2023 - Citric Acid - Properties, Microbial Production, and Applications

molecules
Review
Citric Acid: Properties, Microbial Production, and Applications
in Industries
Ewelina Ksiażek
˛
Department of Agroenginieering and Quality Analysis, Faculty of Production Engineering, Wroclaw University
of Economics and Business, Komandorska 118–120, 53-345 Wrocław, Poland; ewelina.ksiazek@ue.wroc.pl;
Tel.: +48-697-377-695 or +48-71-36-80-289
Abstract: Citric acid finds broad applications in various industrial sectors, such as the pharmaceutical,
food, chemical, and cosmetic industries. The bioproduction of citric acid uses various microorganisms,
but the most commonly employed ones are filamentous fungi such as Aspergillus niger and yeast
Yarrowia lipolytica. This article presents a literature review on the properties of citric acid, the
microorganisms and substrates used, different fermentation techniques, its industrial utilization, and
the global citric acid market. This review emphasizes that there is still much to explore, both in terms
of production process techniques and emerging new applications of citric acid.
Keywords: citric acid; food additives; Aspergillus niger; Yarrowia lipolitica; biosynthesis; citric acid market
1. Introduction
Citric acid (CA), also known as 2-hydroxypropane-1,2,3-tricarboxylic acid, is found
in plant and animal tissues such as blood, bone, and muscle. For living organisms, citric
acid is one of the essential carboxylic acids in the Krebs cycle, a series of reactions that
oxidize glucose into carbon dioxide and water, releasing energy. Due to its harmless nature
and chelating and sequestering properties for metal ions, citric acid has applications in the
food, pharmaceutical, chemical, and even metallurgical industries [1,2]. The annual global
Citation: Ksia˛żek, E. Citric Acid: production of citric acid currently reaches approximately 2.8 million tons, and the citric
Properties, Microbial Production, and acid market is one of the fastest-growing segments in the food additive industry [3]. The
Applications in Industries. Molecules continuous growth in citric acid production is attributed to its wide-ranging applications,
2024, 29, 22. https://doi.org/ not only in the food and pharmaceutical industries but also in biopolymer production,
10.3390/molecules29010022 environmental protection, and biomedicine [4,5].
Academic Editors: Papadopoulou In industrial citric acid production, the dominant method is submerged fermentation
Olga, Patricia Regal, Ðurd̄ica Ačkar involving strains of Aspergillus niger, yeast Yarrowia lipolytica, and some bacterial strains [6].
and Raffaella Boggia Aspergillus niger is considered the best among microorganisms in the commercial synthe-
sis of citric acid due to its high production efficiency [7,8]. The development of citric
Received: 1 November 2023
acid production has significantly increased since the last century, thanks to biotechnology,
Revised: 11 December 2023
which provides knowledge about fermentation techniques and product recovery; biochem-
Accepted: 15 December 2023
istry, which provides insights into various factors influencing citric acid synthesis and
Published: 19 December 2023
inhibition; and molecular regulatory mechanisms and strategies to enhance citric acid
production efficiency [1,9].
In this review, an attempt has been made to gather and update data on the biosynthesis
Copyright: © 2023 by the author. of citric acid by the filamentous fungus Aspergillus niger while also highlighting the differ-
Licensee MDPI, Basel, Switzerland. ences between it and the yeast Yarrowia lipolytica. The review addresses the progress in citric
This article is an open access article acid bioproduction, optimal fermentation strategies, and the utilization of conventional
distributed under the terms and and unconventional carbon sources. Additionally, it discusses the prospects and future
conditions of the Creative Commons trends of the global citric acid market.
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2024, 29, 22. https://doi.org/10.3390/molecules29010022 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, x FOR PEER REVIEW 2 of 40
Molecules 2024, 29, 22 2 of 38
2. Physical and Chemical Properties

2. Physical and Chemical Properties
Citric acid [77–92–2], according to IUPAC nomenclature (International Union of Pure
Citric acid [77–92–2], according to IUPAC nomenclature (International Union of
and Applied
Pure and AppliedChemistry), is also known
Chemistry), as 2-hydroxypropane-1,2,3-tricarboxylic
is also known acid. Cit-
as 2-hydroxypropane-1,2,3-tricarboxylic
ric acid is a polyprotic α-hydroxy acid but can also be classified as
acid. Citric acid is a polyprotic α-hydroxy acid but can also be classified as a β-hydroxya β-hydroxy acid (Fig-
ure 1) [8,10]. It is present in plants, animal cells, and physiological fluids.
acid (Figure 1) [8,10]. It is present in plants, animal cells, and physiological fluids. In smallIn small quanti-
ties, citric acid
quantities, citricis acid
foundis in citrusinfruits,
found citrusespecially lemons and
fruits, especially limes.
lemons andIn limes.
amounts In exceeding
amounts
1% of the dry weight of the product, it is present in lemons (4–8%), blackberries
exceeding 1% of the dry weight of the product, it is present in lemons (4–8%), blackberries (1.5–3.0%),
grapefruitsgrapefruits
(1.5–3.0%), (1.2–2.1%),(1.2–2.1%),
as well as as oranges,
well as raspberries, and strawberries
oranges, raspberries, in the range
and strawberries of
in the
0.6–1.3% [11–13].
range of 0.6–1.3% [11–13].
Figure1.1.Chemical
Figure Chemicalstructure
structureof
ofcitric
citricacid.
acid.
Citric acid is an organic compound, a tricarboxylic hydroxy acid, with three carboxylic
Citric acid is an organic compound, a tricarboxylic hydroxy acid, with three carbox-
functional groups. It is a triprotic compound that undergoes three constant dissociations,
ylic functional groups. It is a triprotic compound that undergoes three constant dissocia-
which allows it to form three types of salts and exhibit buffering properties. The chemical
tions, which allows it to form three types of salts and exhibit buffering properties. The
and physical properties of citric acid are presented in Table 1 [14,15]. Citric acid forms crys-
chemical and physical properties of citric acid are presented in Table 1 [14,15]. Citric acid
talline mono-, di-, and tri-basic salts with various cations. From a technological perspective,
forms crystalline mono-, di-, and tri-basic salts with various cations. From a technological
the most important are calcium citrate, potassium citrate, and sodium citrate [16].
perspective, the most important are calcium citrate, potassium citrate, and sodium citrate
Citric acid is a weak acid in two crystalline forms: Anhydrous citric acid (C6 H8 O7 )
[16].
and monohydrated citric acid (C6 H8 O7 ·H2 O). Anhydrous citric acid crystallizes from a
Citric acid is a weak acid in two crystalline forms: Anhydrous citric acid (C6H8O7)
hot concentrated solution above 36.6 ◦ C, forming a white crystalline powder. On the other
and monohydrated
hand, monohydrated citric acid
citric acid(Ccrystallizes
6H8O7·H2O). Anhydrous citric acid crystallizes from a hot
from a cold solution at temperatures below
concentrated
◦ solution above
36.6 C, forming colorless, transparent 36.6 °C, forming
crystalsa[16,17].
white crystalline
Anhydrouspowder.citric acidOnabsorbs
the othera
hand, monohydrated citric acid crystallizes from a cold solution
small amount of water at 25 ◦ C and relative humidity in the 25 to 50% range. If the humidity at temperatures below
is36.6 °C, forming
between 50% and colorless,
75%, it transparent
absorbs water crystals [16,17]. while
significantly, Anhydrous citric acid
approaching 75%absorbs
relativea
small amount of water at 25 °C and relative humidity in the
humidity takes the form of a monohydrate. The anhydrous form of citric acid is obtained 25 to 50% range. If the hu-
midity is between 50% and 75%, it absorbs water significantly,
when the relative humidity is less than 40%. Monohydrated citric acid slightly absorbs while approaching 75%
relative humidity takes the form
moisture at a relative humidity of 65–75% [17]. of a monohydrate. The anhydrous form of citric acid is
obtained when the relative humidity is less than 40%. Monohydrated
Citric acid is highly soluble in water and organic solvents such as ethanol, 2-propanol, citric acid slightly
absorbs
ether, moisture
ethyl acetate,at a1.4-dioxane,
relative humidity of 65–75% [17].
tetrahydrofuran, acetonitrile, and ethanol-water mix-
tures Citric
[18]. Itacid
has aishigher
highlysolubility
soluble ininwater alcoholand organic
than solvents
in water. such
Adding as ethanol,
alcohol 2-propa-
to an aqueous
nol, ether, ethyl acetate, 1.4-dioxane, tetrahydrofuran, acetonitrile,
solution significantly increases the solubility of citric acid [19,20]. The solubility of citricand ethanol-water mix-
tures [18]. It has a higher solubility in alcohol than in water. Adding
acid in different solvents can be ranked as follows: Tetrahydrofuran < 1.4-dioxane < water < alcohol to an aqueous
solution significantly
2-propanol < ethanol < increases
acetonitrile the solubility
[21]. of citric
Citric acid does acid [19,20]. in
not dissolve The solubility toluene,
chloroform, of citric
acid in different
benzene, solvents can
carbon disulfide, be ranked as follows:
or tetrachloride Tetrahydrofuran
[17]. Its solubility increases< 1.4-dioxane < water
with an increasing
< 2-propanolof< 20.55–60.05
temperature ◦ C [19–21].[21]. Citric acid does not dissolve in chloroform, tol-
ethanol < acetonitrile
uene, benzene,
When heated carbon
to 150 ◦ C, citric
disulfide, oracid
tetrachloride [17]. Itslosing
remains stable, solubility
only increases with water.
its crystalline an in-
creasing
Above 175 ◦ C, it undergoes
temperature of 20.55–60.05
a melting °Cand[19–21].
decomposition process. Dehydration of citric
Whento
acid leads heated to 150 °C,ofcitric
the formation acid remains
trans-aconitic stable,
acid. It islosing
assumedonlythat
its crystalline water.
further thermal
Above 175 °C, itof
transformations undergoes
trans-aconitica melting
acid anddue decomposition
to dehydrationprocess. result inDehydration
the production of citric
of
acid leads
aconitic to the formation
anhydride or a mixture of trans-aconitic
of both isomers acid. It is assumed that further thermal trans-
[15,22].
Citric acid
formations can chelate metal
of trans-aconitic acidions
dueby to forming
dehydrationbonds between
result in thetheproduction
metal, carboxyl, and
of aconitic
hydroxyl
anhydride groups of the citric
or a mixture of both acid molecule.
isomers Citric acid and its salts form complexes with
[15,22].
copper, nickel,
Citric acidiron,
canmagnesium,
chelate metal zinc,
ionsand by tin. This valuable
forming property
bonds between helps
the prevent
metal, changes
carboxyl, and
in chemical potential, precipitation of solids, or changes in chemical
hydroxyl groups of the citric acid molecule. Citric acid and its salts form complexes with properties [15,23].
copper, nickel, iron, magnesium, zinc, and tin. This valuable property helps prevent
Molecules 2024, 29, 22 3 of 38
Citric acid esterifies with alcohols under typical conditions in the presence of catalysts
such as sulfuric acid, p-toluenesulfonic acid, or ion-exchange resin. The esterification
reaction of citric acid with alcohols, occurring at temperatures above 150 ◦ C, does not
require the presence of a catalyst. Citric acid forms polyesters with polyalcohols such as
sorbitol and mannitol. Interrupting the esterification reaction before completion results in
the formation of free carboxylic groups, forming salts [15].
Table 1. Chemical and physical properties of citric acid.
Properties Characteristic References

Anhydrous: 192.12 g·mol−1
Molar mass [11]
Monohydrate: 210.14 g·mol−1
powdery, colorless transparent crystals or white, granular,
fine powder
Appearance and form [14]
Anhydrous: monoclinic holohedral crystals.
Monohydrate: rhombic crystals
Anhydrous: 153◦ C
Melting point [8]
Monohydrate: ≈100 ◦ C
Boiling point None, decomposition into water and CO2 > 175 ◦ C [24]
Vapor pressure 1.7 × 10−8 mmHg at 25 ◦C [18]
Anhydrous: 1.665 g·cm−3
at 20 ◦C
Density [14]
Monohydrate: 1.542 g·cm−3 at 20 ◦ C
Octanol/water partition coefficient logPOW = −1.72 ± 0.4 at 20 ◦ C [10]
Partition coefficient logP = −1.198 ± 0.4 w 25 ◦ C [14]
pK1 = 3.128
dissociation constant pK2 = 4.761 at 25 ◦ C [14]
pK3 = 6.396
Henry’s constant KH = 2.3 ×·10−7 Pam3 ·mol−1 [25]
• In water, the solubility increases with temperature:

• solubility is 54% at 10 ◦ C.
• solubility is 84% at 100 ◦ C.
Solubility • In alcohol, the solubility is 59.1 g per 100 milliliters [14]
(g/100 mL) at 25 ◦ C
• In a mixture of ether and alcohol, the solubility is
162 g per 100 milliliters (g/100 mL) at 25 ◦ C
3. Citric Acid Biosynthesis

