators in pro-
ductive cytoplasm simply permit the accumulation of
citric acid through glycolysis.
Papagianni et al. (2005) refuted the statement of
Ro€hr and Kubicek (1981), that citric acid accumulation
by A. niger was due to the existence of an intracellular
ammonium pool which inhibits the enzyme PFK. During
his investigation he found that instead of accumulation
or deposition inside the cell to make an ammonium
pool, ammonium ions are entered the cell and combine
with glucose to produce glucosamine which is then
released outside the cell or into the fermentation broth
and responsible for PFK inhibition. Hence the details
relationship between glucose, ammonium ion concen-
trations, the enzymes within the TCA cycle and accumu-
lation of citric acid is obscure which certainly needs
further investigation.
Factors affecting citric acid production
The accumulation of citric acid is strongly influenced by
the composition of the medium, especially in sub-
merged fermentation processes. It was shown that the
factors mainly affecting the citric fermentation are the
type and concentration of carbon source, nitrogen and
phosphate limitation, pH, aeration, trace elements con-
centration, and morphology of the producing micro-
organism (Max et al. 2010).
Carbon source
Angumeenal and Venkappayya (2013) showed that due
to extracellular mycelium-bound invertase of A. niger,
sucrose is most suitable carbon source then glucose,
fructose and lactose for citric acid production. Because
invertase can rapidly hydrolyses sucrose at low pH
(Kubicek-Pranz et al. 1990). Other carbon sources such

as sorbose, ethanol, cellulose, manitol, lactic acid, malic
acid, a-acetoglutaric acid, starch, pentoses (xyloses and
arabinoses), sorbitol and pyruvic acid showed very lim-
ited growth of the fungus with minimal yield (Yokoya
1992). In industrial scale, use of pure sucrose or glucose
may not be economically feasible. Therefore, low-grade
carbon sources such as cane and beet molasses are
used after proper pre-treatment using potassium ferro-
cyanide or cation-exchange resins to reduce heavy
metal ion contamination (Angumeenal and
Venkappayya 2013).
The concentration of the carbon source is also crit-
ical to the success of citric acid production (Xu et al.
1989). Xu et al. (1989) reported that A. niger strains
needed an initial sugar concentration of 10-14% as
optimal but no citric acid was produced at sugar con-
centration of less than 2.5% and maximum citric acid
production observed at the concentration 14-–22% of
sugar. It is because the high concentrations of the sugar
reported to suppress a-ketoglutarate dehydrogenase
and results maximum citric acid accumulation (Hossain
et al. 1983). In contrast, at low concentrations of the
sugar the size of the mycelium is reduced, and its shape
is also affected (Papagianni et al. 1999). It has been also
observed that immobilised cells of A. niger needed low
sugar concentrations in compare to free cells for max-
imum citric acid accumulation (Honecker et al. 1989).
Nitrogen limitation
Nitrogen being the part of a cell’s proteins necessary
for cellular metabolism (Ali et al. 2002b) Hence, its con-
centration plays critical role in the synthesis of citric
acid as well as the fungal growth. Basically ammonium
salts such as ammonium nitrate and sulphate, urea,
peptone, malt extract, etc are used in citric acid produc-
tion (Grewal and Kalra 1995). Acid ammonium com-
pounds are preferred because their consumption leads
to pH decrease, which is essential for the citric acid fer-
mentation. Ammonium nitrate promotes reduced vege-
tative growth, while ammonium sulphate promotes
prolonged vegetative growth. Ammonium sulphate is
the preferred choice of salt as it does not produce the
unwanted oxalic acid, while reducing the pH of the
medium as the salt is consumed. Molasses are usually
nitrogen rich and used in industrial fermentation with-
out any additional ammonium salts as supplements.
Nitrogen concentration greater than 0.25% accumulates
oxalic acid in the fermentation medium and decrease
the citric acid yield (Gupta et al. 1975). It has been also
reported that a high nitrogen concentration increases
the consumption of sugar and fungal growth, while
CRITICAL REVIEWS IN MICROBIOLOGY 11
decreasing the amount of citric acid produced (Hang
et al. 1977).
