Received: 29 July 2020 Revised: 11 December 2020 Accepted: 7 January 2021

DOI: 10.1002/fft2.66

REVIEW ARTICLE

Microbial citric acid: Production, properties, application, and

future perspectives

Bikash Chandra Behera1 Rashmiranjan Mishra2 Sonali Mohapatra3

1
School of Biological Sciences, National
Institute of Science Education and Research, Abstract
Bhubaneswar, India
Microbial citric acid is an important organic acid widely used in pharmaceutical food,
2
Department of Biotechnology, MITS School
beverage, detergents, and cosmetics industries. Although citric acid is produced by dif-
of Biotechnology, Bhubaneswar, India
3
Department of Biotechnology, College of
ferent types of microorganism, the filamentous fungus Aspergillus niger is a workhorse
Engineering & Technology, Bhubaneswar, India for the production of citric acid. In the present review, special attention has been paid
to address the advanced literature of citric acid production such as microorganism,
Correspondence substrates, screening methods, different fermentation techniques, different factors
Bikash Chandra Behera, School of Biological
affecting citric acid production, and product recovery as well as numerous biotech-
Sciences, National Institute of Science Edu-
cation and Research, Bhubaneswar, Odisha nological applications of citric acid are also discussed for simple understanding of the
752050, India.
subject.
Email: bikash@niser.ac.in

KEYWORDS
fermentation, genetic engineering, metabolism, microorganism

1 INTRODUCTION 2010). Further, CA is frequently incorporated in facial packs and masks

Citric acid (CA) is an organic acid that is generally found in a variety
of fruits such as limes, lemons, oranges, pineapples, and grapefruits. It
is a natural ingredient that aids in detoxification, maintaining energy
levels, and supporting healthy digestion and kidney function. It has a
slightly tart and refreshing flavor and is employed for balancing the
sweetness in soft drinks, juices, and other beverages. Citric acid used
in food and beverage (F&B) industry due to its antioxidant properties
to preserve the food or as an acidifier enhances the flavors and aro-
mas of fruit juices, ice cream, and marmalades. In the pharmaceutical
industry, it is used as an antioxidant to preserve vitamins, effervescent,
pH corrector, blood preservative, iron citrate tablets as a source of iron
for the body, ointments and cosmetic preparations, and so forth (Max
et al., 2010). In the chemical industry, for softening and treatment of
textiles, it is used as a foaming agent. In metallurgy, certain metals are
utilized in the form of citrate. Because of less eutrophic effect, CA is
used in the detergent industry as a phosphate substitute (Max et al.,
as it naturally brightens and lightens the skin tone, minimizes break-
outs and oiliness, and regenerates the dead skin cells. Currently, the
global CA market is projected to reach USD 3.2 billion by 2023 and
is expected to witness a Compound Annual Growth Rate (CAGR) of
5.1% during the forecast period (Market report world.com, 2020). The
global production of CA is estimated to be around 736,000 tones/year,
and the entire production is carried out by fermentation. In Brazil,
almost the entire demand of CA is met through imports. Due to its
numerous applications, the volume of CA production by fermentation
is continually increasing at a high annual rate of 5% (Finogenova et al.,
2005; Francielo et al., 2008) and also witnessing steadily increasing
demand/consumption. It is accepted worldwide as a GRAS (generally
recognized as safe) as approved by the Joint The Food and Agriculture
Organization (FAO)/World Health Organization (WHO) Expert Com-
mittee on Food Additives (Carlos et al., 2006; Rohr et al., 1996). The
driving factor behind the growth of the global CA market is the expand-
ing application in various industries. Development of CA production

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the original work is properly cited.
© 2021 The Authors. Food Frontiers published by John Wiley & Sons Australia, Ltd and Nanchang University, Northwest University, Jiangsu University, Zhejiang Univer-
sity, Fujian Agriculture and Forestry University

62 wileyonlinelibrary.com/journal/fft2 Food Frontiers. 2021;2:62–76.

