3.

13 Citric Acid
Kohtaro Kirimura and Isato Yoshioka, Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University,
Tokyo, Japan
© 2019 Elsevier B.V. All rights reserved.
This is an update of K. Kirimura, Y. Honda, T. Hattori, 3.13 - Citric Acid, Editor: Murray Moo-Young, Comprehensive Biotechnology (Second Edition),
Academic Press, 2011, Pages 135-142.

3.13.1 Introduction and Scope 158

3.13.2 Properties and Applications of Citric Acid 158
3.13.3 Historical Background of Citric Acid Production 159
3.13.4 Microorganisms and Biosynthesis of Citric Acid 160
3.13.5 Factors Affecting Citric Acid Production by A. niger 160
3.13.6 Fermentation Processes for Citric Acid Production 162
3.13.6.1 Submerged Fermentation Process 162
3.13.6.2 Surface Fermentation Process 162
3.13.6.3 Solid Fermentation Process (Koji Process) 162
3.13.7 Product Recovery 163
3.13.8 Synthetic Biology and Genome Breeding 164
3.13.9 Perspectives for Future 164
References 164

Glossary
Parasexual cycle Despite the absence of meiosis during the life cycle of asexual fungi, recombination and genetic variation can
occur through the parasexual cycle by a mechanism called parasexuality. Different individuals are capable of undergoing cell
fusion through hyphal or protoplast fusion with each other to form a vegetative heterokaryon, where genetically different
nuclei coexist in the common cytoplasm. Genetic exchange is possible at the stage of segregation of stable or transient diploids
derived from the heterokaryons obtained through the parasexual cycle, in spite of usual sexual cycle

3.13.1 Introduction and Scope

Citric acid, 2-hydroxypropane-1,2,3-tricarboxylic acid, is a key metabolic intermediate and is the starting point of the tricarboxylic
acid (TCA) cycle. From the viewpoints of production volume and utility, citric acid is one of the most important bioproducts. The
annual worldwide output has reached 2.1 million ton at 2016 and increased steadily; 0.9 and 1.7 million tons in 2000 and 2010,
respectively. Various plants, especially citrus fruits such as lemon and orange, contain large quantities of citric acid, and citric acid is
ubiquitous in nature because it is an intermediate in aerobic metabolism through the TCA cycle whereby carbohydrates are oxidized
to carbon dioxide. The widespread presence of citric acid in the animal and plant kingdoms is an assurance of its nontoxic nature,
and nowadays citric acid is accepted as generally recognized as safe (GRAS) by the Joint FAO/WHO Expert Committee on Food
Additives. Citric acid and its salt form (citrate) are commodity chemicals and are used in many industrial fields. It has long
been used as an acidulant in the manufacture of soft drinks and foods, as an aid to the setting of jams and in other ways in the
confectionery industry because of its general recognition as safe, pleasant acid taste, and high water solubility. Citric acid has
been also used as a complexing agent in metal treatment, as a monomer for functional and/or biodegradable polymers, and as
a water softener in detergents, because of its organic acid, chelating, and buffering properties.
Today, citric acid is industrially produced by fermentation, and the filamentous fungus Aspergillus niger is exclusively used due to
its high citric acid productivity at low pH without the secretion of toxic by-products. In this chapter, we provide a current article of
the applications of citric acid in the industrial fields and citric acid production by A. niger. Some contents of this chapter are partly
related to the chapter on Gluconic and Itaconic Acids.

3.13.2 Properties and Applications of Citric Acid

Citric acid as a product is sold either in the anhydrous form or as the monohydrate. The transition temperature between the two
forms is 36.6
C. The pKa-values of citric acid, its three dissociation steps, are 3.13, 4.78, and 6.43 (25
C). A 1% solution of citric
acid in pure water has a pH of 2.2. With its three carboxyl and one hydroxyl group, citric acid functions as an excellent complexing
agent for di- and trivalent cations. The equilibrium constants are K1 ¼ 8.2  104, K2 ¼ 1.8  105 and K3 ¼ 1.0  107 (18
C).