Phosphorus
Along with nitrogen and the carbon source, phosphate
is also very essential element for citric acid yield
(Vandenberghe et al. 1999). Low levels of phosphate
are found very suitable for citric acid production
whereas its excess concentration may lead to the for-
mation of certain sugar acids, a decrease in the fixation
of CO2, and the stimulation of growth. (Grewal and
Kalra 1995). For optimal citric acid production, KH2PO4
has been reported to be the most suitable at a concen-
tration of 0.5–5 g/l (Shu and Johnson 1948b). Kubicek
and Rohr (1977) reported that citric acid accumulates
with limited phosphate, even when nitrogen is
not limited.
pH of fermentation medium
The pH of the medium is most important during the ini-
tial stage of fermentation and at the end, before the
recovery of the product during citric acid fermentation
(Papagianni 2007). Initially in the germination stage the
germinating fungal spores in the fermentation medium
after inoculation require a pH greater than 5 in order to
germinate (Papagianni 2007). The germinating spores
absorb ammonia and release protons, thereby increas-
ing the acidity of the medium and favouring the pro-
duction of citric acid (Papagianni 2007). However, the
initial pH of the medium depends on the substrate
used. A pH value 2.5 to 4.0 was found to be optimum
for chemically defined medium (Jernejc et al. 1982)
where as an initial pH value of 6.0 to 7.5 was found to
be best in molasses medium (Berry et al. 1977). Hossain
et al. (1983) observed that an initial pH of 4.5 was opti-
mal in whey permeate for citric acid production, while
Roukas and Alichanidis (1991) suggested an initial pH
value of 3.0 in beet molasses.
On the other hand, the pH for recovery stage of citric
acid should be below 2. At this low pH, the formation
of unwanted products such as oxalic and gluconic acid
is inhibited, and the possibility of contamination by
other microorganisms is also reduced, making recovery
of citric acid easier (Max et al. 2010).
Temperature
Temperature is an extremely important parameter for
citric acid production (Kapoor et al. 1982). Citric acid
production is dependent on glycolysis and TCA cycle
12 B. C. BEHERA
enzyme. These enzymes are temperature dependent. In
higher or lower temperature these enzyme systems will
not work and affect citric acid production. Incubation
temperature ranges from 25 to 30
C was found to be
more suitable for high yields and rapid rates of citric
acid production (Prescott and Dunn 1959). Kapoor et al.
(1982) reported that the citric acid yield decrease above
the temperatures 30
C due to increase oxalic acid pro-
duction, while temperature below 25
C slow the
growth of the organism and thus the fermentation
rates. During submerged fermentation, heat generated
which is controlled by heat exchange facilities, such as
snake tubes and water jackets. In the initial stage of fer-
mentation, warm water is supplied to maintain the
required temperature, while in later stages, when tem-
perature increases, cool water is supplied to remove fer-
mentation heat, thus keeping the temperature in the
required range. However, in solid substrate fermenta-
tion it becomes very difficult to control the fermenta-
tion temperature, due to the solid nature of the
substrate and lack of effective reactors.
Concentration of alcohols
Alcohols act on membrane permeability in microorgan-
isms by affecting their phospholipid composition.
Alcohol such as methanol showed positive effect on cit-
ric acid formation due to its inhibitory effect on metal
ions. The amount of methanol/ethanol required is
depends on the composition of the medium and the
strain of microorganism used (Moyer 1953). Ingram and
Buttke, (1984) reported that alcohols stimulate citric
acid production by affecting growth and sporulation
due to changes in lipid composition of the cell mem-
brane. Kubicek et al. (1985) reported that lower alcohols
added in pure material inhibit citric acid production but
if added into crude carbohydrates these alcohols
enhance citric acid production. Methanol, ethanol, n-
propanol, isopropanol or methylacetate concentration
between 1 to 5% neutralise the negative effect of the
metals ions in citric acid production and favours max-
imum citric acid formation (Kubicek et al. 1985). Moyer
(1953) studied that the addition of ethanol doubles the
citrate synthetase activity and decreases the activity of
aconitase which result in increased citric acid
accumulation.