3.1. The Beginning of Citric Acid Production
For the first time, citric acid was isolated from lemon juice in 1784 in England by
Carl Scheele, who obtained calcium citrate by adding lime to lemon juice [26]. In 1838,
Liebig confirmed the presence of one hydroxyl group and three carboxyl groups in the
structure of citric acid. Since 1860, citric acid production from lemons has been carried
out in the United Kingdom, France, and Germany. Intensive research was conducted to
find an alternative method for obtaining citric acid [27]. In 1893, the German botanist
Wehemer observed that citric acid is formed as a byproduct during the production of
calcium oxalates by Penicillium glaucum [26]. Industrial-scale production of citric acid
involving microorganisms was initiated in 1917 by Currie, who developed a method for
obtaining it from filamentous fungi Sterigmatocystis nigra (currently Aspergillus niger) using
culture media containing sucrose [28,29]. The significant milestones in the discovery and
research of citric acid are shown in Figure 2.
The biochemical foundations of the biosynthesis process of citric acid were elucidated
in the 1950s with the discovery of glycolysis and the tricarboxylic acid cycle [30].
Molecules 2024, 29, 22 4 of 38
•The discovery of citric acid by Islamic alchemist Abu Musa Jabir Ibn
Period 7th century
Hayyan
• Carl Wilhelm Scheele, a Swedish chemist, isolated citric acid from

Year 1784
lemon juice
•Jstus Liebig, a German chemist, recognized that citric acid is

Year 1834
hydroxytricarboxylic acid
•The first chemical synthesis of citric acid was performer by Girmaux

Year 1880
and Adam
•Dunschmann and Pechmann synthesize citric acid from ethanedi

Year 1891
carboxylic acid
•Lawrence used the condensation of sthyl oxaloacetate with ethyl

Year 1897 bromoacetate in several successive reactions to finally obtain citric
acid
•Carl Wehmer discovered that citric acid could be produced from

Year 1893
sugar by the Penicillium mold
•First succesful fermentation proces using Penicillium at Les Produits

Year 1919
Organiques de Tirlemont in Tienen, Belgium
•James Currie and Charles Thom discovered that citric acid could be
Year 1917
produced in large quantities by Aspergillus niger
•The Currie – Thoma fermentation proces was comercialized by

Year 1923
Pfizer Inc.
•Nobel Prize for Hans Krebs and Fritz Lipmann for presenting the
Year 1937
mechanisms of TCA
•Studies on the influence of micro/macroelements in culture media

Period 1937 – 1956
and growth parameters on optimized citric acid fermentation
•Advances in molecular biology and genetics of Aspergillus niger are

Period 1997 – 1996 reflected in the intense focus on genes, cloning and sequence
analyses
Period 1997 – 2017 •Generation of non–homologous mutants
Figure 2. Major milestones in the discovery and research of citric acid [28,31].
Figure 2. Major milestones in the discovery and research of citric acid [28,31].
3.2. Microorganisms Producing Citric Acid

Since discovering the potential for microorganisms to produce citric acid, Aspergillus niger
strains have remained the preferred microorganisms in the production process [26,29,32–35].
In addition to filamentous fungi such as Aspergillus niger, various other microorganisms
have been used to produce citric acid. These include Aspergillus nidulans, Aspergillus ac-
uleatus, Aspergillus fumaricus, Aspergillus carbonarius, Aspergillus awamori, Aspergillus wentii,
Aspergillus saitoi, Aspergillus flavus, Aspergillus foetidus, Aspergillus fonsecaeus, Aspergillus
luchensis, Aspergillus phoenicis, Aspergillus saitoi, Aspergillus usumii, Penicillium janthinellum,
Penicillium restrictum, Trichoderma viride, Mucor piriformis, Talaromyces sp., Eupenicillium
sp., Botrytis sp., Absidia sp., and Ustulina vulgaris. Additionally, promising producers
of citric acid include yeasts such as Yarrowia lipolytica, Candida tropicalis, Candida guillier-
mondii, Candida intermedia, Candida parapsilosis, Candida zeylanoides, Candida fibriae, Candida
subtropicalix, Candida oleophila, and bacteria such as Arthrobacter paraffineus, Bacillus licheni-
formis, and Corynebacterium spp. These microorganisms are all potential sources for citric
acid production [36–39].
Molecules 2024, 29, 22 5 of 38

Bacillus licheniformis, and Corynebacterium spp. These microorganisms are all potential
sources for citric acid production [36–39].
3.2.1. Production of Citric Acid Using Aspergillus niger Fungi

In industrial citric acid production, filamentous fungi, mainly Aspergillus niger, are
[40,41]. These microorganisms offer
used (Figure 3) [40,41]. offer several advantages, including their
ability to quickly adapt and grow on various substrates, regulate and control metabolic
pathways, and regulate
regulate the secretion
secretion of citric acid from both mitochondria
mitochondria and and cytosol.
cytosol.
contributes to
This contributes to citric
citricacid
acidaccumulation
accumulationand andprevents
preventsitsitsdegradation
degradationinin the
the Krebs
Krebs cy-
cycle.
Moreover,
cle. cultures
Moreover, using
cultures Aspergillus
using nigerniger
Aspergillus are characterized
are characterizedby high
by production
high productionefficiency
effi-
and homofermentative
ciency and homofermentative citric acid
citricbiosynthesis [32,42].
acid biosynthesis Aspergillus
[32,42]. niger strains
Aspergillus have have
niger strains been
recognized
been as safe,
recognized as as they
safe, asdo notdo
they produce ochratoxin
not produce under under
ochratoxin controlled cultivation
controlled condi-
cultivation
tions and do
conditions andnot
doelicit strongstrong
not elicit allergic reactions
allergic in humans.
reactions in humans.In addition to citric
In addition acid acid
to citric and
otherother
and organic acid acid
organic biosynthesis, Aspergillus
biosynthesis, nigerniger
Aspergillus is also
is utilized to produce
also utilized enzymes
to produce such
enzymes
as pectinases,
such proteases,
as pectinases, aminoglycosidases,
proteases, aminoglycosidases,catalases, lipases,
catalases, and oxidases
lipases, [43]. [43].
and oxidases
Figure 3. Citric
Figure 3. Citric acid
acid production processes with
production processes with A.
A. niger.
niger.
Until 1980, Aspergillus niger strains used in industrial production were obtained
through screening
screeningand andmutagenesis.
mutagenesis.Mutagenesis
Mutagenesis techniques
techniques areare
stillstill
in usein and
use continue
and con-
to yield
tinue positive
to yield resultsresults
positive in improving biosynthesis
in improving efficiency.
biosynthesis The most
efficiency. commonly
The most used
commonly
used mutagens
mutagens are physical
are physical factorsfactors
(gamma (gamma
and UV and UV radiation),
radiation), chemical chemical
factors,factors, and
and hybrid
hybrid methods
methods that combine
that combine physical physical and chemical
and chemical characteristics
characteristics [44].
[44]. The The development
development of ge-
of genetic
netic engineering
engineering has allowed
has allowed the application
the application of DNA of DNA recombination
recombination technologies
technologies to
to im-
improve
prove strains,
strains, withwith Aspergillus
Aspergillus niger niger being
being used used
as aas a host
host forexpression
for the the expression of heterolo-
of heterologous
gous proteins.
proteins. The improvement
The improvement of Aspergillus
of Aspergillus niger
niger strains
strains is is limitedby
limited bythetherelatively
relatively small
number of available plasmid vectors for these these filamentous
filamentous fungi.fungi. Recently,
Recently, integration and
autonomous replication
autonomous replicationofofplasmid
plasmidvectors
vectorshave
have been used
been to manipulate
used to manipulate the genome with
the genome
targeted gene exchange [1,2,45,46].
with targeted gene exchange [1,2,45,46].
In order
In order toto enhance
enhance the the performance
performance of of species
species such
such as Aspergillus niger,
as Aspergillus niger, research
research has
has
been conducted on the application of genome editing technology
been conducted on the application of genome editing technology in filamentous fungi, in filamentous fungi,
employing CRISPR
employing CRISPR (Clustered
(Clustered Regularly Interspaced Palindromic
Regularly Interspaced Palindromic Repeats) elements with
Repeats) elements with
associated endonucleases
associated endonucleases(such (suchas asCas
Casprotein
proteinfamily
family members).
members). TheThe CRISPR/Cas9
CRISPR/Cas9 sys-
system
tem facilitates chromosome engineering in Aspergillus niger, enabling
facilitates chromosome engineering in Aspergillus niger, enabling genome manipulations genome manipula-
tions with
with high high efficiency
efficiency and and scalability,
scalability, thereby
thereby increasingthe
increasing theimplementation
implementationspeed speed ofof
metabolic engineering cycles [46,47]. In experiments involving the production of natural
metabolites, genome editing disrupted pyrG, which encodes orotidine-50 -decarboxylase,
Molecules 2024, 29, 22 6 of 38
resulting in a 2.17-fold increase in citric acid production compared to the control, suggesting
that inhibiting uridine/pyrimidine synthesis may promote citric acid overproduction [48].
3.2.2. Citric Acid Metabolism in Aspergillus niger

The biochemical mechanism through which Aspergillus niger accumulates citric acid
has been the subject of scientific interest since the 1930s, when cultivation conditions were
established and the impact of various substrate components was assessed for industrial pro-
duction needs. Despite numerous models proposed since 1930 to explain Aspergillus niger’s
ability to accumulate citric acid, many aspects of the biochemical transformations remain
unexplained. Research aimed at understanding the metabolic pathways and properties of
enzymes in Aspergillus niger has made it the best-studied filamentous fungus [49].
The ability of certain strains of Aspergillus niger to biosynthesize citric acid is deter-
mined genotypically while providing appropriate cultivation conditions, and their control
allows for achieving high process yields [50].
In the trophophase, during the growth of Aspergillus niger mycelium, hexoses or
other carbohydrates are taken up through glycolysis and the pentose phosphate pathway
(Figure 4). The involvement of the pentose phosphate pathway in carbohydrate metabolism
is shallow and significantly decreases during citric acid production—the idiophase [51].
Glucose, upon entering the cell, undergoes phosphorylation, converting into glucose-
6-phosphate. This step is catalyzed by hexokinase and glucokinase. Hexokinase is an
enzyme that catalyzes the transfer of phosphate groups from ATP to glucose and fructose.
On the other hand, glucokinase exhibits high affinity only for glucose [52]. The activity of
hexokinase is strongly inhibited by trehalose-6-phosphate. To increase the efficiency of citric
acid biosynthesis, the gene tpsA, responsible for the expression of trehalose-6-phosphate
synthase, was blocked [53,54].
The next step in glycolysis is the isomerization of glucose-6-phosphate to fructose-
6-phosphate. This reaction is catalyzed by phosphoglucose isomerase. Following the
isomerization reaction, a phosphorylation reaction catalyzed by phosphofructokinase,
a key enzyme in the glycolytic pathway, occurs. As a result of this reaction, fructose-
6-phosphate is transformed into fructose-1,6-bisphosphate [55]. Phosphofructokinase
activity is inhibited by a high concentration of ATP, leading to a decrease in its affinity
for fructose-6-phosphate. This means that the activity of phosphofructokinase increases
when the energy charge decreases [56]. Inhibitors of phosphofructokinase also include
a high concentration of manganese, citrate, and phosphoenolpyruvate. Stimulating ef-
fects on phosphofructokinase activity are exerted by NH4+ , Zn2+ , Mg2+ , and adenosine
monophosphate (AMP) [49,57].
The first reaction in the third stage of glycolysis is the conversion of 3-phosphoglyceraldehyde
to 1,3-bisphosphoglycerate. This process is catalyzed by 3-phosphoglyceraldehyde dehy-
drogenase and involves the oxidation of the aldehyde with the participation of NAD+ to
form a carboxylic acid and the production of 1,3-bisphosphoglycerate. In the next stage
of glycolysis, phosphoglycerate kinase catalyzes the transfer of a phosphate group from
1,3-bisphosphoglycerate, forming ATP and 3-phosphoglycerate. The final stage of gly-
colysis involves the conversion of 3-phosphoglycerate to pyruvate, accompanied by the
production of ATP. In this stage, the critical enzyme is pyruvate kinase, which catalyzes the
irreversible transfer of a phosphate group to ATP [58,59].
In the glycolytic pathway, glucose is converted into two molecules of pyruvate. Pyru-
vate transforms precursors such as citrate, oxaloacetate, and acetyl-CoA. Oxaloacetate is
formed through the carboxylation of pyruvate, catalyzed by pyruvate carboxylase. During
this reaction, Aspergillus niger consumes CO2 generated during acetyl-CoA formation [50].
Pyruvate is then transported to the mitochondrion, where it undergoes oxidative decarboxy-
lation to form acetyl-CoA in an irreversible reaction catalyzed by pyruvate dehydrogenase.
The reaction of acetyl-CoA formation from pyruvate serves as a bridge between the gly-
colytic pathway and the citric acid cycle. The citric acid cycle occurs in the mitochondrial
matrix and begins with oxaloacetate, acetyl-CoA, and H2 O condensation to form citrate
Molecules 2024, 29, 22 7 of 38
and CoA.
Molecules 2024, 29, x FOR PEER REVIEW The enzyme catalyzing this reaction is citrate synthase. Subsequently, citrates
7 of 40
undergo isomerization to isocitrate through reactions catalyzed by aconitase [13,60,61].
Figure 4. Overview of pathways leading to citric acid production in A. niger.