158 Comprehensive Biotechnology, 3rd edition, Volume 3 https://doi.org/10.1016/B978-0-444-64046-8.00157-9

 Citric Acid 159

Demands of citric acid worldwide are divided among the principal fields of use approximately as follows: food, confectionery
and beverages (75%); pharmaceutical (10%) and industrial (15%). In food, confectionery and beverages, citric acid is the most
versatile and widely used food acidulant. The use of citric acid as a food acidulent depends in part on its strength as an acid. Its
pleasant taste and its property of enhancing existing flavors have ensured its dominant position in this market. In addition, citric
acid is also used as a preservative.
Citrate is able to complex heavy metals such as iron and copper. This property has led to its increasing use as a stabilizer of oils
and fats where it greatly reduces oxidation catalyzed by these metals. The ability to complex metals combined with its low degree of
attack on special steels allows the use of solutions of citrate in the cleaning of power station boilers and similar installations. Because
citrate is readily able to complex magnesium and calcium ions, trisodium citrate as the water softener agent is widely used in
commercial detergents and cleaners.
Citrate is often used as the anion in the fields of cosmetics, toiletries, and pharmaceuticals; and as the active agent as well as the
stabilizing agent in soluble drug preparations and in preparations employing basic substances. Citrate forms a wide range of
metallic salts, many of which are chemicals of commerce. For example, trisodium citrate is widely used as a blood preservative,
where it prevents clotting by complexing calcium. Ferric ammonium citrate is still used in the treatment of anemia although other
iron salts are increasingly preferred; it is also employed as an aid to emulsification in the manufacture of processed foodstuffs, for
example, cheese. Mixtures of citric acid and its salts have good buffering capacity and are extensively used for this purpose in the
pharmaceutical, toiletry and food industries. Citric acid shows bacteriocidal and bacteriostatic effects, and solutions containing cit-
ric acid are also used as sterilizing agents.
Citrate esters of a wide range of alcohols are known. In particular the triethyl, tributyl and acetyltributyl esters are employed as
non-toxic plasticizers in plastic films used to protect foodstuffs. Monostearyl citrate can be used instead of citric acid as an antiox-
idant in oils and fats. In addition, some polymers containing citrate have been developed and are expected to find use as new bio-
based polymers having biodegradable properties.

3.13.3 Historical Background of Citric Acid Production

The history of citric acid production has been well documented in several useful and comprehensive reviews.1–4
Citric acid was first isolated in 1784 from lemon juice by Scheele, who also established its composition. In 1934, 150 years later,
Krebs discovered that citric acid is a central compound in aerobic metabolism through the TCA cycle. It was first produced commer-
cially by John and Edmund Sturge in England in increasing amounts from 1826 onward and was made from calcium citrate
produced in Italy, where it was derived from lemon juice; this method of manufacture was established in the USA, France, and Ger-
many well before the end of the 19th century. In 1880, the trial for chemical synthesis of citric acid from glycerol was carried out,
following which a number of synthetic methods from other raw materials by different routes were published. However, all these
synthetic methods proved to be unsuitable because of expensive or hazardous raw materials, or an excessive number of reaction
steps leading to low yields of citric acid.
Fermentative production of citric acid was first observed by Wehmer (1893), who discovered that certain species of Penicillium
(Citromyces as termed by Wehmer) were able to accumulate significant quantities of citric acid through cultivation on the media
containing carbohydrates. Although Wehmer attempted to use this finding to establish a commercial citric acid production process
and to run a plant, he was unsuccessful probably due to inevitable contaminations and long duration of fermentation. However, J.
N. Currie (1917) found that several strains of A. niger produced higher amounts of citric acid. His important findings are that A. niger
producing citric acid grows well at pH values around 2.5–3.5, and that conditions with high concentrations of carbohydrates are
suitable for citric acid production. Currie subsequently joined Chas. Pfizer & Co., Inc (USA) with the result that a citric acid plant
using the new method opened in the USA in 1923. Subsequently, industrial scale production using the surface fermentation process
was started in England, Belgium, French, Czechoslovakia, the Soviet Union, and Germany. All these plants used the process in which
A. niger was grown on static medium held in trays housed in ventilated rooms. At first, only media prepared from sucrose and inor-
ganic salts were employed, but soon processes based on the cheaper beet molasses were introduced.
Following World War II, submerged fermentation processes for the production of citric acid using A. niger and media based on
either purified glucose syrups or beet or cane molasses were developed. In 1951, Miles Laboratories, Inc. started industrial scale
production in the USA, using submerged fermentation processes. Since trace elements such as metal ions strongly affect citric
acid production by submerged fermentation process, it was necessary to use strains with low sensitivity to trace elements or to treat
and purify raw materials such as molasses for the removal of trace metals. Today, the submerged fermentation process in batch
system in large-scale tanks is widely used in the USA, Europe, Brazil, and China, and with a yield of 50%–80%, by this fermentation
process with sugar cane or beet molasses, starch and starch hydrolysate as carbon sources.
Solid fermentation process, also known as ‘Koji process’, was first developed in Japan, around the 1950s. In this fermentation
process, the substrate is solid with adequate moisture and acts as both carrier (physical support) and source of nutrients. There are
some similarities between solid and surface fermentations of citric acid, and the presence of trace elements may not affect citric acid
production by solid fermentation so strongly as it does in submerged fermentation process. Agro-industrial residues such as wastes
of sweet potato, cassava, and maize-containing starch as carbon sources have been used for practical production of citric acid. Until
today, in Asian countries such as China and Japan, small-scale plants still use the solid fermentation process for the production of
citric acid by A. niger.
160 Citric Acid