BEHERA ET AL.
increased greatly since the last century due to biotechnology, which
provides proper knowledge of fermentation techniques and product
recovery; biochemistry, which provides knowledge of different factor
that affects synthesis and blockage of CA production; molecular regu-
latory mechanisms; and strategies that enhance CA production. In the
past 60 years, extensive reviews of literature along with more than
thousands of reports have been published in connection to CA produc-
tion (Max et al., 2010; Papagianni, 2007; Prescott & Dunn, 1959; Show
et al., 2015; Tong et al., 2019; Vandenberghe et al., 1999). However, the
enhancement of CA production with the recent advancement of the
last few years has not been updated.
Hence, this paper reviews recent developments on CA production
by presenting a brief summary of the subject, describing microorgan-
isms, production techniques, and substrates, and so forth.
2 PROPERTIES
The citric acid (CA) with systematic International Union of Pure
and Applied Chemistry (IUPAC) name 2-hydroxypropane-1,2,3-
tricarboxylic acid is an intermediate in the CA cycle in the metabolism
of all aerobic organisms. CA is a colorless white crystalline powder that
is practically odorless and exists in anhydrous form (C6 H8 O7 , molec-
ular weight 192.12) or monohydrate form (C6 H8 O7 .H2 O, molecular
weight 210.14). Anhydrous CA is highly soluble in water, freely soluble
in ethanol, and sparingly soluble in ether, whereas monohydrate CA
is soluble in water and sparingly soluble in ether. It is solid at room
temperatures, melts at 153◦ C, and boiling temperature is 310◦ C
(Kubicek, 1998). It decomposes with loss of carbon dioxide (CO2 )
above about 175◦ C. Once dissolved in water, it shows weak acidity
but a strong acid taste that affects sweetness and provides a fruity
tartness for which it is widely used to complement fruit flavors in the
F&B industry. In combination with citrate, the acid shows excellent
buffering capacity, while its excellent metal ions chelating properties
add to the physicochemical properties that make it ideally suited for
food, cosmetic, and nutraceutical and pharmaceutical applications,
whose number testifies to its exquisite versatility (Ciriminna et al.,
2017).
3 GLOBAL CA MARKET
In 1989, the world production of CA and citrate salts amounted to
about 0.5 million tonnes. In 2015, it exceeded 2 million tonnes, with
the global market expected to increase at 3.7% annual rate at least
until 2020 (Markit, 2015). In 2015, China accounted for 59% of world
production and 74% of world exports, hosting the largest produc-
ers. The sudden abundance of the product, with production output
almost doubled in the 2004 to 2013 decade, led to unprecedented low
prices that bottomed out at USD 700/t in 2015 (CCM, 2016). In recent
years, increasing inclination toward ready-to-drink beverages and pro-
cessed foods has been witnessed on account of economic growth, busy
lifestyles, increasing urbanization, and rising disposable incomes. This
63
has led to the heightened demand for CA in different industries. Addi-
tionally, the growing awareness about the adverse impacts caused by
chemicals used in everyday products has resulted in a shift toward
products made using natural ingredients. On account of these factors,
the market volume of CA is projected to reach USD 3.2 billion by 2023
and is expected to witness a CAGR of 5.1% during the forecast period
(Market report world.com, 2020).
Based on the application, the market has been classified into F&Bs,
household detergents and cleaners, pharmaceuticals, and others. At
present, CA is primarily used in the F&B industry for preparing numer-
ous food products such as soups, mayonnaise, juices, creams, and car-
bonated drinks. On the basis of the form, the market has been bifur-
cated into the anhydrous and liquid segments. Currently, anhydrous
CA accounts for the majority of the total market share. Region-wise,
Western Europe represents the largest market for CA across the globe.
Other major markets include the United States, China, Middle East
and Africa, Central/Eastern Europe, Brazil, and India. On assessing the
import and export scenario of the market, it has been found that the
United States and China are the biggest importer and exporter of CA,
respectively. The competitive landscape of the market has also been
examined in the report with some of the key players being Archer
Daniels Midland Company, Cargill Inc., Tate & Lyle PLC, Jungbunzlauer
Suisse AG, Cofco Biochemical (Anhui) Co., Ltd., Huangshi Xinghua Bio-
chemical Co. Ltd., RZBC Group Co. Ltd., Weifang Ensign Industry Co.,
Ltd., Gadot Biochemical Industries Ltd., and S.A. Citrique Belge N.V. The
market in the United States was worth USD 448.4 million in 2016 and
is projected to experience steady growth over the next eight years.
The European region accounted for the largest share in the market in
2014, owing to the heavy demand for functional food additives from
the flourishing processed F&B market of the region. Food as an appli-
cation dominates the market with more than three-fourth share, in
terms of value as well as volume. It is the fastest-growing application
and is expected to continue to be during the forecast years. The mar-
ket for CA for application in the pharmaceutical and personal care sec-
tors is emerging with substantial opportunities and is projected to grow
at a competitive CAGR from 2015 to 2020. The manufacturers are
penetrating various market segments by introducing CA-based prod-
ucts such as confectionery, diabetic baked products, ice creams, low-
calorie jellies, low-calorie sugar, dietary beverages and snacks, and low-
fat dairy products. The rising demand for these products is expected to
boost the demand among the consumers.
Companies in the industry are focusing on increasing their pro-
duction capacities to cater to rising product demand. For instance, in
December 2017, Jungbunzlauer, a major manufacturer of biodegrad-
able ingredients, announced the construction of a new CA plant in
Austria.
According to the WHO, approximately 65–70 million people in
North America experience digestive system problems. This, in turn, has
led to an increase in the demand for digestive CA-based F&Bs and phar-
maceuticals in the region, and thus impacting the industry trend on a
positive note.
According to the German Nutrition Society (DGE), awareness
among consumers in Europe pertaining to the use of CA for maintaining
64 BEHERA ET AL.