Figure 4. Overview of pathways leading to citric acid production in A. niger.
InThe
thefirst reaction
past, it wasinspeculated
the third stage of glycolysis
that is the conversion
the accumulation of 3-phosphoglycer-
of citric acid in the citric acid
aldehyde to 1,3-bisphosphoglycerate. This process is catalyzed by 3-phosphoglyceralde-
cycle occurs due to the inhibition of aconitase, isocitrate dehydrogenase-NADP, and α-
hyde dehydrogenase and involves the oxidation of the aldehyde with the2+ participation of
ketoglutarate dehydrogenase by external factors (metal ions, pH, Cu ) or internal factors
NAD+ to form a carboxylic acid and the production of 1,3-bisphosphoglycerate. In the next
(glycerol,
stage of citrates)
glycolysis,[49,52].
phosphoglycerate kinase catalyzes the transfer of a phosphate group
The accumulation of citricforming
from 1,3-bisphosphoglycerate, acid is associated with the activity of
ATP and 3-phosphoglycerate. Thetricarboxylate
final stage of trans-
porters that compete with aconitase for citrates. The affinity of the tricarboxylate transporter
for citrate is significantly higher than that of aconitase. Consequently, citrates are released
from the mitochondrion without inhibiting the Krebs cycle. The transport of citrates by
Molecules 2024, 29, 22 8 of 38
tricarboxylate carriers operates through exchange with cytosolic malate. Hence, malate can
be considered a potential trigger for the accumulation of citric acid, as an increase in its
concentration precedes the accumulation of citrates [49].
3.2.3. Production of Citric Acid Using Yarrowia lipolytica Yeast

Yeast, especially Yarrowia lipolytica and Candida strains, have been used in citric acid
production since the 1960s [62]. Initially, n-alkanes were used as carbon sources in cultures,
Molecules 2024, 29, x FOR PEER REVIEW
but over time, other substrates such as glucose, acetates, molasses, glycerol, inulin,9 oils,
of 40
and fatty acids were introduced (Table 2; Figure 5) [63–65].
Figure 5.
Figure Citric acid
5. Citric with Y.
acid production processes with Y. lipolytica.
lipolytica.
2. Substrates
TableThe used in citric
main advantages acid production
of Yarrowia by Yarrowia
lipolytica lipolytica strains.
strains compared to filamentous fungi in-
clude better tolerance to high carbon source concentrations, lower sensitivity to heavy
Substrate Strain Citric Acid Cultivation Method References
metal ions, and lower oxygen levels in the growth medium. This allows for the utilization
Yarrowia − 3
g·dm citric acid biosynthesis
of alipolytica
wide range of substrates.121–129
Moreover, SF using Yarrowia[66] lipolytica
Yarrowia lipolytica 49 g · dm −3 SF [67] [40,75–
yeast is characterized by higher efficiency, faster production, and easier control
Glucose
77].lipolytica VKM Y-2373
Yarrowia 80–85 g·dm − 3 FBF [68]
The accumulation
Yarrowia lipolytica NRRL Y-1094 of citric acid
30.31 g·dm in − yeast
3 requires a deficiency
SF in a nitrogen
[69] source
Pure glycerol because
Yarrowia itsNG40/UV7
lipolytica production initiates 115
after
g·dm the−3depletion of available
SF nitrogen. Citrate[70]synthase,
the enzyme converting
Yarrowia lipolytica NG40/UV7
oxaloacetate
112 g·dm
and− 3 acetyl-CoA into citric
SF
acid, is modulated
[70]
by the
Waste glycerol provision of ammonium ions in the medium. Therefore, ensuring a high C/N ratio is cru-
Yarrowia lipolytica SKY7 18.70 g·dm−3 FBF [38]
cial so that the excess carbon is redirected towards citric acid production in the stationary
−3
growth phase (Figure 6) [78,79].75.9 g·dm
Yarrowia lipolytica A101 SF [71]
Yarrowia lipolytica SWJ-1b 85 g·dm−3 SF [72]
Inulin
Yarrowia lipolytica AWG7 INU 8 200 g·dm−3 RBF [73]
Waste cooking oil Yarrowia lipolytica SWJ-1b 31 g·dm−3 SF [74]
The main advantages of Yarrowia lipolytica strains compared to filamentous fungi

include better tolerance to high carbon source concentrations, lower sensitivity to heavy
metal ions, and lower oxygen levels in the growth medium. This allows for the utilization
of a wide range of substrates. Moreover, citric acid biosynthesis using Yarrowia lipolytica
yeast is characterized by higher efficiency, faster production, and easier control [40,75–77].
Molecules 2024, 29, 22 9 of 38
The accumulation of citric acid in yeast requires a deficiency in a nitrogen source

because its production initiates after the depletion of available nitrogen. Citrate synthase,
the enzyme converting oxaloacetate and acetyl-CoA into citric acid, is modulated by
the provision of ammonium ions in the medium. Therefore, ensuring a high C/N ratio is
crucial so that the excess carbon is redirected towards citric acid production in the stationary
growth phase (Figure 6) [78,79].
Figure 6. Metabolic pathways of Y. lipolytica.

Figure 6. Metabolic pathways of Y. lipolytica.
Yeast also
Yeast also offers
offers the
the advantage
advantage of of the
the ease
ease ofof genetic
geneticmodifications
modificationsusing
usingmolecular
molecular
techniques and eliminates the need for prior substrate processing [42]. Tan et
techniques and eliminates the need for prior substrate processing [42]. Tan et al. expressed al. expressed
the pyruvate
the pyruvate carboxylase
carboxylase gene
gene cloned
clonedfrom
fromMeyerozyma
Meyerozymaguilliermondii
guilliermondiiininYarrowia
Yarrowialipolytica
lipolytica
SWJ-1b to enhance citric acid production. The research resulted
SWJ-1b to enhance citric acid production. The research resulted in both increased in both increased py-
pyruvate
ruvate carboxylase activity and citric acid production by the obtained
carboxylase activity and citric acid production by the obtained recombinant Yarrowia recombinant Yar-
rowia lipolytica
lipolytica [63]. [63]. On other
On the the other hand,
hand, LiuLiuet et
al.al.increased
increasedthethe expression
expression of
of the
theICL1
ICL1gene
gene
and reduced the ACL1 gene of the ATP citrate lyase to enhance citric acid production by
Yarrowia lipolytica. The result of these studies was citric acid production reaching 84.0
g·dm−3 within 214 h [80].
A significant challenge in citric acid production with yeast is the concurrent secretion
of isocitric acid, which is undesirable and interferes with crystallization [80]. The amount
Molecules 2024, 29, 22 10 of 38
and reduced the ACL1 gene of the ATP citrate lyase to enhance citric acid production
by Yarrowia lipolytica. The result of these studies was citric acid production reaching
84.0 g·dm−3 within 214 h [80].
A significant challenge in citric acid production with yeast is the concurrent secretion
of isocitric acid, which is undesirable and interferes with crystallization [80]. The amount
of accumulated citric acid depends on the yeast strain and carbon source used. In cul-
ture media containing vegetable oils or n-alkanes as the carbon source, the proportion of
isocitric acid is around 35–45%, while in glycerol-based media, it is about 10–12% [81].
To reduce the presence of isocitric acid in the culture medium, strains have been im-
proved using genetic engineering methods, such as inducing overexpression of isocitrate
lyase, resulting in a significant reduction in isocitrate levels, or increasing the activity of
pyruvate carboxylase [63,75].
The issue of citric acid production by yeast has been extensively described in the work
by Börekçi et al., “Citric Acid Production of Yeasts: An Overview” [69].
4. Production of Citric Acid

4.1. Cultivation Methods and Conditions
Currently, over 90% of the world’s citric acid production is manufactured using three
methods: Submerged fermentation (SF), liquid surface fermentation (LSF), and solid-state
fermentation (SSF) [37]. The advantages and disadvantages of different cultivation methods
used in citric acid biosynthesis are presented in Table 3.
Table 3. Advantages and disadvantages of different cultivation methods used in the biosynthesis of
citric acid by Aspergillus niger.
Type of Cultivation Process Parameters Process Advantages Process Disadvantages References

Long duration
Sensitive to contamination by
other microorganisms
Process Ease of operation Requires large
Surface cultivation in
Duration: 8–12 days Energy-efficient production areas [29,32,82]
liquid substrates (LSF)
Process yield: 70–75% Technically simple Generates large amounts
of heat
Production on a small and
medium industrial scale
Technically and
technologically simple Difficulties in controlling
Low substrate cost process parameters (pH,
Surface cultivation in solid Energy-efficient humidity, temperature)
Process Duration: 4 days [36,83–85]
substrates (SSF) Low risk of contamination High product contamination
Low waste generation High cost of
Low sensitivity to heavy product acquisition
metal pollution
Ability to control Sensitivity to the inhibitory
process parameters effects of trace elements
Submerged fermentation High process efficiency A large amount of waste
Process Duration: 4 days [29,50,82]
cultures (SF) Low production costs is generated
Ease of maintaining 80% of the world’s citric
sterile conditions acid production
4.1.1. Liquid Surface Fermentation Cultures

This method is still used on a small and medium industrial scale due to its simple
technology and low production costs [26]. In surface fermentation cultures, Aspergillus
niger fungi grow on the surface of the growth medium and form a thick mycelial layer. This
process occurs in fermentation chambers on high-quality steel, aluminum, or polyethylene
trays (Figure 7). The fermentation chambers are equipped with an aeration system that
controls temperature and humidity levels. The air supplied to the fermentation chambers
Molecules 2024, 29, 22 11 of 38

is filtered using bacteriological filters to prevent contamination by Penicillium, other strains
of Aspergillus niger, or lactic acid bacteria [4,86].
Figure 7. Schematic of liquid surface fermentation cultures.

cultures.
Surface
Surface cultivation
cultivationgenerates
generatessignificant
significantheat,heat,requiring
requiring a high
a highaeration
aeration raterate
to maintain
to main-
the proper temperature. This process generates significant heat
tain the proper temperature. This process generates significant heat during fermentation, during fermentation,
which is
is controlled
whichFigure 7. Schematicby
controlled by proper
proper
of liquid aeration.
aeration.
surface The
The
fermentation chamber
chamber requires
cultures. requires adequate
adequate ventilation,
ventilation, and
and
fermentation
fermentation chambers
chambers have have ensured
ensured efficient
efficient air circulation passing
air circulationaeration
passing over the substrate’s
over the substrate’s
surface Surfaceacultivation
through generatesfilter
significant heat, requiring a high rate to main-
surface through
tain abacteriological
the proper bacteriological
temperature. This filter
to control
controlhumidity
processtogenerates humidity
significant
and
and
heat
temperature
temperature
during
through
fermentation,through cooling.
cool-
Carbon
ing. Carbondioxide produced
which isdioxide produced
controlled
during the fermentation
during the
by proper aeration. fermentation
The chamber
process inhibits
requiresprocess
the
adequateinhibits
production
theand
ventilation,
of
production citric
of
acid
citricatacid
concentrations
fermentation chambers
at concentrationshigher
have than
ensured
higher 10% [4,5].
efficient
than 10%air[4,5].
circulation passing over the substrate’s
surface through a bacteriological filter to control humidity and temperature through cool-
4.1.2. Solid-State Fermentation
ing. Carbon dioxide produced duringCultures the fermentation process inhibits the production of
4.1.2. Solid-State Fermentation
citric acid at concentrations Cultures
higher than 10% [4,5].
This cultivation method involves the growth of microorganisms on solid substrates.
This cultivation method involves the growth of microorganisms on solid substrates.
Initially, the
4.1.2. appropriate
Solid-State moisture
Fermentation is provided in the form of humidity in the raw material,
Cultures
Initially,
and the appropriate
additional ismoisture
moisturemethod supplied is provided in the form of humidity in the raw material,
This cultivation involvesby thethe air during
growth the process.
of microorganisms Solid-state
on solid substrates.cultivation is
and additional
considered moisture
a reaction
Initially,
is supplied
in a heterogeneous
the appropriate
by
moisture is provided
the air
system during the process.
withofsimultaneous
in the form humidity in the raw
Solid-state
multicomponent
material,
cultivation
mass
is considered
and andtransport
heat a reaction
additional [9,83].inis asupplied
moisture heterogeneous system
by the air during with simultaneous
the process. multicomponent
Solid-state cultivation
massOn and heat
is considered transport
a reaction[9,83].
in a heterogeneous system with simultaneous multicomponent
a laboratory scale, SSF devices consist of media such as petri dishes or flasks in
mass and heat transport [9,83]. devices consist of media such as petri dishes or flasks in
whichOn a laboratory
screening
On a laboratory
scale,
tests can be SSF
scale, performed. On an
SSF devices consist of industrial
media such as scale,
petrivarious
dishes ortypes
flasks inof bioreactors
which
are used,screening
which which tests
differ
screening
can
testsmainly
be performed.
in the presence
can be performed.
On an industrial
or absence
On an industrial
scale,
of mixing
scale, various
various
types ofand types
forced
bioreac-
ofaeration.
bioreac-
tors are
torsused,
are which
used, whichdiffer
differ mainly
mainly inin the
the presence
presence or or absence
absence of mixing
The simplest type is a shelf bioreactor, in which solid material is placed on trays made of mixing
and forced and
aera- forced aera-
tion. The
tion. simplest
The simplesttype
typeis is a
a shelf
shelf bioreactor,
bioreactor, in in
whichwhich
solid solid
material
of metal or plastic. The trays are placed in a chamber where circulating air regulates material
is placed is
on placed
trays on trays
made made
of of metal
metal or or plastic.The
plastic. The trays
trays areareplaced
placedin a in
chamber
a where circulating
chamber where air regulatesair regulates
circulating
temperature and humidity. The second type of culture can take place in packed-bed column
temperature and humidity. The second type of culture can take place in packed-bed col-
temperature
bioreactors. andthird
The
umn bioreactors.
humidity.
Thetype
The
thirdistype
second
stirred drum
is stirred
type
drum
of culture which
bioreactors, can take
bioreactors, which are
are
place
used
used
ininpacked-bed
in SSFs SSFs
re- requiring
col-
umn bioreactors.
slow, continuous The third
mixing
quiring slow, continuous and type is stirred
no forced
mixing and drum
aeration
no forced bioreactors,
(Figure
aeration which are used in SSFs re-
8)8)[87,88].
(Figure [87,88].
quiring slow, continuous mixing and no forced aeration (Figure 8) [87,88].
Figure 8. Schematic of solid-state fermentation cultures.