On the other hand, in the 1960s, Tabuchi et al. reported that eight genera of yeasts including Candida lipolytica (at present
reclassified as Yarrowia lipolytica) produce citric acid from glucose and molasses.5 Moreover, they reported citric acid production
from n-paraffin and discovered the methylcitric acid cycle as a novel metabolic pathway.6 Since that time, many researchers
examined the possibility of industrial scale production of citric acid by yeasts. However, nowadays industrial strains have
been limited to A. niger.
Recent interest about industrial citric acid production by A. niger has been focused on the possibility of using agro-industrial
residues as substrates. Since the market price of citric acid is cheap, it is necessary to seek newer and cheaper “raw materials” usable
as substrates.

3.13.4 Microorganisms and Biosynthesis of Citric Acid

A large number of microorganisms containing filamentous fungi, yeasts, and bacteria have been known to accumulate citric acid
and have been tested for citric acid production.2,3 Among them, A. niger has been recognized as the best producer; the main advan-
tages of the use of A. niger are high-yield production and ease of handling.2,3
For industrial scale production by A. niger, some selected strains with enhanced productivity have been used. Improvement of
citric acid-producing strains has been performed by the conventional method: mutagenesis and selection. Random mutations and
selections according to some working hypothesis have been available for strain improvement.2–4,7–9 The most employed method
has been ‘induced mutations’ in parental strains using mutagens such as UV irradiation, gamma irradiation, and chemical muta-
gens. For selection of mutant strains, the techniques of single-colony (single-spore) isolation and stable long-term maintenance
of isolated strains are the principal ones that are generally employed.2,3
Large quantities of citric acid are secreted by A. niger during and after the late logarithmic growth phase, provided that there is
an excess of carbon source (carbohydrate) and oxygen. The metabolic pathway in relation to citric acid production is shown in
Fig. 1. The yield of citric acid in the fermentation is expressed as kilograms of citric acid monohydrate per 100 kg of carbohy-
drate supplied. It should be noted that the theoretical yields of citric acid monohydrate from sucrose and glucose are 123% and
117%, respectively, based on the assumption that no carbon is diverted to mycelia (biomass), carbon dioxide, or other
byproducts.
The biochemical and physiological mechanisms of citric acid production by A. niger have attracted both biological and biotech-
nological interests, and studies have been performed by many research groups.2–4,7 Roles and metabolic controls of the glycolytic
pathway (Embden–Meyerhof–Parnas pathway) and TCA cycle in relation to the glycolytic flux have been intensively discussed,2–
4,7,9–14
and properties of key enzymes affecting citric acid biosynthesis have been revealed (see Refs. 2–4, 11, 12). Moreover, recent
interests have also focused on the machinery of citrate transport4,12,14,15 as shown in Fig. 2 and metabolic engineering of citric acid-
producing A. niger.4,12–15 In our study, no decrease of citric acid production was observed when ctpA, encoding mitochondrial
malate-citrate shuttle protein, was disrupted.15 Thus, the other organic acids-transport protein(s), but not the CTPA protein,
must have an important role for citric acid production by A.niger.
Hybridization, namely genome shuffling, by protoplast fusion of citric acid-producing strains through parasexual cycle has been
already available for strain improvement.16–18 For example, by intraspecific protoplast fusion between two different citric acid-
producing strains of A. niger, diploid strains16,17 and haploid hybrid (recombinant) strains17 with increased citric acid production
have been generated. Moreover, the hybrid strains have also been generated by interspecific and intergeneric protoplast fusion.18
On the other hand, various genes encoding key enzymes in citric acid production by A. niger have been cloned and functionally
characterized10,13,19–25; [see Refs. 4, 11, 12]. Based on the molecular genetics researches, molecular breeding of citric acid-producing
strains has been examined by several research groups. Although some trials for strain improvement by genetic engineering have
been performed and continued, the genetic engineered strains with much improved properties have not been obtained.4,11,12,14
Some fundamental understanding of the citric acid fermentation might be necessary for breeding an epoch-making citric acid-
producing strain.