TA B L E 1 Citric acid (CA) producing microorganism

Microbes CA producing species References

Bacteria Bacillus licheniformis, Kroya Fermentation
Arthrobacter Industry (1970),
paraffinens, Sardinas (1972),
Corynebacterium sp., Fukuda et al. (1970)
Bacillus subtilis,
Brevibacterium
flavum,
Corynebacterium sp.,
and Penicillium
janthinellum
Fungi Aspergillus niger, A. Karow and Waksman
aculeatus, A. awamori, A. (1947), Roukas (1991),
carbonarius, A. wentii, A. El Dein and Emaish
foetidus, Penicillium (1979), Grewal and
janthinelum Kalra (1995), Chen FIGURE 1 Overview of strain improvement
(1994)
Yeast Saccahromicopsis Gutierrez et al. (1993),
lipolytica, Candida Kapelli et al. (1978), Oh era, Saccharomyces, Zygosaccharomyces, and Yarrowia. Although there
tropicalis, C. Oleophila, et al., (1973), Omar and
are reports of CA production by yeast (André et al., 2007; Mattey,
C. Guilliermondii, C. Rehm (1980), Rane and
Parapsilosis, C. Sims (1993), Uchio 1999; Rymowicz et al.,2010), one drawback of yeast fermentation is
Citroformans, Hansenula et al., 1975 that they produce substantial quantities of Iso-citric acid (isoCA), an
anamola, Yarrowia undesired by-product.
lipolytica, Torulopsis,
Fungi that are reported to produce CA are Aspergillus. niger, A.
Hansenula,
Debaromyces, Torula, awamori, A. clavatus, A. nidulans, A. fonsecaeus, A. luchensis, A. phoeni-
Pichia, Kloekera, and cus, A. wentii, A. saitoi, A. flavus, Absidia sp., Acremonium sp., Botrytis sp.,
Zygosaccharomyces Eupenicillium sp., Mucor piriformis, Penicillium citrinum, P. janthinellu, P.
luteum, P. restrictum, Talaromyces sp., Trichoderma viride, and Ustulina vul-
garis (Dhillon et al., 2011).
digestive health increased to 49% in 2016 as compared to 44% in 2015.
The organization also suggested that there has been a rise in the num-
ber of people consuming CA-based yogurt to maintain digestive health. 4.1 Improvement of strains for CA production
Rising awareness in the Middle East about CA-based pharmaceuticals
and F&Bs is driving the market growth in the region. A number of inter- Several methods such as mutations, protoplast fusion, recombinant
national market participants are establishing offices and plants in the DNA technology, and gene cloning are carried out as shown in Figure 1
region in an attempt to increase their footprint. For instance, in January for improvement of industrially important microorganisms (Parekh
2017, Pfizer inaugurated a new pharmaceutical manufacturing plant et al., 2000). Among these, random mutagenesis and protoplast fusion
in Saudi Arabia. China’s CA market is estimated to witness the high- are the simpler and commonly used techniques. The mutagenic pro-
est growth rate in the Asia Pacific over the forecast period. The market cesses involve physical, chemical, and site-directed mutagenesis for
is mainly driven by factors such as the rising geriatric population and strain improvement. The overproduction of industrial products by
prevalence of lifestyle diseases such as cardiovascular diseases, mental strain improvement has been considered in the commercial fermenta-
health problems, and gut health-related issues. tion process (Parekh et al., 2000; Vu et al., 2010). Improvement of strain
can be also achieved by modifying the metabolism of the microorgan-
ism by inducing mutations in them through physical or chemicals muta-
4 MICROORGANISMS KNOWN TO PRODUCE gens (Swain et al., 2012). Among physical mutagens, the most common
CA mutagens used are gamma and ultraviolet (UV) radiations (Pelechova
et al., 1990). Among chemical mutagens, diethyl sulfonate, N-methyl-
Different types of microorganisms such as bacteria, fungi, and yeast N-nitroso-guanidine, ethidium bromide aziridine, N-nitroso-N-methyl
have been reported for the bio-production of CA, as shown in Table 1 urea, and ethyl methane-sulfonate are very common (Musilkova et al.
(Crolla & Kennedy, 2001; Kuforiji et al., 2010; Papagianni, 2007). 1983). Strain improvement through the parasexual cycle was first
Bacteria, such as Arthrobacter paraffinens, Bacillus licheniformis, and described by Pontecorvo et al. (1953). Diploids displayed higher CA
Corynebacterium sp. (Carlos et al., 2006), are known to produce large yields compared to their parent haploids and was reported by Das and
amounts of CA. In addition to bacteria, some yeasts that can produce Roy (1978). Genetic manipulation of A. niger with respect to CA produc-
CA are Candida, Hansenula, Pichia, Debaromyces, Torula, Torulopsis, Kloek- tion through protoplast fusion was described by Kirimura et al. (1988a).
BEHERA ET AL.
The other two methods such as the single-spore technique and pas-
sage method are well-known alternative methods for the selection of
improved strains (Soccol et al., 2006).
5 SUBSTRATES AND PRETREATMENT
Substrate plays a very critical role to reduce cost and get optimal yield
in fermentation conditions. Hence, substrate is more important for pro-
ductivity and fermentation yield (Lesniak, 1999). Increases in yield and
reduction in fermentation time directly depend on the purity of sub-
strate used (Lesniak, 1999). Aspergillus niger ferment molasses, sucrose,
syrups of beet or cane sugar, hydrol obtained as a by-product dur-
ing crystalline glucose and palm oil for CA production (Gutcho, 1973;
Kutermankiewicz et al., 1980; Lesniak, 1999; Lesniak et al.,1986). Sub-
strate used in CA fermentation requires pretreatment for the removal
of trace metals (Kristiansen et al., 1999). It has been found that cane
molasses used in fermentation contains calcium, magnesium, man-
ganese, iron, and zinc, which have a retarding effect on the synthe-
sis of CA. Potassium ferrocyanide is generally used for chemical pre-
treatment of substrate before fermentation, which can precipitate zinc
and iron effectively. Enzymatic hydrolysis was also carried out for pre-
treatment of starch material such as corn wheat and potato for pro-
duction of CA to reduce the heavy metals concentration below critical
levels (Pietkiewicz et al., 1996).
To reduce the cost of production, agricultural waste and by-products
are used in CA production. The commonly used agricultural residues
including coffee husk, rice bran, wheat bran, carrot waste, cassava
bagasse, banana peel, vegetable wastes, sugarcane bagasse, tapioca,
cheese whey, rice straw, coconut husk, brewery wastes, decaying fruits,
corn cob, orange peel, kiwifruit peel, pineapple peel, pomaces of grapes,
and apples (Dutta et al., 2019; Sawant et al., 2018; Soccol et al.,
2006).
6 BIOCHEMICAL ASPECT OF CA PRODUCTION
During glycolysis, pyruvate produced is oxidized and combined with
coenzyme A to form CO2 , acetyl coenzyme A (Acetyl-CoA), and nicoti-
namide adenine dinucleotide (NAD) + hydrogen (H) (NADH). Subse-
quently, the Acetyl-co-A formed is combined with the oxaloacetate to
produce citrate (Figure 2). Besides, pyruvate produced during glycol-
ysis can also be carboxylated by pyruvate carboxylase (PC) to form
oxaloacetate. CA produced by the combination of Acetyl-co-A and
oxaloacetate is then transformed through a reaction sequence that
yields two molecules of CO2 and regenerates the four-carbon oxaloac-
etate again. Thus, at each turn of the cycle, one molecule of acetic
acid enters, two molecules of Adenosine Triphosphate (ATP) and CO2
are formed, and a molecule of oxaloacetate is utilized to form citrate
(Prescott & Dunn, 1959).
Enzymes play a very crucial role during CA formation. Pentose phos-
phate and glycolytic pathways act as a channel by A. niger to metabolize
glucose and accumulation of CA (Cleland & Johnson, 1954; Martin &
65
F I G U R E 2 Production of citric acid by Aspergillus niger
(PFK = phosphofructokinase, PC = pyruvate carboxylase,
ACO = aconitase
Wilson, 1951). Mischak et al. (1985) reported that citrate synthase is
an enzyme responsible for reversible catalysis between Acetyl-CoA
and oxaloacetate favoring citrate production. According to Ramakr-