This method can use waste from agriculture and industry, such as fruit and vegeta
Molecules 2024, 29, 22 processing waste, as substrates. Drum, column, and rotary bioreactors are12used of 38 for cit
acid production on solid-state substrates [89].

One clear advantage of this method is its low energy consumption and minim
This method can use waste from agriculture and industry, such as fruit and vegetable
wasteprocessing
generation, which is environmentally friendly. Additionally, the process tak
waste, as substrates. Drum, column, and rotary bioreactors are used for citric
aboutacid
four days under
production optimalsubstrates
on solid-state conditions,
[89]. significantly shorter than submerged and l
uid surface
Onefermentation
clear advantagecultures [32,90,91].
of this method is its low energy consumption and minimal waste
generation, which is environmentally friendly.
Solid-state cultures of Aspergillus niger have Additionally,
gainedthe process takes
importance inabout
recentfouryears. St
days under optimal conditions, significantly shorter than submerged and liquid surface
due to the low level of process automation and a need for improvements in bioreac
fermentation cultures [32,90,91].
design, they are only
Solid-state marginally
cultures usedniger
of Aspergillus in industrial
have gainedcitric acid production
importance [92].
in recent years. Still,
due to the low level of process automation and a need for improvements in bioreactor
4.1.3.design,
Submerged
they areFermentation
only marginally used in industrial citric acid production [92].
Around 80% ofFermentation
4.1.3. Submerged the world’s citric acid production is achieved using submerged f
mentation. Citric80%
Around acidof production through
the world’s citric batch culture
acid production is carried
is achieved using out in tankfer-
submerged bioreacto
of high-quality corrosion-resistant
mentation. Citric steel equipped
acid production through batch culturewith aeration
is carried out in and mixing systems
tank bioreactors of (F
high-quality
ure 9). The most corrosion-resistant
commonly used steelcarbon
equippedsource
with aeration and mixing
for citric systems (Figure
acid production is 9).
sucrose,
The most commonly used carbon source for citric acid production is sucrose, as well as
well as by-products of its production, such as molasses [13,32].
by-products of its production, such as molasses [13,32].
FigureFigure
9. Schematic of of
9. Schematic submerged fermentation.
submerged fermentation.
The advantages of submerged culture over surface culture include lower costs, low
The advantages
contamination risk, of submerged
a high culture over
level of automation, surface
and higher culture
process include
yield. lower
To achieve highcosts, lo
contamination risk, a high
citric acid production level
yields of automation,
in submerged and
cultures, higher
control processparameters
of process yield. To and
achieve hi
citriccareful
acid production yields
substrate selection are in submerged
crucial [93]. cultures, control of process parameters a
The periodic
careful substrate batch culture
selection is the most
are crucial frequently used method in industrial citric acid
[93].
production. Other methods include fed-batch and semicontinuous cultures [94]. In fed-
The periodic batch culture is the most frequently used method in industrial citric ac
batch fermentation, sterilized nutrients are added to the fermenter during biomass growth
production. Other
(Figure 10). methodsfermentation,
In continuous include fed-batch and semicontinuous
sterilized liquid cultures
nutrients are introduced [94]. In fe
into the
batchfermenter
fermentation, sterilized
at the same flow ratenutrients are added
as the fermenting wort to the fermenter
leaving the system. during biomass
Parameters such grow
as temperature, pH, oxygen consumption, and carbon dioxide production
(Figure 10). In continuous fermentation, sterilized liquid nutrients are introduced into t are measured
and controlled to optimize the fermentation process [95].
fermenter at the same flow rate as the fermenting wort leaving the system. Paramet
such as temperature, pH, oxygen consumption, and carbon dioxide production are me
ured and controlled to optimize the fermentation process [95].
MoleculesMolecules
2024, 29, x FOR
2024, 29, 22PEER REVIEW 13 of 38 1
Figure
Figure10.
Figure 10.Schematic
10. Schematic ofof
Schematic of fed-batch
fed-batch
fed-batch fermentation.
fermentation.
fermentation.
4.2. Factors Influencing Citric Acid Production

4.2.4.2. Factors Influencing Citric Acid Production
Factors Influencing Citric Acid Production
The course of Aspergillus niger cultivation and the rate of citric acid biosynthesis in
The course of Aspergillus niger cultivation and the rate of citric acid biosynthesis in
The course
submerged
submerged culture ofare
culture Aspergillus
areinfluenced
influencedby niger
bymany
many cultivation
factors, and the
factors,including
including the rateofofof
thetype
type citric
carbon
carbon acidand
source
source biosynthe
its concentration,
submerged the
culture are
and its concentration, type and concentration
influenced
the type of
by many
and concentration metal ions present
factors,
of metal in
ions includingthe culture
present in thethe media,
type
culture me- low
of carbon s
molecular
dia, low weight alcohols,
molecular weight fungal morphology,
alcohols, fungal as well as
morphology, as well
temperature,
as pH, aeration
temperature, pH, rate,
aer-
and
and
its concentration, the type and concentration of on
metal ions present in the cultur
ation rate, and mixing rate. A brief summary of the impact of factors on citric acid biosyn- in
mixing rate. A brief summary of the impact of factors citric acid biosynthesis
dia, low molecular
different
thesis incultivation
weightmethods
methods
different cultivation isalcohols,
presented fungal morphology,
in Figurein
is presented 11. The impact
Figure
asfactors
of
11. The impact
well as
thattemperature,
stimulate
of factors that
pH
ation
the rate, and
citric acid
stimulate mixing
the biosynthesis rate. A brief
process has
citric acid biosynthesis summary
been has
process widely of the
beenresearched. impact
widely researched. of factors on citric acid b
thesis in different cultivation methods is presented in Figure 11. The impact of facto
stimulate the citric acid biosynthesis process has been widely researched.
Figure11.
Figure 11.Summarizing
Summarizing the impact
impact of
of factors
factorsthat
thatstimulate
stimulatethe
thecitric acid
citric biosynthesis
acid process.
biosynthesis process.
4.2.1.
4.2.1.Nitrogen
Nitrogen
The
Theconcentration
concentration and source of
and source ofnitrogen
nitrogenhave
havea afundamental
fundamental impact
impact ononthethe growth
growth
ofofAspergillus
Aspergillusniger
niger and
and the biosynthesis
biosynthesis ofofcitric
citricacid
acidininboth
bothsubmerged
submerged andand solid-state
solid-state
cultures.
cultures. The
The most
most preferred nitrogensources
preferred nitrogen sourcesare arenitrogen
nitrogensalts,
salts, including
including ammonium
ammonium
nitrate,
nitrate,ammonium
ammoniumsulfate,
sulfate,and
andammonium
ammoniumchloride.
chloride.Among
Amongother othernitrogen
nitrogen sources, urea,
sources,
Figure
urea,11.
peptone, Summarizing
and
peptone,yeast yeast the
andextract canimpact
becan
extract ofdistinguished
factors[4].
distinguished
be thatAccording
stimulate tothe
[4]. According citric
theto acid ammonium
literature, biosynthesis
the literature, am- proce
nitrate
monium is considered the most the
nitrate is considered favorable nitrogennitrogen
most favorable source [96–100].
source [96–100].
4.2.1. Nitrogen
Ammonium compounds lead to an advantageous reduction in the pH of the culture
medium to a level lower than 2, which is a necessary condition for citric acid production.
The concentration and source of nitrogen have a fundamental impact on the g
of Aspergillus niger and the biosynthesis of citric acid in both submerged and solid
cultures. The most preferred nitrogen sources are nitrogen salts, including ammo
Molecules 2024, 29, 22 14 of 38
Ammonium compounds lead to an advantageous reduction in the pH of the culture

medium to a level lower than 2, which is a necessary condition for citric acid production.
Limiting the nitrogen source during cultivation inhibits fungal biomass growth and in-
creases citric acid production [50]. The optimal concentration of the nitrogen source should
be around 0.2%, as it promotes the biosynthesis of citric acid by Aspergillus niger. The high-
est citric acid biosynthesis efficiency is achieved when the intracellular concentration of
ammonium ions is 2–3 mM·g−1 . However, process efficiency decreases when the intracellu-
lar nitrogen ion concentration is 1 mM·g−1 of biomass [97–99]. Culture media are supplied
during cultivation to increase the volumetric citric acid biosynthesis rate. In addition to the
proper amount of added nitrogen source, the timing of the addition and supplying it at the
wrong fermentation phase can reduce the citric acid accumulation rate [26].
In the case of yeast, citric acid biosynthesis begins after the nitrogen source is depleted.
Limiting the nitrogen concentration at a high substrate concentration for yeast is crucial
because citric acid is released through a specific, energy-dependent transport system
induced by intracellular nitrogen restriction [62].
4.2.2. Phosphorus
The presence of phosphorus in the culture medium also influences the efficiency
of citric acid biosynthesis. The best sources of phosphorus are KH2 PO4 and K2 HPO4 .
Limiting phosphorus concentration in the culture medium, similar to nitrogen, critically
impacts citric acid production. Increasing the process efficiency is allowed by a phosphorus
concentration in the range of 0.006 to 0.32 g·dm−3 . Fungal mycelium requires the presence
of phosphorus at concentrations ranging from 0.01 to 0.02% for proper growth [90,101].
High phosphorus concentrations stimulate mycelial growth, induce secondary enzy-
matic reactions, and inhibit citric acid production [98].
4.2.3. Trace Elements

Trace elements are a crucial factor affecting the efficiency of citric acid biosynthesis.
Manganese, zinc, copper, and iron are of great importance [101]. To achieve high process
efficiency, especially in submerged cultures, it is necessary to use culture media with
controlled trace element content. This is due to their significant influence on the growth
and physiology of Aspergillus niger and the efficiency of citric acid biosynthesis [50].
Magnesium is essential for the proper growth of Aspergillus niger and citric acid
production due to its role as a cofactor in enzymatic reactions. Maximum citric acid
biosynthesis efficiency is achieved at magnesium sulfate concentrations ranging from 0.020
to 0.025% [29,90].
The addition of manganese to the culture medium plays a significant role in the
accumulation of citric acid, cell wall synthesis, sporulation, and the production of secondary
metabolites [50]. At a concentration of 10 mg·dm−3 , manganese limits the efficiency of
citric acid biosynthesis. However, at concentrations lower than 3 µg·dm−3 , it significantly
reduces the process efficiency. Manganese deficiencies contribute to reduced lipid synthesis
and increased cell membrane permeability due to decreased concentrations of certain
enzymes involved in anabolic processes [102–104].
In citric acid production, limiting the presence of iron in the culture medium is crucial.
Approximately 1 mg of iron per liter of culture medium is needed to achieve high process
efficiency. Higher iron concentrations can lead to the accumulation of oxalic acid [90].
The presence of copper ions reduces the harmful effect of excess iron ions. Furthermore,
copper is known to inhibit manganese ions. The optimal concentration of CuSO4 ·7H2 O
in the culture medium should be 78 mg·dm−3 [101]. The presence of copper at various
concentrations affects the morphology of Aspergillus niger mycelium. Therefore, its presence
determines the attainment of the appropriate mycelial structure, allowing for high efficiency
in citric acid biosynthesis [50,105].
Molecules 2024, 29, 22 15 of 38
4.2.4. Low-Molecular-Weight Alcohols