3.13.5 Factors Affecting Citric Acid Production by A. niger

Citric acid production by A. niger is strongly affected by the compositions of media and cultivation conditions. Types and concen-
trations of carbon, nitrogen, and phosphorus sources are important factors affecting citric acid production, as well as trace elements,
pH, temperature, oxygen supply, and additives as promoting substances.2–4,7,11,12 In brief, for high-yield production of citric acid,
the following conditions are favorable: excess amounts of readily metabolized carbon sources (carbohydrates) with low concentra-
tions of growth-promoting elements such as nitrogen, phosphorus, and trace elements containing heavy metal ions.2–4,7,11,12
Among the carbon sources, sucrose and glucose are readily metabolized and favorable carbon sources for high-yield production
of citric acid.2–4,7,11,12 Various carbohydrates containing cellobiose,8 cellulose hydrolysate,26 and xylan27 are usable for citric acid
production by A. niger, but use of galactose and lactose gives low yields. Citric acid production is influenced by the nature and
concentration of nitrogen source. Physiologically, ammonium salts, but not nitrate or nitrite salts, are preferred.2–4,7,9,11,12 Acid
 Citric Acid 161

Figure 1 Citric acid production in relation to glycolytic pathway (Embden–Meyerhof–Parnas pathway) and tricarboxylic acid (TCA) cycle. Dotted
arrows indicate electron transfer. Cyt., cytochrome; Frc-1,6-P2, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; UQ, ubiquinone. CS,
citrate synthase (EC 2.3.3.1); GAPDH, glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); ICDH, isocitrate dehydrogenase (NADþ, EC
1.1.1.41; NADPþ, EC 1.1.1.42); PC, pyruvate carboxylase (EC 6.4.1.1); PDH, pyruvate dehydrogenase (EC 1.2.4.1).

Figure 2 Proposed scheme for machinery of citrate transport in Aspergillus niger. CA-TP, citrate transport protein in plasma membrane (unidenti-
fied); CTP, mitochondrial citrate transport protein (unidentified); X and Y, postulated counter compounds such as organic acids (unidentified).

ammonium compounds such as ammonium sulfate and ammonium nitrate are preferred because their consumption leads to pH
decrease, which is favorable for high-yield production of citric acid.2–4,7,11,12 Moreover, in the mycelia of citric acid-producing
strains, higher amounts of ammonium ion are detected, probably due to imperfect metabolism of carbon and/or nitrogen sour-
ces.2,3,7,9,11 The concentrations of nitrogen sources are usually 0.1–0.4 g L1 of medium, and higher concentrations of nitrogen
sources lead to excess growth and consumption of carbon source and decrease the amount of citric acid produced.2–4,7,9,11 It is note-
worthy that the balance of carbon per nitrogen sources (C/N ratio) is also an important factor.2,3,7,9,11
On the other hand, low levels of phosphate have a positive effect on citric acid production because excess amounts of phos-
phorus readily lead to excess growth and formation of by-products such as oxalic acid.2–4,7,11,12,25 Addition of lower alcohols,
162 Citric Acid

Table 1 Industrial fermentation processes for citric acid production

Process Substrate Concentration of initial sugar (%(w/v)) Cultivation period (days) Yield (%) Running cost

Submerged fermentation Molasses 10–20 7–14 55–80 High

Starch hydrolysate 12–16 5–10 50–80 High
Surface fermentation Molasses 10–20 5–14 60–65 Low
Solid fermentation Starch residue 35–40 4–5 50–60 Low

especially methanol, at concentrations of 2%–4% (v/v) or 2%–5% (v/w), increases the amount of citric acid produced.2,3,7 Much
oxygen supply is necessary for citric acid production, and respirations and functions of respiratory systems affect citric acid produc-
tion.2–4,7,10–12,19,22 Besides the cytochrome pathway, the cyanide-insensitive alternative respiration for reoxidation of NADH is
functioning in the mitochondria of citric acid–producing A. niger.2–4,10–12,19,22