ishnan et al. (1955), CA accumulation only occurred when enzymes
such as aconitase (ACO), isocitrate dehydrogenase, and succinic dehy-
drogenase were severely inhibited during the tricarboxylic acid (TCA)
cycle. However, Kubicek & Rohr (1977) reported the presence of these
enzymes in very less amounts throughout the CA fermentation period.
Hence, rather than inhibited degradation, CA accumulation may be the
result of enhanced biosynthesis (Max et al., 2010). In A. niger, outside
the cell, a membrane-bound enzyme invertase converts sucrose into
glucose and fructose that are then transported into the cell (Rubio &
Maldonado, 1995) via glucose transporters. Inside the cell, hexokinase
converted the transported glucose into glucose-6-phosphate and
initiates the process of glycolysis. Inhibition of some enzyme during
glycolysis causes high flux through glycolysis that results in accumu-
lation of CA by A. niger (Rohr et al., 1992). For example, deficiency of
manganese, or phosphate and nitrogen limitation, inhibits anabolism
of the fungus and results in degradation of proteins and leads to
increased intracellular NH4 + concentration (Habison et al.,1983;
Rohr & Kubicek, 1981). This high concentration of intracellular NH4 +
inhibits the enzyme phosphofructokinase (PFK), an essential enzyme
with magnesium as a cofactor, and changes fructose 6-phosphate into
fructose 1,6-bisphosphate in glycolysis (Figure 2). Inhibition of PFK
leads to a flux through glycolysis results in the accumulation of CA. In
contrast, Papagianni et al. (2005) reported that CA accumulation by
A. niger was due to the existence of an intracellular ammonium pool
that inhibits the enzyme PFK. During their investigation, they found
that instead of accumulation or deposition inside the cell to make an
66
ammonium pool, ammonium ions entered the cell and combined with
glucose to produce glucosamine that is then released outside the cell
or into the fermentation broth and responsible for PFK inhibition.
Hence, the detailed relationship between glucose, ammonium ion
concentrations, the enzymes within the TCA cycle, and accumulation
of CA is obscure and certainly needs further investigation.
Isocitrate dehydrogenase is an NADP+ dependent enzyme found
both in mitochondria and cell cytoplasm activated in the presence
of Mg2+ and Mn2+ . Inhibition of this enzyme by α-ketoglutarate
and citrate favours citric acid accumulation (Ratledge & Kristiansen,
2001). High concentrations of glucose and ammonium pool repress
the synthesis of α-ketoglutarate dehydrogenase, an important enzyme
required to regulate TCA cycle and thus inhibited the CA cycle move-
ment, resulting in the accumulation of CA (Rohr & Kubicek, 1981).
7 PRODUCTIONS
Choosing fermentation for CA production depends upon the raw mate-
rial and type of microorganism used. Rohret al. (1983) classified raw
materials used for CA production into two groups: (i) With a low ash
content from which the cations could be removed by standard proce-
dures (e.g., cane or beet sugar, dextrose syrups, and crystallized dex-
trose); (ii) raw materials with a high ash content and high amounts
of other nonsugar substances (e.g., cane and beet molasses, crude
unfiltered starch hydro-lysates). A wide range of substrates could be
employed for the efficient and feasible production of CA according to
the type of fermentation. Industrial CA fermentation can be carried out
with different substrates in three different ways such as surface, sub-
merged, and solid-state fermentation with some advantages and dis-
advantages (Table 2). However, each of these fermentation methods
requires media preparation, inoculation, fermentation, and recovery of
the CA.
7.1 Surface fermentation
Surface fermentation is a stationary batch fermentation process
known as the beginning of CA fermentation process completed within
8 to 12 days. It is carried out in fermentation chambers, with a number
of trays arranged in shelves made in stainless steel (Bauweleers et al.,
2014). The fungal mycelium develops on the surface of the medium
on those trays. After sterilizing the fermentation medium contained
in trays (Manzoni, 2006), it is inoculated with spore suspension and
incubated at 28–30◦ C (Marzona, 1996) for 24 h. Spores germination
started with drops in pH of the medium from 6.0–6.5 to 1.5–2.0, which
can be visualized with naked eyes with continuous mycelium on the sur-
face. In this process, a huge amount of heat is generated during fer-
mentation, which is controlled with proper aeration. The CO2 gener-
ated during the fermentation process would inhibit the production of
CA in concentrations higher than 10%. Since the chamber needs to be
effectively ventilated, the fermentation chambers are provided with an
effective air circulation, which passes over the medium surface through
BEHERA ET AL.
a bacteriological filter in order to control humidity and temperature by
evaporative cooling (Max et al., 2010; Soccol et al., 2006).
However, surface fermentation has some disadvantages such as low
yield and labor-intensive with higher maintenance cost compared to
submerged fermentation (Drysdale & McKay, 1995). It is also sensitive
to changes in the composition of the media (Benghazi et al., 2014). The
advantages of surface fermentation are lower energy consumption and
foam free (Moyer, 1953).
7.2 Submerged fermentation
In the submerged fermentation process, the broth medium inside
the nutrient substratum is in liquid form and the organism can grow
throughout the broth medium (Reddy, 2002). The fermentation is car-
ried out in bioreactors and completed within 5–12 days (Socool et al.,
2006). The organism, after 1 to 2 days of inoculation, grows as pellets
of approximately 0.5 cm diameter and suspended freely in the medium.
Thus, the organism with a huge contact surface area can take up the
nutrients and oxygen. The air flow is introduced into the vessel at high
speed, and the agitation equipment mixes and breaks the air bubbles
to increase oxygen levels. The carbon source present are decomposed
anaerobic or partially anaerobically by microorganisms present in the
medium (Swain et al., 2012). The advantages of submerged fermenta-
tion include better control of the fermentation process and the maxi-
mum use of a wide range of substrates (Max et al., 2010). It provides
higher yields; lower capital, maintenance, labor costs, and contamina-
tion risks make it more suitable for CA production (Rohr et al., 1983).
In contrast, the disadvantage of submerged fermentation is the forma-
tion of foam, which can be avoided using antifoam agents such as ani-
mal or vegetable fats and chambers with a volume of up to one-third of
the total fermenter volume.
7.3 Solid-state fermentation
In solid-state fermentation, microorganisms are grown in a low-water
containing insoluble material, which acts as physical support and a
source of nutrients as well. (Pandey, 1992; Vandenberghe et al., 2000).
The condition parameters for solid-state fermentation are substrate
used is solid and moistened to about 70% moisture, pH between
4.5 and 6.0, and a temperature between 28–30◦ C. The process is
completed within 4–5 days (Drysdale & McKay, 1995). The major
advantages of solid-state fermentation are low energy requirements,
higher yield, low risk of contamination, less efforts in downstream
processing, less effluent generation, simple operation, operable under
less water, and less operating costs as compared to submerged fer-
mentation. Besides, in solid-state fermentation, cheap and widely
available agro-industrial substrates can be easy utilized without any
pre-treatment as the system is less sensitive to the presence of trace
elements compared to submerged fermentation (Berovic & Legisa,
2007). On the other hand, this method has some disadvantages
such as difficulties to scale up, low amenability of the process to
BEHERA ET AL. 67