Potential stimulators of citric acid fermentation include low-molecular-weight alcohols,
such as methanol, ethanol, and n-propyl alcohol [106,107]. Ethanol added to culture media
inhibits mycelial growth and sporulation, reduces substrate consumption, increases cell
membrane permeability, and influences an increase in citrate synthase activity and a
reduction in aconitase activity [108–110].
Methyl alcohol, unlike ethanol, is not assimilated by Aspergillus niger and does not
undergo conversion to acetyl-CoA, which is a precursor of the Krebs cycle. The mechanism
of methanol’s interaction with citric acid biosynthesis in synthetic and natural media
has not been fully elucidated. Methyl alcohol in high-purity media disrupts metabolic
processes and biomass growth, reducing citric acid production. It also leads to disturbances
in the synthesis of cellular proteins in the early stages of cultivation [111,112]. Methyl
alcohol affects the permeability of cell membranes, which may be due to changes in the
composition of phospholipids and triglycerides [113]. Phospholipids play a significant
role in regulating membrane permeability for citric acid. Methyl alcohol may disrupt the
formation of mycelial structure through chelation effects on metal ions such as copper
(II), which play a significant role in regulating the content of fatty acids in glycolipids
and phospholipids [114].
In the study by Maddox et al. [115], methanol in synthetic media containing galactose
as a carbon source had a toxic effect by limiting fungal growth and reducing substrate
consumption. However, it simultaneously increased the efficiency of citric acid production.
Furthermore, methyl alcohol inhibited the activity of 2-oxoglutarate dehydrogenase, re-
sulting in increased citric acid accumulation. Similarly, Yaykaşlı et al. [116] demonstrated
that methanol in the biosynthesis process using immobilized conidia of Aspergillus niger in
media with sucrose contributed to an increase in citric acid production.
Most of the available literature data report a positive impact of lower concentrations
of methyl alcohol on the efficiency of citric acid production by Aspergillus niger in natural
media characterized by lower purity, such as molasses-based media [106,111,113,117–121].
The stimulating effect of methyl alcohol added to natural media results from its limitation
of the negative impact on the biosynthesis process of metal ions such as manganese, iron,
and zinc, which strains of Aspergillus niger are highly sensitive to. Methyl alcohol, on the
other hand, increases the tolerance of Aspergillus niger to the content of iron, manganese,
and zinc ions present in the media [117]. Methyl alcohol induces changes in the normal
carbohydrate metabolism pathway by increasing glycolytic capabilities, leading to citric
acid accumulation. In natural media, it stimulates citric acid production by affecting fungal
growth and altering the composition of the cell wall lipids [121].
4.2.5. The pH Value

The pH value plays a significant role during spore germination, where the pH should
be higher than 5, and during citric acid production, when a low substrate pH is required
(pH ≤ 2). Most fungal mycelia grow in the pH range of 3 to 6, and their metabolic activity
mainly depends on the culture medium’s pH [122–124].
During the production of citric acid by Aspergillus niger in submerged and surface
cultures, a pH range of 2 to 6 is utilized. The low pH level reduces the risk of culture
contamination by other microorganisms. Furthermore, a pH below 3 prevents the produc-
tion of oxalic and gluconic acids [124]. Conversely, increasing the pH to 4.5 can lead to a
significant reduction in citric acid production efficiency by up to 80% [50,125].
The pH of the culture medium can also affect the morphology of Aspergillus niger
mycelia. At pH values of 2.0 to 2.2, the mycelia assume the desired form of small aggregates
and short hyphae, which is most desirable for citric acid production. At a pH of 1.6,
the morphological development of mycelia is disrupted, and process efficiency decreases
significantly. In a medium with a pH of 3.0, mycelia form larger aggregates, favoring oxalic
acid biosynthesis [50].
Molecules 2024, 29, 22 16 of 38
4.2.6. Aeration and Mixing Rate

The appropriate dissolved oxygen concentration in the cultivation medium is a criti-
cal factor influencing the efficiency of citric acid biosynthesis [26,113]. The aeration rate
significantly affects citric acid biosynthesis, especially in submerged cultivation methods.
In submerged cultivations, the process efficiency increases with higher aeration rates and
pressures (0.10–0.17 MPa) [90]. In cases of insufficient oxygen in the medium or interrup-
tions in oxygen supply, citric acid production may be inhibited, and fungal growth may
be affected [29,126,127]. The proper concentration of dissolved oxygen in the cultivation
medium influences the rate of glycolysis and the respiratory chain, leading to high ATP
levels and increased citrate secretion [57].
In industrial production, the aeration rate is adjusted according to the current fungal
demand. Initially, the aeration rate is around 0.1 vvm, and during the phase of intensive
fungal growth, it is increased to approximately 0.5 vvm [26]. To enhance the efficiency
of citric acid biosynthesis in submerged cultivations, aeration rates ranging from 0.9 to
1.3 vvm have been applied [39]. Inappropriate aeration rates can have an adverse impact
on the efficiency of the bioprocess [50].
Proper aeration, combined with high dissolved oxygen levels, contributes to the
reduced activity of cytochrome-dependent enzymes and the increased activity of alternative
oxidase, consequently favoring the alternative respiratory pathway. This process conserves
energy by bypassing the need to generate ATP. As a result, citric acid fermentation requires
continuous cooling because the free electron transport generates heat [49].
An essential parameter associated with aeration is the mixing rate, which affects
fungal dispersion, dissolved oxygen concentration in the medium, and even enzyme
activity, such as citrate synthase, aconitase, and isocitrate dehydrogenase. The activity of
citrate synthase decreases with increasing mixing rates, while the activity of aconitase and
isocitrate dehydrogenase increases with higher mixing rates. The optimal mixing rate in
laboratory bioreactors is 300 to 1000 rpm [57].
Intense mixing also leads to the development of fungal mycelia in short, branched
hyphae characterized by high citric acid overproduction. It also contributes to mycelial
fragmentation and regrowth. This phenomenon can be beneficial because metabolically in-
active and highly vacuolated mycelial fragments are the most susceptible to fragmentation,
and this process generates new Aspergillus niger mycelial fibers [105].
4.2.7. Temperature
Temperature is another parameter that affects enzymatic activity, microbial transport
systems, and consequently, the efficiency of citric acid biosynthesis. In the production of
citric acid, the optimal temperature falls within the range of 28 ◦ C to 30 ◦ C. Numerous
studies have shown that the highest process efficiency was achieved at a temperature of
30 ◦ C [122,128–131]. Temperatures above 30 ◦ C lead to the denaturation of citrate synthase,
limiting citric acid accumulation and biomass growth in the medium while promoting oxalic
acid production. Cultures at lower temperatures decrease enzymatic activity [122,131].
4.3. Substrate
The search for citric acid biosynthesis substrates involving Aspergillus niger has been
the subject of extensive research [54,123,132–141]. High-purity substrates such as sucrose,
glucose, fructose, and maltose can be utilized in citric acid production. Examples of using
high-purity raw materials in the citric acid production process involving Aspergillus niger
are presented in Table 4.
Sucrose is the most favorable carbon source due to its low molecular weight, facilitating
transport across microbial cell membranes, and rapid hydrolysis by invertase activated in
low-pH environments [101,142]. High-purity substrates allow for the control of the citric
acid production process with a yield greater than 70% [26,90]. However, using pure sugar in
industrial citric acid production is associated with high costs, as the raw material cost often
exceeds the obtained product’s price [143]. Therefore, in industrial citric acid production
Molecules 2024, 29, 22 17 of 38
involving Aspergillus niger, cheap and renewable carbon sources such as agro-industrial
waste materials are employed [2,94].
Examples of using agro-industrial waste materials in the citric acid production pro-
cess with Aspergillus niger are presented in Tables 5 and 6. Environmental concerns and
the high pollution control costs drive the interest in utilizing waste and industrial ef-
fluents in citric acid biosynthesis. Industrial by-products used in citric acid production
include beet molasses [144–146], sugarcane molasses [147], cellulose [148], lipids [137,149],
whey, fruit pomaces [92,150,151], inulin [152], starch materials [121,134,153,154], sweet
potatoes [155,156], cassava [4,136], seaweed [122], and glycerol [157,158]. Industrial waste
is considered the best carbon source in solid-state cultures [32].
The primary raw materials in citric acid production involving Aspergillus niger are cane
and beet molasses, primarily due to their low cost [50,93]. Molasses is characterized by a
high carbohydrate content, approximately 50%, mainly in sucrose, glucose, and fructose.
However, its chemical composition is variable and heterogeneous, which could hinder the
biosynthesis process. To enhance the quality of the culture medium, molasses undergoes
various treatments involving ferrocyanide, hydrochloric acid, tricalcium phosphate with
hydrochloric acid, ammonium oxalate, ammonium dihydrogen phosphate, and lime, as
well as fractionation [119,159–161].
Table 4. High-purity substrates used in citric acid production by Aspergillus niger strains.
Cultivation
Substrate Strain Substrate Concentration Yield References
Method
Aspergillus niger C–12 150 g·dm−3 SF 77.7% (m/m)
[26]
Sucrose Aspergillus niger C–12 150 g·dm−3 SF 81.2% (m/m)
Aspergillus niger NCIM705 60 g·dm−3 SF 30.7 g·dm−3 [162]
Aspergillus niger PM–1 150 kg·m−3 SF 121 kg·m−3 [86]
Glucose
Aspergillus niger Yang no. 2. 0.12 mg·dm−3 SF 15.4 mg·mL−1 [112]
Aspergillus niger ATCC 12846 100 g·dm−3 SF 0.3%
Aspergillus niger ATCC 26036 100 g·dm−3 SF 0.4%
Galactose Aspergillus niger ATCC 26550 100 g·dm−3 SF 0.1% [115]
Aspergillus niger IMI 31821 100 g·dm−3 SF 2.3%
Aspergillus niger IMI 83856 100 g·dm−3 SF 1.5%
Starch hydrolysates Aspergillus niger UE–1 15% (glucose equivalent) LSF 490 g·kg−1 [163]
Aspergillus niger GCB–47 150 g·dm−3 SF 45.1 g·dm−3
Starch [164]
Aspergillus niger GCMC 150 g·dm−3 SF 69.5 g·dm−3
Anhydrous glycerol Aspergillus niger W78B 150 g·dm−3 SF 59.0 g dm−3 [165]
Anhydrous glycerol Aspergillus niger PD66 100 g·dm−3 SF 64.2% (m/m) [166]
Anhydrous glycerol + sucrose Aspergillus niger PD66 135 g·dm−3 +15 g·dm−3 SF 95.80% (m/m) [167]
Table 5. Agricultural and industrial wastes used in citric acid production by Aspergillus niger strains.
Strain Cultivation
Substrate Yield References
Aspergillus niger Method
Aspergillus niger ATCC 9142 SSF 97.81 g·kg−1 [168]
Sugarcane bagasse Aspergillus niger 14/20 SSF 50.01 µg·g−1 [147]

Aspergillus niger DS 1 SSF 31.8% [169]
Aspergillus niger ATCC 9142 SF 106.65 g dm−3 [170]
Sugarcane molasses Aspergillus niger EB–3 SSF 0.112 mg·dm−3 [171]
Aspergillus niger GCMC–7 SF 96.1 g dm−3 [161]
Molecules 2024, 29, 22 18 of 38
Table 5. Cont.
Strain Cultivation
Aspergillus niger A20 SLF 29.7 g dm−3
[172]
Beet molasses Aspergillus niger A20 SF 8.6 g dm−3
Aspergillus niger W78B SF 110 g dm−3 [173]
Aspergillus niger FUO–2 SF 88.73 g dm−3 [136]
Cassava
Aspergillus niger NRRL 2001 SSF 88.0 g·kg−1 [36]
Pineapple waste Aspergillus niger DS 1 SSF 54.2% [174]
Aspergillus niger NRRL 567 SSF 65.6
Apple waste [151]
Aspergillus niger NRRL 567 SF 8.3 g dm−3
Fruit waste–
Aspergillus niger SF 1.15 g dm−3 [123]
Parkia biglobosa
Palm oil Aspergillus niger IBO–103MNB SSF 337.94 g·kg−1 [149]
Starch Aspergillus niger ATCC 9142 SF 2.7 g dm−3 [139]
Whey Aspergillus niger ATCC 9642 SFC 2.43 g dm−3 [175]
Date syrup Aspergillus niger J4 SF 56.7 g dm−3 [176]
Peat Aspergillus niger NRRL 567 SF 82.0 g·kg−1 [177]
Aspergillus niger ATCC 9142 SSF 6.15 g·kg−1 [178]
Distillery stillage
Aspergillus niger ATCC 201122 SF 71.63% [179]
Molasses (14%) + corn starch (14%) +
Aspergillus niger NCIM 1055 SF 0.13 mg·dm−3 [121]
sucrose (5%)
Corn starch + sucrose (15%) Aspergillus niger SSF 138.24 g·kg−1 [134]
Date waste + whey Aspergillus niger ATCC 6275 SF 32.4 g dm−3 [180]
Orange waste + cane molasses Aspergillus niger von Tiegh 1867 SF 640 g·kg−1 [181]
Grape waste + sucrose (15%) 34.4 g·kg−1
Aspergillus niger SSF [182]
Lime waste + sucrose (15%) 28.6 g·kg−1
Apart from cane and beet molasses, starch-based and lignocellulosic products are
considered inexpensive and readily available carbon sources due to their high carbohydrate
content [106,121,183,184]. However, these resources are limited due to contamination with
heavy metals, amino acids, or proteins and the need for hydrolysis. Citric acid biosynthesis
from non-hydrolyzed starch-based materials can be conducted using amylolytic strains
of Aspergillus niger. This process, however, is characterized by low efficiency [90,106]. For
starch-based materials, enzymatic hydrolysis includes treatment with α-amylase, amy-
loglucosidase, isoamylase, or pullulanase [90]. For cellulose hydrolysis, β-endoglucanase,
β-exoglucanase, and β-glucosidase are used [183].
There are reports in the literature on using Aspergillus niger strains for citric acid
biosynthesis using glycerol as the sole carbon source, but this issue has not been widely
studied [107,157,158,160,165]. The use of pure glycerol as the primary carbon source was
investigated by Foryś et al. Their studies obtained 59.0 g·dm−3 of citric acid produced with
a yield of 0.39 g·g−1 [136].
In studies using glycerol as a carbon source in Aspergillus niger cultures, it was mainly
used with other substrates. Schneider et al. used glycerol as an additive in concentrations
ranging from 0 to 40% in solid substrates composed of waste from tung oil production as
the primary carbon source. They achieved the highest yield (350.0 g·kg−1 ) after seven days
of cultivation with a 20% glycerol addition [158].
Bauwelers and Grosenker, in surface cultures using molasses as the primary carbon
source with a 30% addition of waste glycerol, which had been previously treated with CaO
at a concentration of 3.0 g·kg−1 , achieved a 95% yield in citric acid biosynthesis. In their
Molecules 2024, 29, 22 19 of 38
submerged cultures, using substrates containing 70% cassava flour, 20% cornmeal, and
10% waste glycerol, they obtained an 88% yield in citric acid biosynthesis. However, in
substrates containing 60% cassava flour, 20% cornmeal, and 20% waste glycerol, the process
yield was slightly lower, at 85% (Table 6) [157].
Table 6. Examples of using glycerol for citric acid biosynthesis by strains of Aspergillus niger.
Strain Cultivation
Molasses (70%) + waste glycerol (30%) SLF 95%
Cassava flour (70%) + corn flour (20%)
SF 88%
+ waste glycerol (10%)
Aspergillus niger [157]
Cassava flour (60%) + corn flour (20%)
SF 85%
+ waste glycerol (20%)
Glucose (80%) + waste glycerol (20%) SF 90%
Waste glycerol Aspergillus niger PD66 SF 6.2% (m/m) [166]
Waste glycerol Aspergillus niger PD66 SF 114.14 g dm−3 [185]
There is limited scientific literature on the biosynthesis of citric acid by Aspergillus