Trace elements
Divalent metal ions have been found to play very crit-
ical role in the yield of citric acid as well as for growth
of A. niger (Dronawat et al. 1995). The metals ions that
are most sensitive with regard to citric acid production
include Fe2þ, Cu2þ, Zn2þ and Mn2þ hence, must be lim-
iting and have received much research attention. The
interdependence of medium constituents has to be
taken into account as it is crucial to citric acid produc-
tion. Therefore, for the optimum citric acid yield, control
of these trace elements are necessary especially during
submerged fermentation (Shu and Johnson 1948b).
Trace element or the metal ion manganese has con-
sidered as most important as other metal ion.
Concentrations of Mn2þ at less than 3 lg/l has been
shown to drastically reduce the yield of citric acid pro-
duction (Clark et al. 1966) where as Mattey and Bowes
(1978) reported that the addition of 10 mg Mn2þ per
litre reduced accumulation of citric acid by 50% relative
to control culture. Hence, care must be taken when
choosing broth ingredients and even the materials of
construction of the bioreactor vessels, to ensure that
traces of manganese do not reduce the yield from the
fermentation. Jernejc et al. (1989) found that in the
presence of Mn2þ, the amount of total lipids in mycelia
was up to two times higher than in conditions favour-
ing citric acid production. Manganese is also important
in synthesis of cell wall, sporulation and secondary
metabolites production (Shu and Johnson 1948b;
Tomlinson et al. 1950). Cellular anabolism of A. niger is
impaired under manganese deficiency and protein
breakdown results in a high intracellular ammonium.
This high intracellular ammonium concentration inhib-
its the enzyme phosphofructokinase, (an essential
enzyme in the conversion of glucose and fructose to
pyruvate) leading to a flux through glycolysis pathway
and the formation of citric acid.
In addition to the above, deficiency of Mn2þ causes
many physiological and metabolic changes in A. niger
during citric acid production such as lower levels of
pentose phosphate pathway and tricarboxylic acid cycle
enzymes (Kubicek and Rohr 1977), higher levels of pyru-
vate and oxaloacetate (Kubicek and Rohr 1978), ele-
vated levels of amino acids during idiophase (Kubicek
et al. 1979), elevation of cell wall chitin and decreased
b-1, 3-glucan (Kisser et al. 1980), increased monosome
portion of ribosomes and elevated proteinase activity
as well as rate of protein degradation (Ma et al. 1985)
which directly affect citric acid production (Xu
et al. 1989c).
Copper was found to complement the ability of iron
at optimum level, to enhance the biosynthesis of citric
acid. The toxic effect of Fe2þ can be eliminated by high
concentration of Cu2þ (Rohr et al. 1983). 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 cit-
ric acid production (Benuzzi and Segovia 1996).
Zn2þ plays critical role during initiation of citric acid
accumulation. Tomlinson et al. (1950) deduced that the
optimum concentrations of zinc are 0.3 ppm for citric
acid production. Zinc favoured the production of citric
acid if added with KH2PO4 (Vandenberghe et al. 1999).
On the other hand, its excess presence could favour the
fungal growth only without any accumulation of citric
acid (Grewal and Kalra 1995). Citric acid accumulation
decreased by the addition of iron, which also had some
effect on mycelial growth. Optimum concentrations of
iron for citric acid production are 1.3 ppm (Tomlinson
et al. 1950). Magnesium is required both for growth as
well as for citric acid production. Optimal concentration
of magnesium sulphate for citric acid production was
found in the range of 0.02-0.025% (Kapoor et al. 1982).
Nickel, molybdenum and cobalt are some other trace
metals also reported to affect the citric acid accumula-
tion in A. niger (Habison et al. 1983).
Aeration
Citric acid formation is depending upon the dissolved
oxygen concentration directly. Vandenberghe et al.
(1999) reported that oxygen concentration above 25%
is required for better citric acid production. It has been
observed that the critical dissolved oxygen tension is
9–12% of air saturation for growth phase and 12–13%
of air saturation for the production phase (Grewal and
Kalra 1995). When the organism turns into develop-
ment of filaments, the dissolved oxygen tension rapidly
falls to less than 50% of its previous value, even if the
dry mass has not increased by more than 5%.