TA B L E 2 Comparative analysis of CA production by different fermentation technique

Quantity of Type of
Fermentation Advantage Disadvantage Material CA microorganism References
d
SSF Lower energy and cost Difficulties in scale-up, Pineapple 124 A. niger DS-1 Kumar et al. (2003)
requirements, higher yield, difficult control of waste
low-cost natural media, process parameters
Apple pomace 51.4a A. niger BC-1 Shojaosadati and
better oxygen circulation, higher recovery
Babaeipour
less susceptible to trace product costs
(2002)
elements inhibition, low
risk of bacterial Banana peels ∼180 gd A. niger MTCC Karthikeyan and
contamination, less 282 Sivakumar (2010)
amount of post-recovery Coffee husk 150 gd A. niger CFTRI Shankaranand and
waste 30 Lonsane (1994)
Corn cob 254 gd A. niger Hang and Woodams
(1998)
Grape pomace 60a A. niger NRRL Hang and Woodams
567 (1985)
Corn husk 259 ± 10 gd A. niger NRRL Hang and Woodams
2001 (2000)
Kiwi fruit 60%a A. niger NRRL Hang et al., 1987)
567
SF Less effort in operation, Large amount of heat Brewery waste 78.5a A. niger ATCC Roukas and
installation, and energy generation, 9142 Kotzekidou
cost time-consuming and (1986, 1987)
needs large area/space,
Turnip whey 27-46a A. niger NCIM Chanda et al. (1990)
sensitive to
(supplemented 595
contamination by
with
Penicillia, other
molasses)
Asperigilli, yeasts, and
lactic acid bacteria Sweet potato 45.90±4.2e A. niger IIB-A6 Anwar et al. (2009)
starch
hydrolysate
SMF Beet molasses 68.7 Y. lipolytica Lesniak et al. (2002)
A-101
Sophisticated control High media cost, sensitive Cane molasses 114 g/L A. niger GCMC 7 Haq et al. (2004)
mechanisms, lower labour to trace metal
Date syrup 50 ± 1.5% A. niger ATCC Roukas and
cost, higher productivity inhibition, high waste
9142 Kotzekidou
and yield water generation
(1997)
Corn cobs 603.5d A. niger NRRL Hang and Woodams
2001 (2001)
Coconut oil 99.6c C. lipolytica Soccol et al. (2006)
c
Soybean oil 115 C. lipolytica Soccol et al. (2006)
N-5704
Orange peel 53%a A. niger Rivas et al. (2008)
CECT-2090

a, e: based on sugar consumed, b, d: based on dry matter, c: based on oils and fatty acids.