niger strains on glycerol-based substrates, even though they are considered the best acid
producers and are used in industrial production. The lack of interest is likely due to the
belief that glycerol slows down the growth rate of filamentous fungi and is not conducive
to citric acid production by Aspergillus niger [186].
The intensive growth of research into glycerol biotransformation by microorganisms is
driven by the challenge of managing the glycerol phase resulting from biodiesel production
and the increasing demand for industrially valuable products, such as citric acid, docosa-
hexaenoic acid, propionic acid, lactic acid, and dihydroxyacetone. Glycerol metabolism
is of great importance due to the production of double the amount of reducing equiva-
lents compared to glucose metabolism, indicating that glycerol provides more energy for
further conversions [187].
In addition to the type of carbon source, its concentration also plays a significant role
in the process of citric acid biosynthesis. High efficiency and rapid citric acid biosynthesis
are achieved with carbohydrates rapidly taken up and metabolized by filamentous fungi.
The effect of carbon source concentration and type on citric acid accumulation depends
on the properties of phosphofructokinase-1. Under physiological conditions, its activity is
inhibited by citric acid at concentrations of 1–5 mM. However, this inhibition of enzyme
activity does not occur during fermentation. This is because high concentrations of sucrose
or glucose are used, which leads to an increase in fructose-2,6-bisphosphate, a potent acti-
vator of phosphofructokinase-1. High carbohydrate concentrations also induce premeases,
allowing for the rapid uptake of carbon and, as a result, glycolysis. Also, high carbon
source concentrations significantly regulate pyruvate carboxylase activity [49,188,189].
Carbohydrates in concentrations exceeding 200.0 g·dm−3 lead to a reduction in the rate
of citric acid biosynthesis. This reduction may be due to an increase in fungal biomass
concentration, elevated medium viscosity, and the synthesis of polyalcohols. On the other
hand, carbohydrate concentrations below 50.0 g·dm−3 result in low citric acid biosynthesis
efficiency and the accumulation of oxalic acid [50,190,191].
5. Application of Citric Acid in the Food Industry

From a health quality perspective, citric acid, when used as a food additive, has been
approved as generally recognized as safe (GRAS) by the FAO/WHO Expert Committee
on Food Additives, and its Acceptable Daily Intake (ADI) does not require limitation [54].
Derivatives of citric acid, such as calcium citrate, iron citrate, manganese citrate, potassium
citrate, sodium citrate, diammonium citrate, isopropyl citrate, and stearyl citrate, have also
received GRAS status as food additives [13].
Molecules 2024, 29, 22 20 of 38
Citric acid is characterized by its low production cost, easy accessibility, non-toxicity,
biocompatibility, universality, and the safety of its decomposition products. It finds wide
applications in the food, pharmaceutical, biomedical, chemical, agricultural, and environ-
mental protection industries [92]. Examples of citric acid applications in the food industry
and other sectors are presented in Tables 7 and 8. In food products, citric acid serves various
functions, including acidity regulation, preservation, antioxidant properties, emulsification,
flavor and aroma enhancement, buffering, and antibacterial activity. Citric acid’s ability to
chelate metal ions and its buffering properties, when combined with citrates, make it an
ideal additive in food and nutraceutical production [42,192,193].
Citric acid plays a significant role as an antioxidant in oil production and in limiting
the oxidation of lipids in meat processing. It inhibits lipid oxidation by forming bonds
between pro-oxidative metal ions and the carboxyl or hydroxyl groups of the acid. The
antioxidant activity of citric acid in food depends on the dose applied and increases with
higher acid concentrations [194].
In meat processing, citric acid reduces the pink color and increases the brightness of
heat-treated meat. In cooked meat, citric acid limits the endogenous pink color and the
color induced by sodium nitrite and nicotinamide. Reduction of the pink color in meat may
also result from the chelation of heme iron by citric acid, preventing heme from binding
with ligands that cause a pink color [195,196].
Table 7. Examples of citric acid applications in the food industry.
Industry Application References

Beverages—wines, juices, non-alcoholic Used as an acidity regulator in carbonated and
[12,197]
beverages, syrups non-carbonated beverages, a buffering agent, pH regulator
Used as an antioxidant, antibacterial agent, controlling sugar
Sweets—jams, jellies,
inversion and product pH for optimal gelling, preservative, [192]
candies
providing a bitter taste and enhancing flavor
Sodium citrate is used in cream production to stabilize casein,
prevent the formation of creams during hot milk beverage
Dairy products production, and act as an emulsifier to stabilize the water and [14]
oil phases in cheese production. Aqueous solutions of citric
acid are used for milkstone removal from equipment
It acts as a chelating agent, helping maintain the natural color
and prevent discoloration of preserved meats; acts as an
Meat products antioxidant and synergist for antibacterial agents; [32]
Sodium citrate is used in slaughterhouses to prevent
coagulation or clotting of fresh blood
Citric acid, along with ascorbic acid, inhibits enzyme activity
Fruit and vegetable industry and oxidation reactions that may deteriorate [192]
colors and flavors
Used in the deodorization and hydrogenation of oil to chelate
metal ions, catalyze the rancidity of fats, interrupt the
Oils [37]
formation of peroxides and other oxidation products in the
auto-oxidation of oils
Prevents discoloration and the development of unwanted
Seafood [12]
odors by chelating metals
Citric acid and its salts are widely used in the food industry to prevent enzymatic
browning [198]. Enzymatic browning of fruits and vegetables is a phenomenon that reduces
shelf life and influences consumer decisions [199,200].
Citric acid is used as an additive in rinse water before deep freezing and in fruit
syrups [201]. Adding citric acid to stored fruits and vegetables positively affects color
retention and organoleptic quality and extends their shelf life. It can also be combined with
other anti-browning agents, such as ascorbic acid [202].
The inhibition of citric acid’s polyphenol oxidase (PPO) activity is due to its pH-
lowering capacity. PPO activity gradually decreases with increasing citric acid concen-
trations. However, the citric acid concentration needed to inhibit PPO activity varies
Molecules 2024, 29, 22 21 of 38
depending on the PPO activity and buffer solutions used [198]. Radish slices immersed in a
0.3% aqueous citric acid solution showed no browning during storage. Querioz et al. found
that a citric acid concentration of 100 mM inhibited the PPO activity of cashew apples [199].
However, a 10 mM citric acid solution inhibited banana PPO [148].
Citric acid also contributes to a decrease in the thermodynamic parameters of polyphe-
nol oxidase. This is believed to be due to a reduction in the stability of PPO and the number
of non-covalent bonds in the enzyme’s structure, leading to changes in the protein’s sec-
ondary and tertiary structure [198].
Citric acid can be considered a substance capable of controlling cellular respiration and
contributing to the better preservation of fruits and vegetables during storage [200,203].
Table 8. Examples of citric acid applications in various industries.
Industry Application References

It is used as an anticoagulant, effervescent in
Medicines, pharmaceutical combination with bicarbonates or carbonates, a
Pharmaceutical industry [8,14,204,205]
preparations, blood banks flavoring agent, and a stabilizer. It imparts the desired
sour taste, which helps mask medicinal flavors
It is added to hair care products, cosmetics, and
detergents for pH regulation and used as a stabilizer,
Cosmetics industry Detergents, cosmetics [14,206]
buffering agent, and chelating agent to
prevent discoloration
Enhances the bioavailability of mineral nutrient
chelates, improves taste, regulates stomach pH, and
Animal feeds [207]
enhances the effectiveness of animal feeds; used as a
flavor enhancer in pet food
Agriculture
Forms chelate with Fe, Cu, Mg, and Zn, used for soil
correction, increase phosphorus availability to plants,
Fertilizers are employed to remove lead from contaminated soils, [208]
and are used for copper chelation in algaecides for
water reservoirs
It is used for pH regulation, as a buffer, and as a
Textile industry
chelating agent in the dyeing process
[37]
Cleans steam boiler from metal oxides and purifies
Metallurgical industry
iron and copper oxides used in nuclear reactor welding
It is used as a chelating agent to control the metal
Other applications Electroplating
deposition rate on substrates
in industry
Utilized as a copolymer in nanomaterials to
Biomedical engineering [209]
encapsulate biologically active compounds
Solutions of citric acid are used to remove iron,
Water purification calcium, and other cations that damage cellulose [210]
acetate membranes used in reverse osmosis systems
5.1. Newly Emerging Applications of Citric Acid

Research into new applications of citric acid in various industries is currently the
subject of many studies [211–216]. One of the new applications of citric acid is the pro-
duction of household detergents. Citric acid chelates Mg2+ and Ca2+ ions responsible for
water hardness and does not contribute to the eutrophication of aquatic systems, unlike
phosphates used in detergents [217]. New and innovative applications of citric acid in the
food industry and beyond are expected to lead to increased production.
5.1.1. Cross-linking Agent and Plasticizer