3.13.6 Fermentation Processes for Citric Acid Production

Citric acid fermentation by A. niger is the most economical and exclusively used method for the production of citric acid,1–4,7,11,12
with the use of three different processes: submerged, surface, and solid fermentations including semi-solid fermentation.2,3,7 For
reference, typical data for fermentations using raw materials are shown in Table 1. Since different methods of fermentation can
lead to different yields of citric acid production by the same strain, it should be noted that one strain that gives high yields in
the submerged fermentation is not necessarily a good producer in either the surface or solid fermentation processes. On the other
hand, possible organic acids as by-products in the fermentation processes are oxalic and gluconic acids,2–4,7,11,12,25 resulting from
inadequate cultivation conditions: inadequate compositions of cultivation media, harmful effects of trace elements, uncontrolled
pH, and physical stress to mycelia.

3.13.6.1 Submerged Fermentation Process

The submerged fermentation process is widely used for citric acid production.1–4,7,11,12,14 This process is employed in large scale
and requires more sophisticated facilities and precise control. On the other hand, it provides several advantages such as higher
productivity and yields, lower labor costs, and lower contamination risk. Generally, submerged fermentations can be performed
in batch, fed-batch, or continuous systems, and for citric acid production, the batch system is most frequently used. Citric acid
production is carried out at 28–30
C with the cultivation media containing 100–150 g L1 carbon sources such as sucrose and
glucose (starch hydrolysate) and it lasts 5–14 days, depending on the process conditions. Generally, the yields of citric acid based
on supplied carbohydrates (usable carbon sources) are 50%–60% under the conditions with vigorous growth using conidia as
starting inoculation and 80%–90% under the conditions with limited growth using pregrown mycelia as seed culture,
respectively.
In the submerged fermentation process as well as shake flask cultivation, the viscosity of culture broth increases with fermenta-
tion time, due to the production of amylose-like polysaccharides by A. niger.7,28 Addition of viscous substances to the cultivation
media at the starting point and/or reduction of physical stress on mycelia is effective for decrease of production of amylose-like
polysaccharides.28

3.13.6.2 Surface Fermentation Process

The surface fermentation (shallow pan) process is the traditional method for citric acid production.1–3,7 However, industrial
production by this process is still being carried out at a small scale in some areas of Europe because the operation is low cost, simple,
and requires only small-scale facilities.
This process is performed as follows: the acid-resistant trays in a sterile compartment are filled with the medium containing sugar
(beet molasses) and inoculated with spores of A. niger. Under stationary conditions but with a high degree of aeration, a mycelial
mat is formed on the surface of the medium. After the fermentation period ends, the contents of the tray are separated into crude
fermentation fluid and mycelial mats. The latter is washed and extracted with hot water for recovery of citric acid in the intracellular
fraction. Citric acid production is carried out at 28–30
C with the cultivation media containing 100–200 g L1 of carbon sources
such as beet molasses in 8–14 days, and yields of citric acid based on supplied carbohydrates are 50%–60%.

3.13.6.3 Solid Fermentation Process (Koji Process)

The solid fermentation (or solid-state fermentation) process is the simplest method for citric acid production.2,3,7,8,26,27 Solid fermen-
tation is performed under conditions with a low water activity using insoluble materials that act as both physical support and source of
nutrients, for example, wastes of sweet potato, cassava, and maize. One of the advantages of the solid fermentation process is that agro-
industrial residues with moistures, probably 40%–75%, are usable for this process. The semisolid fermentation process is a variant of
 Citric Acid 163

the solid fermentation process and is performed using a combination of the insoluble carrier and liquid nutrients.7,8,26,27 The carrier
acts as physical (solid) support adsorbing liquid nutrients as substrates. Bagasse impregnated with cane molasses and ground corncobs
impregnated with starch hydrolysate syrup were used for the industrial semi-solid fermentation process.
For the solid fermentation process, substrates are adequately moistened by the addition of water, to approximately 70% mois-
ture, and the pH of liquid nutrients is adjusted to 4.0–6.0. Suspension of spores is dispersed and mixed with solid media containing
10%–20%(w/w) carbon sources, and citric acid production is carried out under stationary conditions at 28–30
C for 3–5 days.
After the fermentation period ends, hot water is added to the total cultures followed by filtration on cloth to recover the filtrates
containing citric acid. Yields of citric acid based on supplied carbon sources are 50%–60%. The advantages of this process are
that fermentation periods are relatively short, and that the yields are high. However, one of the major disadvantages of this process
is that the wastes and residues are formed in large quantities after the fermentations.