standardization, difficult control of process parameters, and problems
with heat build-up. It cannot utilize available nutrients completely
owing to poor heat and oxygen transfer in the substrate (Kapilan,
2015).
8 RECOVERY OF CA
CA contain various undesirable by-products such as mycelium, other
organic acids, mineral salts, proteins, and other impurities at the end of
the fermentation. Hence, there is a need to obtain pure CA only (Gre-
wal & Kalra, 1995). Recovery of CA from the fermented broth is gener-
ally performed through three major procedures such as precipitation,
extraction and purification.
It has been observed that oxalic acid is formed during the CA fer-
mentation, which can be removed by increasing the pH up to 3.0 with
calcium hydroxide at 72–75◦ C (Sawant et al., 2018). Subsequently, cal-
cium oxalate is formed, which can be further precipitated and elimi-
nated by centrifugation or filtration process. The CA that remained in
68
the form of calcium salt (calcium citrate) in the original solution can be
recovered through precipitation by adding calcium oxide at 90◦ C and
pH nearly equal to 7.0 (Soccol et al., 2006). Tri-calcium citrate tetrahy-
drate is formed, which is then treated with 70% sulfuric acid to form CA
and insoluble calcium sulfate (gypsum). After filtering off the gypsum, a
solution of 25%–30% of CA is obtained. To remove residual impurities,
the filtrate is treated with activated carbon or can be purified in ion-
exchange columns. Further, it is concentrated by evaporation in a vac-
uum at 40◦ C, and crystals of CA monohydrate can be formed in a vac-
uum crystallizer at a temperature of 20–25◦ C or anhydrous CA can be
formed at crystallization temperatures above 36.5◦ C (Grewal & Kalra,
1995; Kubicek, 1986).
Purification and crystallization of CA from fermentation broth
can be alternatively performed by solvent extraction method in
which insoluble or sparingly soluble solvent such as n-octyl alcohol,
tridodecylamine, and isoalkane (Soccol et al., 2006), alanine 336 in
heptane or xylene (Sirman et al., 1990), mixture of butylacetate and
N, N-disubstituted alkylamide (Yi et al., 1987), aliphatic alcohols,
ketones, ethers or esters (Kasprzycka et al., 1989), organophosphorus
compounds, such as tri-n-butylphosphate (Pagel & Schwab, 1950),
alkylsulphoxides (Grinstead, 1976), and water-insoluble amines or
a mixture of two or more of such amines are used (Prochazka et al.,
1994). The advantage of the solvent extraction method is to prevent
the use of lime and sulfuric acid (H2 SO4 ), and thus the production of
gypsum can be avoided (Grewal & Kalra, 1995). In this process, CA is
recovered from the aqueous solution by washing off the extract with
water, subsequently crystallized and concentrated. The concentrated
CA solution dissolved in acetone is passed with compressed CO2 .
The anti-solvent effects of CO2 remove the residual impurities and
food-grade CA is obtained by simple decolorization and crystallization
(Shishikura et al., 1992).
9 FACTORS AFFECTING CA PRODUCTION
The factors mainly affecting the citric fermentation are carbon source,
nitrogen and phosphate limitation, pH, aeration, trace elements con-
centration, and morphology of the producing microorganism (Max
et al., 2010).
The pH of the medium is most important during the initial stage of
fermentation and at the end, before the recovery of the product during
CA fermentation (Papagianni, 2007). Initially, in the germination stage,
the germinating fungal spores in the fermentation medium after inoc-
ulation require a pH greater than 5 in order to germinate (Papagianni,
2007). The germinating spores absorb ammonia and release protons,
thereby increasing the acidity of the medium and favoring the produc-
tion of CA (Papagianni, 2007). However, the initial pH of the medium
depends on the substrate used. A pH value from 2.5 to 4.0 was found
to be optimum for chemically defined medium (Jernejc et al., 1982),
whereas an initial pH value from 6.0 to 7.5 was found to be the best
in molasses medium (Berry et al., 1977). On the other hand, the pH for
the recovery stage of CA should be below 2. At this low pH, the for-
mation of unwanted products such as oxalic and gluconic acid is inhib-
BEHERA ET AL.
ited, and the possibility of contamination by other microorganisms is
also reduced, making recovery of CA easier (Max et al., 2010).
CA production is dependent on glycolysis and TCA cycle enzyme.
These enzymes are dependent on temperature. In higher or lower
temperature, these enzyme systems will not work and affect CA pro-
duction. Incubation temperature ranges from 25 to 30◦ C were found
to be more suitable for high yields and rapid rates of CA production
(Prescott & Dunn, 1959). Kapoor et al. (1982) reported that the CA
yield decrease above the temperature of 30◦ C due to increase in oxalic
acid production, while temperature below 25◦ C slow the growth of the
organism and the fermentation rates.
Angumeenal and Venkappayya (2013) showed that because of
extracellular mycelium-bound invertase of A. niger, invertase can
rapidly hydrolyze sucrose at low pH; thus, sucrose is the most suit-
able carbon source than glucose, fructose, and lactose for CA pro-
duction (Kubicek-Pranz et al.,1990). The concentration of the carbon
source is also critical to the success of CA production. Xu et al. (1989)
reported that A. niger strains needed an initial sugar concentration of
10% to 14% as optimal, but no CA was produced at a sugar concen-
tration of less than 2.5%, and maximum CA production was observed
at the concentration 14%–22% of sugar. It is because of the high con-
centration of the sugar reported to suppress α-ketoglutarate dehydro-
genase, resulting in maximum CA accumulation (Hossain et al., 1983).
In contrast, at low concentration of sugar, the size of the mycelium is
reduced, and its shape is also affected (Papagianni et al., 1999). It has
been also observed that immobilized cells of A. niger needed low sugar
concentrations in comparison to free cells for maximum CA accumula-
tion (Honecker et al., 1989).
Nitrogen concentration greater than 0.25% accumulates oxalic acid
in the fermentation medium and decreases the CA yield (Gupta, Hed-
ing, & Jorgensen, 1975). It has been also reported that a high nitrogen
concentration increases the consumption of sugar and fungal growth
while decreasing the amount of CA produced (Hang et al., 1977). Basi-
cally, ammonium salts such as ammonium nitrate and sulfate, urea, pep-
tone, malt extract, and so forth are used in CA production (Grewal &
Kalra, 1995). Acid ammonium compounds are preferred because their
consumption leads to pH decrease, which is essential for citric fermen-
tation. Ammonium sulfate is the preferred choice of salt, as it does not
produce the unwanted oxalic acid while reducing the pH of the medium
as the salt is consumed. Molasses are usually nitrogen-rich and used in
industrial fermentation without any additional ammonium salts as sup-
plements.
Low levels of phosphate are found to be very suitable for CA pro-
duction, whereas its excess concentration may lead to the formation
of certain sugar acids, a decrease in the fixation of CO2 , and the stim-
ulation of growth (Grewal & Kalra, 1995). For optimal CA production,
potassium dihydrogenphosphate (KH2 PO4 ) has been reported to be
the most suitable at a concentration of 0.5–5 g/L (Shu & Johnson,
1948).
Moyer (1953) studied that the addition of ethanol doubles the cit-
rate synthetase activity and decreases the activity of ACO, which
results in increased CA accumulation. Alcohols such as methanol
showed a positive effect on CA formation due to its inhibitory effect
BEHERA ET AL.
on metal ions. The amount of methanol/ethanol required depends on
the composition of the medium and the strain of microorganism used
(Moyer, 1953). Ingram & Buttke (1984) reported that alcohols stimu-
late CA production by affecting growth and sporulation due to changes
in the lipid composition of the cell membrane. Kubicek & Röhr (1986)
reported that lower alcohols added in pure material inhibit CA produc-
tion but when added into crude carbohydrates, these alcohols enhance
CA production. Methanol, ethanol, n-propanol, isopropanol, or methy-
lacetate concentration between 1% and 5% neutralizes the negative
effect of the metals ions in CA production and favors maximum CA for-
mation (Kubicek & Röhr, 1986).
The metal ions that are most sensitive with regard to CA produc-
tion include ferrous ion (Fe2+ ), copper ion (Cu2+ ), zinc ion (Zn2+ ), and
manganese (Mn2+ ) and hence must be limiting (Dronawat et al., 1995).
Concentrations of Mn2+ at less than 3 μg/L has been shown to dras-
tically reduce the yield of CA production (Clark et al., 1966), whereas
Mattey & Bowes (1978) reported that the addition of 10 mg Mn2+ per
liter reduced the accumulation of CA by 50% relative to control cul-
ture. Cellular anabolism of A. niger is impaired under manganese defi-
ciency and protein breakdown results in high intracellular ammonium.
This high intracellular ammonium concentration inhibits the enzyme
PFK (an essential enzyme in the conversion of glucose and fructose to
pyruvate), leading to a flux through glycolysis pathway and the forma-
tion of CA.
Presence of different copper concentrations in the pellet formation
medium is very important in order to enhance a suitable structure,
related to cellular physiology, for CA production (Benuzzia & Segovia,
1996). The toxic effect of Fe2+ can be eliminated by a high concentra-
tion of Cu2+ (Rohr et al., 1983). Tomlinson et al. (1950) deduced that
the optimum concentrations of zinc are 0.3 ppm for CA production.
Zinc favored the production of CA if added with KH2 PO4 (Vanden-
berghe et al., 1999). On the other hand, its excess presence could favor
fungal growth only without any accumulation of CA (Grewal & Kalra,
1995). CA accumulation decreased by the addition of iron, which also
had some effect on mycelial growth. Optimum concentrations of iron
for CA production are 1.3 ppm (Tomlinson et al., 1950). Magnesium is
required both for growth as well as for CA production. Optimal con-
centration of magnesium sulfate for CA production was found in the
range of 0.02–0.025% (Kapoor et al., 1982). Nickel, molybdenum, and
cobalt are some other trace metals that are also reported to affect the
CA accumulation in A. niger (Habison et al., 1983).
Vandenberghe et al. (1999) reported that oxygen concentration
above 25% is required for better CA production. It has been observed
that the critical dissolved oxygen tension is 9–12% of air saturation
for the growth phase and 12–13% of air saturation for the production