Therefore, small compact pellets are the preferred
mycelial forms of A. niger during the citric acid produc-
tion. Low oxygen environment is directly involved in
the growth limitation, which is crucial for citric acid pro-
duction where as strongly aerated cultures (0.3 m3/kg
dry CB/h) increase sporulation hence decrease, the
accumulation of citric acid (Vandenberghe 2000). Hence
the amount of oxygen supplied during citric acid fer-
mentation is a critical factor and variations in the rate
of aeration can have a detrimental effect on yield of cit-
ric acid (Grewal and Kalra 1995). Increase in aeration
rates, may reduce partial pressure of the dissolved car-
bon dioxide in the medium. Carbon dioxide is import-
ant substrate for pyruvate carboxylase that replenishes
the supply of oxaloacetate for citrate synthase.
Sufficient CO2 is produced by the reaction catalysed by
pyruvate decarboxylase, but excessive aeration leads to
some losses. In contrast, increased levels of carbon
CRITICAL REVIEWS IN MICROBIOLOGY 13
dioxide are damaging to the final biomass and concen-
trations of citrate (McIntyre and McNeil 1997). It has
been observed that high partial pressure of CO2 prob-
ably retards spore liberation of the filamentous fungi
and helps in citric acid accumulation. Therefore, envir-
onment with high concentrations of CO2 has a positive
effect on citric acid synthesis (Vandenberghe 2000). On
the other hand, during the growth phase, high aeration
rates create foams inside the medium and antifoaming
agents required which may increase the cost of produc-
tion. It can be avoided through gradual increase in the
aeration rate. Li et al. (2014) recommend that aeration
should be performed evenly across the medium with
similar intensity and any interruptions in aeration may
irreversibly destroy the organism’s stability to synthe-
sise and accumulate citric acid (Kubicek et al. 1980).
Morphology of the fungus
Morphology of fungus during citric acid fermentation is
an important parameter. It has been observed that the
formation of compact aggregates or pellets by the fun-
gus during citric acid fermentation favours more citric
acid production than filamentous form. Agitation rate,
pH of the medium, composition of the medium and
inoculums concentration are the major factors that
affecting the morphology of A. niger in submerged fer-
mentation (Papagianni 2007). Small aggregates of short
filaments form are found at pH values around 2.0 ± 0.2
and associated with an increase citric acid production.
At lower pH (pH 1.6), the aggregated short filamentous
form of the fungus changed to bulbous hyphae and
results very less citric acid production whereas at higher
pH (pH > 3.0) aggregates had longer perimeters and
oxalic acid formation was observed (Papagianni 2007).
Other factors
Lipids, such as groundnut oil (Souza et al. 2014) and
sodium mono-fluoro acetate have also effects on citric
acid production (Meixner-Monori et al. 1984). Lipid can
improve the yield of citric acid with no effect of the dry
weight of mycelium (Millis et al. 1963). Kareem et al.
(2010) reported the effects of calcium fluoride, sodium
fluoride and potassium fluoride on the industrial pro-
duction of citric acid.
Enhanced citric acid production through
metabolic engineering
Increasing global market demand for citric acid has
attracted many researchers since the last century to
14 B. C. BEHERA

Figure 3. Diagrammatic representation of metabolic engineering to enhance citric acid production in A. niger. The round cross
( ) symbolises deletion of the corresponding gene. The genes in italic blue represented the targets required to be enhanced.
The red dashed line represented the feed-back inhibition, i.e., Trehelose-6-Phosphate (TRh6p) inhibited the activity of hexokinase
(Hxk)., OAA oxaloacetate, MAL malate, OA oxalic acid, CIT citric acid, ICIT isocitric acid, KGA, a-ketoglutarate, SUC succinate, FUM
fumarate, GlaA glucoamylase, AgdA alpha-1,4-glucosidase, GoxC glucose oxidase, GgsA trehalose-6-P synthase, Pfk phosphofructo-
kinase, Pyc pyruvate decarboxylase, OahA oxaloacetate acetylhydrolase, Mdh malate dehydrogenase, Fum fumarase, Frds fumarate
reductase, CitA citrate synthase, AOX alternative mitochondrial oxidase.