3.13.7 Product Recovery

The recovery of citric acid from the broth or crude fermentation fluid is generally performed through three procedures: precipitation,
solvent extraction, and separation by ion-exchange chromatography. Industrially, the recovery processes using precipitation and
solvent extraction are used.2,3 For both of these methods, it is necessary to remove beforehand mycelial debris and insoluble resi-
dues from the fluid by either filtration or centrifugation. One of the problems in citric acid production by fermentation continues to
be the recovery process, especially the separation and purification steps. In addition, the separation by the ion-exchange chroma-
tography, the method of adsorption using ion-exchange resins, for recovery of citric acid produced has been studied, and its indus-
trial application is at a test stage.
The method of precipitation is the classical one, and its scheme is shown in Fig. 3. It is most frequently used and applicable to all
types of fermentation processes. It is performed by addition of calcium oxide hydrate (lime). The acid is transformed into tricalcium

Figure 3 Schematic flow of citric acid production and recovery. In this figure, citric acid production from molasses and the precipitation method for
recovery of citric acid are shown.
164 Citric Acid

citrate tetrahydrate, which is slightly soluble. The precipitate is recovered by filtration, treated with sulfuric acid forming calcium
sulfate (gypsum), which is filtered off. The mother liquor of citric acid solution is treated with active carbon (activated charcoal)
and/or passed through cation and anion exchange resins to allow for crystallization of pure citric acid. Finally, the liquor is concen-
trated in vacuum crystallizers at 20–25
C, forming citric acid monohydrate. The precipitate of anhydrous citric acid is obtained at
crystallization temperature below 36.5
C. The disadvantage of this method is formation of gypsum in large amounts, resulting in
high costs for wastewater treatment.
The method of solvent extraction is another alternative for purification and crystallization of citric acid from the crude fermen-
tation fluid, and it requires a fermentation fluid with low impurities. Generally, it consists of sequestering citric acid in the broth or
crude fermentation fluid with trilauryl amine and extracting the complex with a mixture of alkanes and 1-octanol. This method has
the advantage of avoiding the use of calcium hydroxide and sulfuric acid, which are used in large amounts, and the formation of
gypsum as an unnecessary by-product. Another advantage is that solvents and sequestering agents (trilauryl amine, alkanes, and 1-
octanol) can be recovered in this process.

3.13.8 Synthetic Biology and Genome Breeding

The complete genome sequences of A. niger and comparative genomics studies have been reported,29,30 thus making the organism
susceptible to targeted improvement by genetic engineering and metabolic engineering. Molecular breeding of citric acid producing
strains, in other words, generation of the strains with desired properties, has been examined in several research groups. The homol-
ogous transformation system with high frequencies has been developed for gene disruption and targeted gene replacement of citric
acid-producing A. niger.31 Moreover, genome editing system with CRSPR/Cas9 has also been developed for citric acid-producing
A. niger (Yoshioka et al., unpublished results). Such gene knock-out and knock-in systems are very useful for genomic approaches
and genome manipulations in understanding the mechanism and machinery of citric acid production.
To our knowledge, at present there is no experimental report describing synthetic biology of filamentous fungi containing citric
acid–producing A. niger. However, genome breeding of citric acid–producing A. niger by genome editing and synthetic biology
might become one of the strategies for strain improvement.

3.13.9 Perspectives for Future

As descried in a previous chapter,32 the demand of citric acid has been increasing, and there is an urgent need for cost-effective and
environmentally sustainable production technology for citric acid manufacture. Therefore, from the viewpoints of bioeconomy as
well as green (white) biotechnology, it is important to develop a novel bioprocess for citric acid production with higher yields and
less impact on the environment. Many studies have been performed on citric acid production by A. niger, and new facts come to
light. On the other hands, it is certain that there is still plenty of room for improvement of the industrial production process, espe-
cially in the fermentation process and the product recovery steps. Moreover, it remains still unclear why citric acid, an intermediate
in the TCA cycle, is overproduced; there are still many problems to solve (Refs. 2–4, 11, 12, 14, 32). In our opinion, metabolic engi-
neering by genome editing and bio-imaging analysis are powerful techniques for fundamental studies of citric acid production and
should be useful for establishing a fundamental understanding of the citric acid fermentation. Moreover, based on the fundamental
understanding, unlocking the biochemical networks and the regulatory mechanisms in relation to the machinery of citric acid trans-
fer may provide an opportunity for improvement of extant industrial strains and of the process for citric acid production.

See Also: 3.14 Gluconic and Itaconic Acids.

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