Citric acid can successfully be used in the process of cross-linking proteins [218],
polysaccharides [219], and hydroxyapatite [220]. A breakthrough in using citric acid as a
cross-linking agent came with the discovery by Rothenberg and Alberts from the University
of Amsterdam. They demonstrated that glycerol and citric acid can polymerize, creating a
water-soluble, biodegradable, and thermosetting resin. The combination of citric acid and
Molecules 2024, 29, 22 22 of 38
glycerol at temperatures above 100 ◦ C and below 130 ◦ C under normal conditions leads to
the formation of polyester resins through the Fischer esterification reaction [221].
The use of citric acid as a compatibilizer for various polysaccharides, including starch, ther-
moplastic starch, cotton, chitosan, and cellulose, is justified by its multi-carboxyl structure [222].
This allows it to be used as a cross-linking agent, plasticizer, and |hydrolyzing agent [223].
The mechanism of the cross-linking reaction is based on the well-known Fischer
esterification reaction between the carboxyl groups of citric acid and the hydroxyl groups
of starch [224]. Citric acid can react with all three hydroxyl groups of starch. Esterification
between starch and citric acid leads to mono-, di-, and tri-esters forming. Esterification
primarily occurs within the branching points of amylopectin [225]. The formation of ester
bonds can be catalyzed by reducing the pH or adding Lewis acids, which are chemical
compounds capable of accepting an electron pair from a base [224]. In many previous
studies on starch cross-linking, temperatures above 100 ◦ C have initiated the cross-linking
process [226]. Heating citric acid causes dehydration and the formation of an anhydride,
which can react with starch to form starch citrate. With further heating, the citrate undergoes
dehydration, and cross-linking can occur [227,228].
It is possible to conduct cross-linking reactions at lower temperatures (around 70 ◦ C)
using higher concentrations of citric acid. However, the efficiency of the reaction under
these conditions is low because only a tiny amount of added citric acid participates in
the cross-linking reaction. Furthermore, citric acid that has not reacted can act as a plas-
ticizer [225]. As a plasticizer, citric acid increases the tensile strength of starch films. The
improvement in tensile strength is more significant than when glycerol is used as a plas-
ticizer [229]. Excessive citric acid concentration does not interact with starch molecules
but can react with water, disrupting hydrogen bonds and reducing the matrix’s cohe-
sion. This results in increased water solubility, susceptibility to deformation, and reduced
thermal resistance [153].
FTIR spectroscopy and X-ray diffraction of starch films with citric acid have shown
that citric acid can effectively inhibit starch recrystallization or retrogradation due to
strong hydrogen bonding between starch and citric acid [224]. Additionally, citric acid-
cross-linked starch films exhibit significantly higher tensile strength, up to 150% more
than non-cross-linked films. However, achieving the appropriate increase in material
strength requires an optimal amount of citric acid [230]. Citric acid concentrations below
5% act as a cross-linking agent and enhance the tensile strength of starch films. When
the concentration increases from 5% to 30%, tensile strength decreases, but flexibility and
material adhesiveness increase. This suggests that excess free citric acid is a plasticizer [231].
The main disadvantage of citric acid as a cross-linking and plasticizing agent in starch
barrier films is starch degradation due to acid hydrolysis. Acid hydrolysis of starch glyco-
sidic bonds involves the cleavage of these bonds, resulting in the protonation of oxygen
and the addition of a water molecule, leading to the formation of a reducing sugar group.
Effectively preventing starch hydrolysis during cross-linking in the presence of citric acid
can be achieved by maintaining a pH of 4 or lower and a temperature below 105 ◦ C [224].
As a cross-linking agent, citric acid strengthens bonds by incorporating covalent bonds
that complement intermolecular hydrogen bonds, improving the resistance of starch films
to moisture. Strong hydrogen bonds between the carboxyl groups of citric acid and the
hydroxyl groups of starch result in improved interactions between molecules and reduced
solubility of the films in water [153].
When citric acid is added in the range of 1% to 10% to thermoplastic starch, it sig-
nificantly reduces water vapor permeability. This effect is due to replacing hydrophilic
groups with hydrophobic ester groups that impede the diffusion of water vapor molecules
through the matrix. However, water vapor permeability increases when the citric acid
concentration exceeds 10%. This can be attributed to the plasticizing effect caused by an
excess of citric acid. Increased citric acid concentration leads to enhanced chain mobility
and increased interchain spaces, resulting from the attachment of free citric acid to the
Molecules 2024, 29, 22 23 of 38
polymer chain. As a result, the water vapor diffusion coefficient increases, accelerating
water vapor penetration through the starch films [232–234].
Starch films cross-linked with citric acid have higher thermal resistance [226]. The
reason for this is that the cross-links are responsible for the resistance of cross-linked films.
Cross-linked starch films exhibit significantly improved thermal resistance at temperatures
above 320 ◦ C [230].
The three carboxyl groups and one hydroxyl group in citric acid also allow it to
cross-link glycerol, cellulose, and sebacic acid through condensation reactions, forming
ester copolymers capable of drug delivery [8]. Gentamicin incorporated into the poly-
mer effectively kills bacteria. Citric acid delivers ketoconazole as a cross-linking agent
for beta-cyclodextrins on hydrogel hydroxypropylmethylcellulose (HPMC) membranes.
The formation of drug-cyclodextrin complexes contributes to increased solubility and
bioavailability of poorly soluble drugs [235].
5.1.2. Citric Acid in the Synthesis of Deep Eutectic Solvents

Deep eutectic solvents (DES) are homogeneous mixtures of two or more components
capable of interacting with each other as a hydrogen bond acceptor (HBA) and a hydrogen
bond donor (HBD). Deep eutectic solvent mixtures are formed by mixing two or more
components in the appropriate molar ratio in the presence of heat. Additionally, this process
does not require an additional purification step [236,237]. Deep eutectic solvents are one
of the most promising discoveries in “green chemistry”. They can serve as an alternative
to conventional organic solvents and have numerous advantages, such as renewability,
reusability, biodegradability, non-toxicity, widespread availability, shallow vapor pressure,
low flammability, and ease of preparation. Moreover, the components that produce deep
eutectic solvents are inexpensive and safe [238,239].
Deep eutectic solvents have been divided into four types depending on their composi-
tion. Types I, II, and IV contain metal salts and are considered toxic and less sustainable
than type III deep eutectic solvents. Type III deep eutectic solvents are synthesized from
readily biodegradable and regenerable raw materials such as feed additive (choline chlo-
ride, ChCl), fertilizer (urea), antifreeze (ethylene glycol), sweetener (glycerol), and plant
metabolites (sugars, sugar alcohols, and organic acids) [240]. The most frequently studied
eutectics in the literature are type III, based on the combination of quaternary ammonium
salts and a compound serving as a hydrogen bond donor. Type III is the most commonly
used deep eutectic solvent due to the strong interaction of hydrogen bonds between the
hydrogen bond acceptor (HBA) and the hydrogen bond donor (HBD). Many compounds
have been successfully utilized to create deep eutectic solvents. HBAs are mainly quater-
nary ammonium or phosphonium salts, while HBDs are most commonly amides, alcohols,
and carboxylic acids. Citric acid is one of the most commonly employed HBDs among
carboxylic acids [237]. The most popular systems for producing deep eutectic solvents
using choline chloride and citric acid are 1:1, 1:2, and 2:1 [241,242]. The presence of hy-
droxyl and carboxyl groups allows for the formation of sufficiently strong hydrogen bonds.
Deep eutectic solvents based on choline chloride and carboxylic acids demonstrate greater
extraction efficiency than traditional solvents such as water and ethanol [243].
Various molar ratios of choline chloride and citric acid monohydrate significantly
influence the physicochemical properties of deep eutectic solvents. Adding citric acid
monohydrate increases viscosity, surface tension, and density. Deep eutectic solvents with
a higher molar ratio of choline chloride exhibit a higher melting point. Citric acid-based
deep eutectic solvents can find broad industrial applications, particularly in extracting
hydrophilic components from plant or animal materials [243].
In the studies conducted by Kurtulbaş et al., deep eutectic solvents were intentionally
designed, incorporating a hydrogen bond donor (HBD) (glycerol and ethylene glycol) and a
hydrogen bond acceptor (HBA) (citric acid) in a specified molar ratio (1:4) for the extraction
of bioactive compounds (phenols and anthocyanins). In the current investigation, Hibiscus
sabdariffa was extracted using microwave-assisted extraction (MAEX). The most effective
Molecules 2024, 29, 22 24 of 38
extract from Hibiscus sabdariffa was obtained from a mixture of citric acid and ethylene
glycol through microwave-assisted extraction [239].
Hu et al., investigated the molecular mechanisms of isoliquiritigenin extraction using
deep eutectic solvents of choline chloride and citric acid. The results indicated that deep
eutectic solvents exhibited higher efficiency in isoliquiritigenin extraction as an extraction
solvent than ethanol with water. Additionally, the increased efficiency in isoliquiritigenin
extraction was primarily attributed to the strong interaction between isoliquiritigenin and
the extraction solvent and the rapid diffusion of isoliquiritigenin [237].
5.1.3. Antibacterial Agent

Using organic acids to control bacterial flora in food, extend shelf life, and improve
the safety of plant- and animal-derived products has become a common practice in the
food industry. In Europe, the legal basis for using organic acids as agents contributing to
the safety of animal-derived products is Regulation 853/2004 of the European Parliament
and the Council [244–246].
Citric acid effectively combats pathogenic microflora in fresh and processed pork,
beef, poultry, and fresh vegetables and fruits [247–251]. Its antibacterial activity involves
penetrating through the cell membranes, where the pH is higher than in the surrounding
environment. The mechanism of citric acid’s antibacterial action is related to acidifying the
cytoplasm, disrupting metabolic processes, or accumulating the dissociated acid anion to a
toxic level (Figure 12) [252,253]. Organic acids are weak acids, so they do not completely
dissociate in an aqueous environment, and their microbiological activity depends on the
degree of dissociation and the pH of the food product. Reducing the pH increases the
concentration of the acid, reduces the polarity of the molecules, improves acid diffusion
through microbial cell membranes into the cells, and consequently increases antibacterial
activity [253,254]. The effectiveness of organic acids also depends on the acid concentration,
acid properties, temperature, exposure time, and microbial susceptibility [251,255].
Figure 12. The mechanism of citric acid’s action on a bacterial cell [253].
Figure 12. The mechanism of citric acid’s action on a bacterial cell [253].
Citricacid
Citric acidinin concentrations
concentrations ranging
ranging from
from0.1 0.1toto3.0%
3.0%restricts the
restricts thegrowth
growth of bacteria
of bac-
suchsuch
teria as Listeria monocytogenes,
as Listeria Escherichia
monocytogenes, coli, coli,
Escherichia Salmonella typhimurium,
Salmonella typhimurium, and and
Vibrio para-
Vibrio
haemoliticus [244,247,249,251].
parahaemoliticus [244,247,249,251].
Citricacid
Citric acidexhibits
exhibitssynergistic
synergistic effects
effects when
when usedused in mixtures
in mixtures withwith
otherother organic
organic ac-
acids.
ids. A mixture of caprylic and citric acids significantly inhibits the growth
A mixture of caprylic and citric acids significantly inhibits the growth of bacteria. The of bacteria. The
synergybetween
synergy betweencitric
citric and
and caprylic acids is
is associated
associatedwith withthe
theloss
lossofofcell membrane
cell membrane in-
tegrity and changes in its permeability. The mechanism of the synergistic action of both
acids involves damaging or destabilizing the cell membrane, leading to increased perme-
ability and, consequently, cell death. Damage to the bacterial membrane allows hydrogen
ions to penetrate, resulting in a strong bactericidal effect [248,256,257].
Combining citric acid with other decontamination methods, such as ozonation, UV-
Molecules 2024, 29, 22 25 of 38
integrity and changes in its permeability. The mechanism of the synergistic action of
both acids involves damaging or destabilizing the cell membrane, leading to increased
permeability and, consequently, cell death. Damage to the bacterial membrane allows
hydrogen ions to penetrate, resulting in a strong bactericidal effect [248,256,257].
Combining citric acid with other decontamination methods, such as ozonation, UV-C
radiation, and ultrasonication, can significantly impact the inactivation of microorganisms
in fresh food [41].
The effectiveness of citric acid’s antibacterial action varies and depends on many
factors. Citric acid exhibits optimal antibacterial effects in a low pH environment, at low
temperatures, and when used in high concentrations. Available literature sources report
that using citric acid at concentrations above 2% may cause adverse sensory changes in
food products [253,258]. The antibacterial effectiveness of citric acid also depends on the
initial amount of microflora on the product’s surface. Depending on the initial bacterial
count, citric acid reduces the number of microorganisms by 1 to 2 log cfu·g−1 [244].
5.1.4. Deamidation of Gluten

Wheat gluten is widely used in the food industry, serving various purposes, such as
emulsifiers and imparting cohesiveness and elasticity. However, its utility is limited due to
its low solubility under neutral conditions. A practical method to enhance the properties of
gluten is deamidation using carboxylic acids, including citric acid [259,260].
The deamidation reaction transforms amidic groups into carboxylic groups, mainly
glutamine and asparagine residues. This transformation results in increased electrostatic
repulsion, the disruption of hydrogen bonds, and the dissociation of polymers, ultimately
improving the solubility of gluten. While treating gluten with citric acid, the availability
of peptide bonds and hydrogen ions influences the competition between hydrolysis and
deamidation. The degree of hydrolysis of deamidated gluten decreases with increased
citric acid concentration and treatment time, contributing to an increase in soluble protein
content after deamidation [215,261]. Deamidation by citric acid increases the solubility
of gluten to around 70% at pH 7. The shift of the protein’s isoelectric point towards
acidic pH confirms that deamidation increases the quantity of protein polyelectrolytes,
resulting in improved solubility at neutral pH. Deamidation involves the cleavage of
peptide bonds, indicating that it is primarily responsible for increasing protein solubility
rather than hydrolysis [261,262].
Deamidation of gluten with citric acid significantly enhances its emulsifying, foaming,
and elasticity properties. Emulsions stabilized with deamidated gluten feature smaller
emulsion droplet sizes, indicating their ability to reduce surface tension. High emulsion
stability results from the increased flexibility of the gliadin molecule or its molecular
rearrangement [261]. The improvement in the foaming capacity of deamidated gluten is
attributed to the increased molecule flexibility, leading to enhanced protein adsorption
and anchoring at phase boundaries [263,264]. Deamidation also leads to changes in the
secondary conformation of the protein, driven by increased electrostatic repulsion and a
reduction in hydrogen bonds [262]. The secondary structure of gluten consists of 34.5%
α-helices, 17.3% β-turns, and 44.8% β-sheets. Deamidation increases α-helices and β-turns
while reducing β-sheets [260,263].
Citric acid exhibits a solid capability to break peptide bonds in gluten, which leads to
a transformation in the tertiary structure of the protein. Protein fractions of gluten with
a higher molecular weight are more susceptible to degradation than those with a lower
molecular weight. After deamidation with citric acid, the presence of sulfhydryl groups in
gluten has been confirmed, while the tertiary structure becomes less compact [263,264].
Deamidation with citric acid can contribute to the improvement of the nutritional
properties of gluten. From a nutritional standpoint, gluten is not considered a good protein
source due to its deficiency in lysine and threonine. Deamidation with citric acid increases
the overall quantity of essential amino acids, including lysine [261].
Molecules 2024, 29, 22 26 of 38
5.1.5. Extractant
Citric acid can be an effective pectin extractor from fruit pomace instead of toxic
mineral acids such as sulfuric or nitric acid. Pectin extraction using citric acid can reduce
waste from fruit and vegetable processing and limit the harmful environmental impact of
wastewater from conventional extraction methods [265,266].
The process of pectin extraction typically occurs at around 97 ◦ C with a pH of 2.5
using water acidified with citric acid [193,265].
Extracting pectin from citrus fruit peels using citric acid allows for obtaining pectins
with a high galacturonic acid content, essential for their application as a gelling agent.
Pectins also have a high molecular weight, indicating a high content of neutral sugars.
The viscosity of the extracted pectins increases with a higher concentration of citric acid.
Although the extraction process may yield lower efficiency than traditional methods, it
results in pectins of high purity [267].
The pectins extracted from cocoa husks were characterized by a high degree of acety-
lation, contained rhamnogalacturonan, and had side chains rich in galactose. Despite their
high degree of acetylation, these pectins formed gels in a low pH environment and at a high
glucose concentration, suggesting their potential use as an additive in acidic products [268].
5.1.6. Inhibition of Protein Adhesion