involve themselves in search and development of
potential strain that can over-producing citric acid
through genomic manipulations as called metabolic
engineering of A. niger. Several hit and trail methods of
metabolic engineering have been adopted to enhance
the citric acid production by modifying the genes and
metabolic pathways which regulates citric acid produc-
tion (Ruijter et al. 1997,1999; Yin et al. 2015). The meta-
bolic engineering techniques adopted for enhancement
of citric acid production are summarized in Figure 3
and explained below.
Engineering for residual substrate
As already discussed A. niger has remained the organ-
ism of choice for commercial production due to its
powerful polymer degrading enzyme systems to hydro-
lyse many polymeric substrates into monomer.
However, when the fermentation process carried out
with a carbon source such as liquefied corn starch, it
has been observed that at the end of the fermentation
some of the residual sugar such as Iso-maltose remains
unutilised (Wang et al. 2016). In day to day large scale
production process this residual sugar raised as a great
loss and affects the production profit. It has been
detected that this residual sugar (Iso-maltose), is syn-
thesised by an enzyme a-glucosidase, during citric acid
fermentation and deletion of the a-glucosidases encod-
ing gene agdA could efficiently reduce the iso-maltose
concentration when using corn starch as the raw car-
bon source (Wang et al. 2016). It is also observed that
deletion of a-glucosidases encoding gene agdA with
over-expression of glucoamylase glaA, could decrease
88.2% of the residual sugar hence increased the citric
acid production 16.9% (Wang et al. 2016).
Precursor supplement pathway engineering
Systems metabolic engineering has been proved to be
an important tool through which an entire new

biosynthetic pathway can be developed and introduced
in A. niger to enhance citric acid production (Tong
et al. 2019).
Acetyl-CoA is a molecule which oxidised and deliv-
ered its acetyl group for citric acid synthesis. Several
enzyme systems responsible for Acetyl-CoA synthesis
includes pyruvate dehydrogenase (PDH), cytosolic
Acetyl-CoA synthetase (ACS) and ATP-citrate lyase (ACL)
(Chen et al. 2014). There are some controversy on the
activity of the enzyme ATP-citrate lyase (ACL). Some
authors explained that the production of Acetyl-CoA by
ACL, consumes citrate, therefore, decrease citric acid
production. During their experiment, Meijer et al. (2009)
found that the deletion of gene acl1 in A. niger which is
responsible for ATP-citrate lyase synthesis, could
increase the citric acid production. In contrast to the
report of Meijer et al. (2009), Chen et al. (2014) showed
that not only citric acid production in A. niger decreased
with deletion of two cytosolic ACL subunits (Acl1 and
Acl2) but also this inhibit conidial germination, vegeta-
tive growth, pigmentation and asexual development.
Another substrate for citric acid synthesis is oxaloace-
tate which is formed by pyruvate carboxylation in the
cytoplasm and subsequently converted into malic acid.
Malic acid is reconverted into oxaloacetate after entering
into mitochondria through a malate–citrate shuttle. Then
after oxaloacetate takes part in citric acid synthesis. de
Jongh and Nielsen (2008) engineered the cytosol reduc-
tive TCA (rTCA) cycle by inserting heterogeneous malate
dehydrogenase (mdh2), fumarase (FumR) and fumarate
reductase (Frds1). Malate dehydrogenase (Mdh2) over
expressing strain could accelerate the citric acid produc-
tion at initial stage. It has been found that over expres-
sion of cytosolic FumR converts mitochondrial fumarate
to malate, provided more substrate to the mitochondrial
malate-citrate antiporter and thereby improved the citric
acid secretion and productivity while over expression of
Frds1, converts fumarate to succinate which is also a
potential substrate for the mitochondrial citric acid anti-
porter. Hence, succinate can be used for the mitochon-
drial antiport of citrate although malate is the preferred
substrate. It has been observed that more citric acid can
be achieved when co-expressing both the gene in a
same strain, which creates a cytosolic pathway from mal-
ate to succinate and can be used to exchange mitochon-
drial citric acid (de Jongh and Nielsen 2008).