phase (Grewal & Kalra, 1995). When the organism turns into the devel-
opment of filaments, the dissolved oxygen tension rapidly falls to less
than 50% of its previous value, even if the dry mass has not increased
by more than 5%. Therefore, small compact pellets are the preferred
mycelial forms of A. niger during the CA production. Low oxygen envi-
ronment is directly involved in the growth limitation, which is cru-
cial for CA production, whereas strongly aerated cultures (0.3 m3/kg
dry CB/h) increase sporulation hence decreasing the accumulation of
69
CA (Vandenberghe, 2000). CO2 is an important substrate for PC that
replenishes the supply of oxaloacetate for citrate synthase. Sufficient
CO2 is produced by the reaction catalyzed by pyruvate decarboxylase,
but excessive aeration leads to some losses. In contrast, increased lev-
els of CO2 are damaging to the final biomass and concentrations of cit-
rate (McIntyre & McNeil, 1997).). It has been observed that high partial
pressure of CO2 probably retards spore liberation of the filamentous
fungi and helps in CA accumulation. Therefore, the environment with
high concentrations of CO2 has a positive effect on CA synthesis (Van-
denberghe, 2000).
It has been observed that the formation of compact aggregates or
pellets by the fungus during CA fermentation favors more CA produc-
tion than filamentous form. Agitation rate, pH of the medium, compo-
sition of the medium, and inoculums concentration are the major fac-
tors that affect the morphology of A. niger in submerged fermentation
(Papagianni, 2007). Small aggregates of short filaments form are found
at pH values around 2.0 ± 0.2 and associated with an increase in CA
production. At lower pH (pH 1.6), the aggregated short filamentous
form of the fungus changed to bulbous hyphae and results in very less
CA production, whereas at higher pH (pH > 3.0), aggregates had longer
perimeters, and oxalic acid formation was observed (Papagianni, 2007).
Lipids, such as groundnut oil (Souza et al., 2014) and sodium mono-
fluoro acetate have also effects on CA production (Meixner-Monori
et al., 1984). Lipid can improve the yield of CA with no effect of the dry
weight of mycelium (Millis et al., 1963). Kareem et al. (2010) reported
the effects of calcium fluoride, sodium fluoride, and potassium fluoride
on the industrial production of CA.
10 METABOLIC ENGINEERING OF CA
PRODUCTION
Several methods of metabolic engineering have been tried by modify-
ing the genes and metabolic pathways to increase CA production (Rui-
jter et al., 1997; 1999; Yin et al., 2015). An important tool through
which an entire new biosynthetic pathway can be developed and intro-
duced in A. niger to enhance CA production is systems metabolic engi-
neering (Tong et al., 2019). Deletion of gene acl1 in A. niger that is
responsible for ATP-citrate lyase synthesis and could increase the CA
production in A. niger was reported by Meijer et al. (2009). Chen et al.
(2014) found that with deletion of two cytosolic ATP citrate lyase (ACL)
subunits (Acl1 and Acl2), not only CA production in A. niger decreased
but also inhibits vegetative growth, pigmentation, conidial germina-
tion, and subsequently asexual development.
The cytosol reductive TCA reverse tricarboxylic acid cycle (rTCA)
cycle was engineered by de Jongh & Nielsen (2008) by inserting
heterogeneous malate dehydrogenase (mdh2), fumarase (FumR) and
fumarate reductase (Frds1). It has been observed that the mdh2
overexpressing strain could accelerate the CA production at the ini-
tial stage. Overexpression of cytosolic FumR converted fumarate to
malate, provided more substrate to the mitochondrial malate-citrate
antiporter, and thereby improved the CA secretion and productivity,
while overexpression of Frds1 converted fumarate to succinate, which
70
is also a potential substrate for the mitochondrial CA antiporter. When
both the FumR and Frds1 genes were co-expressed in a same strain,
more citric acid production has been observed (de Jongh & Nielsen,
2008).
Conversion of fructose 6-phosphate into fructose 1,6-bisphosphate
is carried out by the essential enzyme PFK, with magnesium as a cofac-
tor, and considered a crucial controlling step for glycolysis metabolic
flux via the allosteric inhibition or activation. Legisa and Mattey (2007)
showed that shorter PFK1 fragment is not only found to be resis-
tant to citrate inhibition but also more susceptible to positive effec-
tors, such as adenosine monophosphate (AMP), ammonium ions, and
fructose 2,6-bisphosphate, which suppresses the ATP inhibition. It
has been found that A. niger strain with an active shorter PFK1 frag-
ment mtpfkA10 with T89D single-site mutation (to elude the phos-
phorylation requirement) exhibited 70% more CA production than the
control strain (Capuder et al., 2009). Ruijter et al. (1999) reported
that an A. niger mutant strain lacking both glucose oxidase (goxC) and
oxaloacetate acetylhydrolase (OAH) (prtF) could efficiently produce
CA instead of oxalic acid in the medium at pH 5, and under these condi-
tions, production was completely insensitive to Mn2++ .
It has been observed that at the end of the fermentation, some
of the residual sugar such as iso-maltose remains unutilized. In day
to day large-scale production process, this residual sugar raises great
loss and affects the production profit. It has been detected that this
residual sugar (iso-maltose) is synthesized by an enzyme α-glucosidase
during CA fermentation, and deletion of the α-glucosidases encoding
gene agdA could efficiently reduce the iso-maltose concentration when
using corn starch as the raw carbon source. It is also observed that dele-
tion of α-glucosidases encoding gene agdA with overexpression of glu-
coamylase glaA could decrease 88.2% of the residual sugar, and hence
increased the CA production 16.9%.
Similarly, hexokinase is the enzyme that converts glucose to glucose-
6-phosphate and fructose-6-phosphate. The increase in sugar uptake
results in an increase in the concentration of glucose 6-phosphate
and consequently also trehalose 6-phosphate, capable of inhibiting
hexokinase and CA accumulation (Arisan-atac, Wolscheck, & Kubicek,
1996). However, deletion of the gene GgsA, which encodes trehelose-
6-phosphate synthase, decreased the level of trehelose-6-phosphate
and CA accumulation initiated earlier (Arisan-atac et al., 1996).
Hou et al. (2018) modified A. niger strain CGMCC10142 by deleting
and overexpressing mitochondrial AOX gene aox1 and uncovered that
the overexpression of aox1 gene improve CA production, and cyanide-
resistant respiration (CRR) respiration was detected in mycelia of A.
niger under CA production conditions. The presence of AOX thus alle-
viates the repression of PFK due to excess ATP and relieving the dam-
age caused by ROS as well (Campos et al., 2015). When CA begins to
accumulate, cytochrome-dependent respiration is thus replaced by the
alternative route, CRR pathway, which enables the NADH oxidization
without concomitant ATP production (Papagianni, 2007).
During submerged fermentation, mycelial morphology of A. niger
plays an important role in CA production. RNA interference through
the silencing of the chitin synthase gene (chsC) was investigated by Sun
et al. (2018). They reported that the mutant strain chsC -3 of A. niger,
BEHERA ET AL.
produced after silencing of chsC gene, could exhibit a significant change
in the mycelial morphology, and thereby showed 42.6% more CA pro-
duction in comparison to the original strain (Sun et al., 2018).
Similarly, the gene that is involved in the regulation of morphology
formation of A. niger in response to Mn2+ is Brsa-25 gene. Dai et al.
(2004) showed that a 10% increase in CA production can be achieved
by downregulation of the Brsa-25 gene expression through antisense
RNA. This downregulation mechanism facilitated pelleted growth and
enhanced CA production in the presence of Mn2+ .
The orotidine-5′-decarboxylase gene (pyrG) is an essential enzyme
involved in uridine biosynthesis (van Hartingsveldt et al., 1987). It has
been reported that gene disruption and downregulation of pyrG sig-
nificantly increases of industrial CA production during submerged fer-
mentation (Zhang et al., 2020). Zhang et al. (2020) disrupted both
pyrG and ku70 homologous gene (kusA) in CA producing isolates wild
type (WT-D) and D353, by a highly efficient clustered regularly inter-
spaced short palindromic repeats (CRISPR)/CRISPR associated protein
9 (Cas9) system based on ribosomal 5s riobo-nucleic acid (5S rRNA)
as a promoter (Figure 3). They observed that pyrG disruption dramat-
ically disturbed the intracellular central metabolism and improved the
intracellular level of the CA and its precursor such as acetyl-CoA and
oxaloacetate, which may lead to the extracellular CA accumulation. In
another study, Zhang et al. (2019) reported disruption and replacement
of agdF by glucoamylase gene using the modified CRISPR/Cas9 sys-
tem to increase the production of glucoamylase 25.9% higher than wild
strain and increase the CA production than the wild strain (Zhang et al.,
2019).
11 EMERGING APPLICATION OF CA
Due to its pleasant taste, high water solubility, chelating and buffering
properties, CA is widely used as a safe acidulant in the food, sugar, con-
fectionery and beverages industry. In carbonated beverages, it is used
to give fruit and berry flavors (Fukui & Tanaka, 1980); in the confec-
tionery industry as flowing agent (Buchard & Merrit, 1979); in the wine
industry to prevent turbidity of wines (Soccol et al., 2006); in candies
to provide dark color and tartness; as an antioxidant synergism in fats,
oils, and fat-containing foods (Buchard & Merrit, 1979); in sherbets as a
flavor adjunct (Buchard a& Merrit, 1979); and in ice cream as an emul-
sifying agent. In addition, esters of CA, such as triethyl, tributyl, and
acetyltributyl, are employed as nontoxic plasticizers in plastic films that
are used to protect foodstuffs (Buchard & Merrit, 1979).
The chelating and pH adjusting properties of CA are applied in the
food industry to enhance the stability of frozen food products by pre-
venting the deterioration of color and flavor in frozen fruit. It also helps
to prolong the shellfire of frozen fish and shellfish.
Due to their sequestering action, such as stabilization of ascorbic
acid and good buffering capacity, CA and its salts are widely used in the
pharmaceutical industries as oral pharmaceutical liquids, elixirs, and
suspensions to buffer and maintain the stability of active ingredients of
the pharmaceutical product (Moledina et al., 1977). Trisodium citrate
is widely used as a blood preservative, where it prevents clotting by
BEHERA ET AL. 71