The issue of protein adhesion to steel surfaces poses numerous challenges in food
production and processing. Proteins adhering to equipment surfaces can serve as a nutrient
source for microorganisms, leading to product contamination. Removing allergens and
preventing cross-contamination is a critical point in the food production process. One
method to inhibit the adhesion of chicken egg white proteins to stainless steel surfaces is to
use a citric acid solution [269].
The effect of inhibiting protein adhesion by citric acid involves changing the surface
charge of steel from positive to negative in an environment with a pH of 7.4 due to the
attachment of dissociated carboxyl groups from the acid. This leads to the repulsion of
negatively charged protein molecules such as ovalbumin or ovomucoid [216,269].
6. Global Citric Acid Market

Acidity regulators are a significant part of the food additives industry, as they im-
pact the taste of food and contribute to its shelf life. The acidity regulators market is
estimated to reach USD 7.29 billion in 2023, with a CAGR of 7.09%. Citric acid dominates
the acidity regulators market due to its wide applications in the non-alcoholic beverage
industry and the increasing societal preference for safe food [270]. In 2016, approximately
67% of the citric acid produced worldwide was used in the food industry, 16% in the
chemical industry, 8% in pharmaceuticals and cosmetics, and 7% in other sectors such as
textiles and metallurgy [271].
In the 1930s, American companies Miles and Pfizer led citric acid producers with
4900 tons [27]. However, by 1978, the combined production of Pfizer and Miles had reached
approximately 70,000 tons. In 1990, the annual production of citric acid had already reached
170,000 tons. Even though the largest citric acid producers in the 1990s were located in the
United States, Europe produced around 255,000 tons annually. At that time, North Amer-
ica had 215,000 tons, Asia produced 66,000 tons, Africa produced 14,000 tons, Australia
produced 8000 tons, and South America produced 7000 tons of citric acid per year [272].
The current citric acid market is considered the fastest-growing segment in the food
additives industry, driven by its wide range of applications in various industries and the
increasing use of citric acid as a cleaning agent. The global citric acid market increased
from USD 3.87 billion in 2022 to USD 4.2 billion in 2023. The Russia-Ukraine war has
ruined, at least in the short term, the chances of reviving the global economy after the
COVID-19 pandemic. The war between the two countries has led to economic sanctions on
many countries, rising commodity prices, and supply chain disruptions, causing inflation
in goods and services and affecting many markets worldwide. The citric acid market is
The current citric acid market is considered the fastest-growing segment in the food
additives industry, driven by its wide range of applications in various industries and the
increasing use of citric acid as a cleaning agent. The global citric acid market increased
from USD 3.87 billion in 2022 to USD 4.2 billion in 2023. The Russia-Ukraine war has ru-
ined, at least in the short term, the chances of reviving the global economy after the
Molecules 2024, 29, 22 COVID-19 pandemic. The war between the two countries has led to economic sanctions 27 of 38
on many countries, rising commodity prices, and supply chain disruptions, causing infla-
tion in goods and services and affecting many markets worldwide. The citric acid market
is expected
expected to grow
to grow to USD
to USD 5.525.52 billion
billion in 2027
in 2027 (Figure
(Figure 13) [95].
13) [95]. TheThe global
global citric
citric acidacid mar-
market
ket reached
reached a volume
a volume of 2.8 million
of 2.8 million tons intons
2022,inand
2022, and forecasts
forecasts suggest suggest that by
that by 2028, the2028,
market the
market
will reachwill
3.3 reach 3.3tons,
million million tons, indicating
indicating a CAGR of a CAGR of 2.78%
2.78% for for 2022–2028
2022–2028 [270]. [270].
GLOBAL CITRIC ACID MARKET

FORECAST
5.52
4.20
3.87
Billion USD
2022 2023 2027
Figure13.
Figure 13.Global
Globalcitric
citricacid
acidmarket
marketforecast.
forecast.
Theglobal
The global citric
citric acid
acid market
market is divided
is divided among
among North
North America,
America, Latin
Latin America,
America, West-
Western
ern Europe,
Europe, Eastern
Eastern Europe,Europe, and Asia-Pacific,
and Asia-Pacific, excluding
excluding Japan (APEJ).
Japan (APEJ). In 2016,In
the2016,
APEJthe APEJ
region
dominated the citric acid market in regards to value. Regarding market size, Western
Europe (including the United Kingdom, Germany, Italy, France, and Spain) led with
production exceeding 500,000 tons. Following it were the APEJ region, North America
(including the United States, Canada, and Mexico), and the rest of the world. The global
COVID-19 pandemic significantly impacted the citric acid market in 2020. This resulted in
a sharp increase in the sales of detergents and cleaning products and a substantial increase
in the demand for citric acid [270]. The citric acid market is experiencing significant
growth in Western Europe, driven by the established food and beverage sector, where
citric acid is widely used as a preservative and flavor enhancer. The growing trend toward
natural and organic products in countries such as Germany, France, and the UK creates
opportunities for citric acid. Furthermore, the pharmaceutical sector in Western Europe is
highly advanced and rigorously regulated, creating demand for trusted and high-quality
ingredients, including citric acid, for use in medicines and dietary supplements [3]. On a
global scale, the Asia-Pacific region is the largest consumer of citric acid, accounting for
28%. North American countries, with a consumption rate of 23%, come second, while
Western Europe, with a share of 22%, ranks third [271].
Key players in the citric acid market include Archer Daniels Midland Company
(Chicago, IL, USA), Cargill Inc. (Shanghai, China), Tate & Lyle PLC (London, UK), Jung-
bunzlauer Suisse AG (Des Plaines, IL, USA), Cofco Biochemical (Anhui) Co., Ltd. (Bengbu,
China), Huangshi Xinghua Biochemical Co. Ltd. (Huangshi, China), RZBC Group Co.
Ltd. (Rizhao, China), Weifang Ensign Industry Co., Ltd. (Weifang, China), Gadot Bio-
chemical Industries Ltd. (Haifa, Israel), S.A. Citrique Belge N.V. (Tienen, Belgium) [273].
Manufacturers focus on diversifying and delivering high-quality products that meet cus-
tomer requirements to maintain a competitive edge. Furthermore, companies compete
by investing in developing next-generation products with high solubility and acceptable
taste. An example is Gadot Biochemical Industries, which introduced a new product
called Cal2 Mg in 2022, a combination of calcium and magnesium citrate. In the same year,
Jungbunzlauer introduced a new product called monomagnesium citrate, a one-to-one
molar ratio magnesium salt mainly used as a mineral source in functional foods, beverages,
and dietary supplements [274].
Molecules 2024, 29, 22 28 of 38
Currently, a challenge in the citric acid market is the pressure from imports originating
in Asian countries, which has decreased selling prices. Additionally, European producers
contend with high production costs. In 2021, the European Commission reaffirmed deci-
sions made in 2016 and re-imposed anti-dumping duties on importing citric acid from the
People’s Republic of China and Malaysia [275].
7. Conclusions
This literature review discusses the properties of citric acid, cultivation conditions
primarily for Aspergillus niger, differences between filamentous fungi Aspergillus niger
and yeast Yarrowia lipolytica, extensive applications of citric acid in the industry, and the
global market for citric acid. The review indicates a growing interest in the microbiolog-
ical production of citric acid and technological advancements in this area, aligned with
market demands.
Additionally, the article emphasizes the use of waste substrates, reflecting the de-
velopment of a closed-loop economy model. Such a model can reduce economic and
environmental costs for producing enterprises. The aspect of exploring new, more cost-
effective substrates from the agri-food industry could contribute to cost reduction and
solutions for waste disposal issues.
A multifaceted view of citric acid arises from its wide-ranging applications in various
industrial sectors and the exploration of new uses. The continuous increase in demand for
citric acid is associated with the most economically viable industrial production process,
characterized by high sensitivity, complexity, and dependence on parameters such as
microorganisms used in substrates and fermentation techniques. In the bioproduction of
citric acid, there is significant interest in improving process efficiency at relatively low costs.
This serves as an incentive for the development of new technologies and innovations in the
design of bioreactors, process scaling, and control. Metabolic engineering can also improve
fermentation parameters, requiring substantial research efforts.
The review highlights the importance of Aspergillus niger as a versatile industrial
microorganism, which, in biotechnological studies, will undergo further investigations into
secondary metabolism, fermentation conditions in citric acid production, and enhancing
efficiency through CRISPR-Cas9 genome editing.
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The author declares no conflict of interest. The author alone is responsible for
the content and writing of this article.
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molecules

Molecules 2024, 29, 22. https://doi.org/10.3390/molecules29010022 https://www.mdpi.com/journal/molecules

2. Physical and Chemical Properties

Table 1. Chemical and physical properties of citric acid.

Properties Characteristic References

• In water, the solubility increases with temperature:

3. Citric Acid Biosynthesis

• Carl Wilhelm Scheele, a Swedish chemist, isolated citric acid from

•Jstus Liebig, a German chemist, recognized that citric acid is

•The first chemical synthesis of citric acid was performer by Girmaux

•Dunschmann and Pechmann synthesize citric acid from ethanedi

•Lawrence used the condensation of sthyl oxaloacetate with ethyl

•Carl Wehmer discovered that citric acid could be produced from

•First succesful fermentation proces using Penicillium at Les Produits

•The Currie – Thoma fermentation proces was comercialized by

•Studies on the influence of micro/macroelements in culture media

•Advances in molecular biology and genetics of Aspergillus niger are

Period 1997 – 2017 •Generation of non–homologous mutants

3.2. Microorganisms Producing Citric Acid

Molecules 2024, 29, 22 5 of 38

3.2.1. Production of Citric Acid Using Aspergillus niger Fungi

3.2.2. Citric Acid Metabolism in Aspergillus niger

Figure 4. Overview of pathways leading to citric acid production in A. niger.

3.2.3. Production of Citric Acid Using Yarrowia lipolytica Yeast

and fatty acids were introduced (Table 2; Figure 5) [63–65].

The main advantages of Yarrowia lipolytica strains compared to filamentous fungi

The accumulation of citric acid in yeast requires a deficiency in a nitrogen source

Figure 6. Metabolic pathways of Y. lipolytica.

4. Production of Citric Acid

Type of Cultivation Process Parameters Process Advantages Process Disadvantages References

4.1.1. Liquid Surface Fermentation Cultures

Molecules 2024, 29, x FOR PEER REVIEW 12 of 40

Molecules 2024, 29, x FOR PEER REVIEW 12 of 40

Figure 7. Schematic of liquid surface fermentation cultures.

Figure 8. Schematic of solid-state fermentation cultures.

Figure 8. Schematic of solid-state fermentation cultures.

acid production on solid-state substrates [89].

Molecules 2024, 29, x FOR PEER REVIEW 14 of 40

4.2. Factors Influencing Citric Acid Production

Ammonium compounds lead to an advantageous reduction in the pH of the culture

4.2.3. Trace Elements

4.2.4. Low-Molecular-Weight Alcohols

4.2.5. The pH Value

4.2.6. Aeration and Mixing Rate

Sugarcane bagasse Aspergillus niger 14/20 SSF 50.01 µg·g−1 [147]

There is limited scientific literature on the biosynthesis of citric acid by Aspergillus

5. Application of Citric Acid in the Food Industry

Table 7. Examples of citric acid applications in the food industry.

Industry Application References

Table 8. Examples of citric acid applications in various industries.

Industry Application References

5.1. Newly Emerging Applications of Citric Acid

5.1.1. Cross-linking Agent and Plasticizer

5.1.2. Citric Acid in the Synthesis of Deep Eutectic Solvents

5.1.3. Antibacterial Agent

5.1.4. Deamidation of Gluten

5.1.6. Inhibition of Protein Adhesion

6. Global Citric Acid Market

GLOBAL CITRIC ACID MARKET