The putative methyl transferase encoding LaeA is a
universal regulator of metabolic processes in filament-
ous fungi is required for the expression of a putative
citrate exporter encoding cexA gene, which is critical for
citric acid production. It was found that laeA is required
for citric acid production through the regulation of cexA
CRITICAL REVIEWS IN MICROBIOLOGY 15
and its disruption caused a significant decrease in citric
acid production (Kadooka et al. 2020).
Feedback inhibition engineering
Phosphofructokinase (PFK), an essential enzyme with
magnesium as a cofactor, changes fructose 6-phos-
phate into fructose 1,6-bisphosphate and considered as
a crucial controlling step for glycolysis metabolic flux
via the allosteric inhibition or activation. As discussed
earlier, increased ATP and citric acid synthesis inhibits
expression of the enzyme PFK. Legisa and Mattey
(2007) showed that spontaneous post-translational
modification plays a key role in keeping the high activ-
ity of A. niger PFK1. They observed that the native PFK1
(85 kDa) was cleaved to an inactive fragment (49 kDa)
which could be reactivated again by PKA phosphoryl-
ation. This shorter PFK1fragment is not only found to
be resistant to citrate inhibition but also more suscep-
tible to positive effectors, such as AMP, ammonium ions
and fructose 2,6-bisphosphate, which suppresses the
ATP inhibition. It has been found that A. niger strain
with an active shorter PFK1 fragment mtpfkA10 with
T89D single site mutation (to elude the phosphorylation
requirement) exhibited 70% more citric acid production
than the control strain (Capuder et al. 2009).
It has been reported that instead of citric, oxalic acid
is produced in the fermentation medium by A. niger
when the pH of the medium is 3.0. or above 3.0 (Ruijter
et al. 1999). The enzyme oxaloacetate acetylhydrolase
(OAH) is responsible for oxalic acid production in A.
niger. Ruijter et al. (1999) reported that an A. niger
mutant, strain lacking of both glucose oxidase (goxC)
and OAH (prtF) could efficiently produce citric acid in
the medium of pH 5 and under these conditions pro-
duction was completely insensitive to Mn2þþ.
Hexokinase is the enzyme which convert glucose to
glucose-6-phosphate and fructose-6-phosphate. The
increase in sugar uptake results in an increase in the
concentration of glucose 6-phosphate and conse-
quently also trehalose 6-phosphate, capable of inhibit-
ing hexokinase and citric acid accumulation (Arisan-atac
et al. 1996). However, deletion of the gene GgsA, which
encodes Trehelose-6-phosphate synthase, decreased
the level of Trehelose-6-phosphate and citric acid accu-
mulation was initiated earlier (Arisan-atac et al. 1996).
Respiratory chain regulation
Alternative oxidase (AOX) or cyanide-resistant terminal
oxidase (EC 1.10.3.11) located on the inner mitochon-
drial membrane is a component of the cyanide-resistant
16 B. C. BEHERA
respiration (CRR) pathway (an alternative to the cyto-
chrome dependent respiration pathway) (Siedow and
Umbach 2000). It performs a significant role in the re-
oxidation of NADH (Sakajo et al. 1991). Fermentation
environments with vigorous aeration and agitation are
an absolute necessity for A. niger to accumulate citric
acid and oxidative stress resulting from the frequent
use of enriched oxygen as fermentation gas leads to
extensive damage. In fungi, AOX was reported to be
involved in reducing the damage that results from oxi-
dative stress or minimise the toxic effects caused by
reactive oxygen species (ROS) (Angelova et al. 2005). In
the process of citric acid production, the mitochondrial
electron transport chain, which transfers electrons from
NADH to molecular oxygen and in combination with
NADH dehydrogenases, AOX can re-oxidise NADH with
less concomitant ATP synthesis through CRR pathway
(Li et al. 2009). In the process of CRR pathway, ubiquin-
one oxidoreductase (complex I), oxidises NADH to
NAD þ and reduces ubiquinone to ubiquinol by translo-
cating four protons. These fewer protons move across
the inner mitochondrial membrane to generate a pro-
ton gradient, and thus provides less ATP by oxidative
phosphorylation (Figure 3). Electrons transfers directly
take place from the ubiquinone pool to oxygen, bypass-
ing complex III and IV as like normal electron transport
chain. Thus, it supports the need of A. niger for citric
acid accumulation by producing less ATP. Because, in
the conversion of glucose into citric acid, one molecule
of ATP and three molecules of NADH generated. In
cytochrome-dependent respiration the excess ATP gen-
erated due to the NADH oxidisation inhibited the
enzyme, PFK and impaired the glycolysis flux and citric
acid accumulation. Hou et al. (2018) modified A. niger
strain CGMCC10142 by deleting and over expressing
the mitochondrial AOX gene aoxl, and uncovered that
the over expression of aox1 gene improve citric acid
production and CRR respiration was detected in mycelia
of A. niger under citric acid production conditions. The
presence of AOX thus alleviates the repression of PFK
due to excess ATP and relieving the damage caused by
ROS as well (Campos et al. 2015). When citric acid
begins to accumulate, cytochrome-dependent respir-
ation is thus replaced by the alternative route, CRR
pathway which enables the NADH oxidisation without
concomitant ATP production (Papagianni 2007).
Engineering mycelia morphology
During submerged fermentation, mycelial morphology
of A. niger plays an important role in citric acid produc-
tion. RNA interference through the silencing of chitin
synthase gene (chsC) was investigated by Sun et al.
(2018). The mutant strain chsC  3 of A. niger produced
after silencing of chsC gene, could produce tiny and
dense mycelial pellets, which reduced the viscosity of
the culture medium, leading to increased oxygen mass
transfer and thereby showed 42.6% more citric acid
production in comparison to the original strain (Sun
et al. 2018).
Similarly, trace heavy metal such as Mn2þ deficiency
plays a crucial role in A. niger metabolism by preventing
DNA, protein and lipid synthesis, inhibiting citrate reutil-
ization, enhancing protein degradation and there by
intracellular NH4þ concentration, altering fatty acid in
the plasma membrane, modifying the polysaccharide
concentration of the cell wall and influencing morph-
ology (Papagianni 2007) which directly affects the citric
acid accumulation (Tong et al. 2019). The gene which is
involved in the regulation of morphology formation of A.
niger in response to Mn2þ is Brsa-25 gene. Dai et al.
(2004) showed that a 10% increase in citric acid produc-
tion can be achieved by down-regulation of the Brsa-25
gene expression through antisense RNA. This down-regu-
lation mechanism facilitated pelleted growth and
enhanced citric acid production in the presence of Mn2þ.
Enhancement of citric acid production through
CRISPR technology
Genetic engineering of the metabolic pathways of A.
niger through genome-editing system alters the native
genome in a very precise manner to change the physio-
logical characters and trigger the biosynthesis of
secreted proteins and secondary compounds which
could provide new fungal strains with enhanced pro-
duction of a particular metabolite (Ulaganathan et al.
2017). By using this approach, a gene can either be
introduced, deleted, up or down regulated at a specific
site within an organism. Clustered Regularly
Interspaced Short Palindromic Repeats/CRISPR associ-
ated protein (CRISPR/Cas) systems have become a very
powerful genome editing technique (Jinek et al. 2014;
Hsu et al. 2014). This high-throughput CRISPR/Cas9 sys-
tem not only helps in A. niger chromosome designing
but also facilitates the manipulations of A. niger gen-
ome by cleaving at the target site. The cleaved DSB at
the target region is then repaired by the non-homolo-
gous end joining (NHEJ) pathway, resulting in gene dis-
ruption with base-pair insertions or deletions (indels). In
the NHEJ pathway both ends of the broken DNA mol-
ecule are captured by a Ku70-Ku80 heterodimer,
brought together and re-ligated by a complex of pro-
teins. In this case the original sequence is either

Figure 4. Schematic representation of CRISPR/CAS9 technology.
restored or small indels and/or nucleotide substitutions
are introduced (Chang et al. 2017). Alternatively, if a
double stranded DNA fragment (containing the desired
alteration) with homology to the targeted locus is sup-
plied into a cell, the DSB may be repaired by the hom-
ology-directed repair (HDR) pathway allowing for
precise replacement or precise genome editing with
appropriate donor DNA (Figure 4). Using this strategy,
gene disruptions have been generated in filamentous
fungi A. niger in a high throughput manner, thereby
enhanced the efficiency and speed of systems meta-
bolic engineering (Tong et al. 2019).
The orotidine-50 -decarboxylase gene pyrG is an essen-
tial enzyme involved in uridine biosynthesis (van
Hartingsveldt et al. 1987). It has been reported that gene
disruption and down-regulation of pyrG significantly
increases of industrial citric acid production during sub-
merged fermentation (Zhang et al. 2020). Zhang et al.
(2020) disrupted both pyrG and kusA in citric acid produc-
ing isolates WT-D and D353, by a highly efficient CRISPR/
Cas9 system based on 5S rRNA as a promoter. They chose
the traditional selection marker pyrG gene and key gene
kusA involved in NHEJ system as targets (Zheng et al.
2019). To simultaneously disrupt these two targets, two
double strand breaks were introduced at respective loci
using Cas9 with corresponding guides and then were
repaired by homologous recombination system (Figure 5).
CRITICAL REVIEWS IN MICROBIOLOGY 17
They observed that pyrG disruption dramatically disturbed
the intracellular central metabolism and improved the
intracellular level of the citric acid and its precursor such
as Acetyl-CoA and oxaloacetate, which may lead to the
extracellular citric acid accumulation. In another study,
Zhang et al. (2019) reported that a-Glucosidase is a by-
product of glucoamylase fermentation and its expression
will reduce the activity and yield in the glucoamylase pro-
duction in A. niger hence may affect the final citric acid
yield. So, disruption and replacement of agdF by glucoa-
mylase gene using the modified CRISPR/Cas9 system
increase the production of glucoamylase 25.9% higher
than wild strain and can increase the citric acid produc-
tion than the wild strain (Figure 6) (Zhang et al. 2019).
Conclusion
This review discussed in details about the biochemistry,
biotechnology and metabolic engineering aspects for
production of citric acid and its enhancement process.
Taking all these aspects into consideration, it becomes
evident that the cost-effective industrial citric acid pro-
duction process is highly complex, sensitive and depend
upon several parameter like choices of microorganism,
raw materials used, types of fermentation technique
employed, designing of appropriate bioreactors with
precise control over process parameters, biochemical
18 B. C. BEHERA

Figure 5. Simultaneous inactivation of pyrG and kusA in A. niger D and D353. a Schematic diagram of simultaneous disrupted muta-
genesis of both pyrG and kusA mediated by integrating the donor DNA with 40-bp micro-homology arms via CRISPR/Cas9 system.
The donor DNAs of MHi-pyrG1-hph and MHi-kusA-hph were co-transformed with linear sgRNA constructs (sgRNA-pyrG1 and sgRNA-
kusA) and Cas9 expression plasmid pCas9-hph into the protoplasts of A. niger D and D353. Two DSBs were generated by the Cas9
under the guide of sgRNA, and then were repaired by HR with the integration of donor DNAs (With prior permission of Zhang
et al. 2020).

Acknowledgement
The author is grateful to the faculty and staff of School of
Biological Sciences, NISER, Bhubaneswar for their help and
cooperation.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 6. Schematic for replacement of agdF by glaA expression
cassette mediated by CRISPR/Cas9 system (Zhang et al. 2019).
pathways, factors affecting citric acid production, quanti-
fication techniques, recovery techniques, strain
Improvement etc. Although adoption of advanced tech-
nology and metabolic engineering helped to overcome
some critical parameter raised during fermentation still
some of them are controversial and significant research
efforts are needed to establish.
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