F I G U R E 3 A Schematic diagram of simultaneously disrupted mutagenesis of both pyrG and kusA mediated by integrating the donor DNA with
40-bp micro-homology arms via CRISPR/Cas9 system in A. niger D and D353. The donor DNAs of MHi-pyrG1-hph and MHi-kusA-hph were
co-transformed with linear sgRNA constructs (sgRNA-pyrG1 and sgRNA-kusA) and Cas9 expression plasmid pCas9-hph into the protoplasts of A.
niger D and D353. Two double-strandedbreaks (DSBs) were generated by the Cas9 under the guide of sgRNA, and then were repaired by
homologous recombination (HR) with the integration of donor DNAs (with prior permission of Zhang et al., 2020)

complexing calcium (Ciriminna et al.,2017; Vandenberghe et al.,1999).
In combination with sodium citrate, acetic acid is used to prevent kid-
ney stones (Gul & Monga, 2014).
As a cross-linker, it has several uses such as in ultrafine protein fibers
for biomedical applications (Reddy et al., 2015), polyols for making
biodegradable films like bio-plastic suitable for eco-friendly packaging
(Seligra et al., 2016), and hydroxyapatite to make bioceramic compos-
ites for orthopedic tissue engineering (Sun et al., 2014).
CA is used is for softening water, which makes it useful in household
detergents and dishwashing cleaners or soap (Ciriminna et al., 2017).
By chelating the metals ions like Ca2 + and Mg2 + ions in hard water,
it helps to produce foam and work better without the need for water
softening.
CA can be used as a cleaning agent due to its excellent metal chelat-
ing properties. It binds to metals and makes them soluble. Hence, solu-
tions of CA are used in the cleaning of power station boilers and similar
installations to remove and discourage the buildup of limescale (Ver-
hoff, 2016).
In 2005, Brazilian researchers first showed that CA can be success-
fully used in place of toxic mineral acids to recover pectin from apple
pomace (Canteri-Schemin et al., 2005). Pectin extraction yield with CA
showed the highest average value (13.75%).
CA has excellent metal chelating properties, and hence widely used
to clean nuclear sites contaminated with radionuclides (Kantar & Hon-
eyman, 2006) and bioremediation of soils contaminated with heavy
metals (Ates et al., 2002). It has been reported that CA in combination
with rhamnolipid biosurfactants affords very excellent results in soil
environmental remediation through bio-based chemical agents. This
combination is not only environmentally compatible but also promote
soil ecological restoration after remediation (Wan et al., 2015).
A combination of ethanol (70%), urea (1%) and CA (1.5%) had a
strong inactivation effect on poliovirus but insufficient for adenovirus
and polyomavirus. Ionidis et al. (2016) found that a combination of
70% ethanol with 2% urea and 2% CA could sufficiently deactivate all
enveloped viruses such as polyomavirus, norovirus and adenovirus vac-
cinia virus on surfaces.
As a component of printing plate emulsions in various bleaches, fix-
ers and stabilizers, CA and its esters are used in photography (France
Patient, 1937), in oil well treatment and cements (Buchard & Merrit,
1979), in the textile industry (U.S. Patent, 1949), in paper and tobacco
industries (Hushedeck, 1965), and the cosmetic industry (Wells et al.,
1972).
12 ECONOMIC ASPECTS
An economic comparison of surface and submerged fermentation pro-
cess for the CA production was carried out by Schierholt (1977).
Surface and submerged fermentation process capacities of 300 and
150 m3 in nine days of fermentation time at the productivity of 72 and
12 tons per day were compared. He observed that the building invest-
ment costs connected with the submerged fermentation are 2.5 times
72
lower than those connected with the surface fermentation process.
However, the equipment expenses costs at submerged fermentation
are considerably higher and more than 60% of those expenses that con-
sist of complicated component such as bioreactors and more sophisti-
cated instrumental control, which are subject to relatively high wear.
For higher capacities, investment costs for the submerged process are
about 25% lower than for surface fermentation, whereas for smaller
capacities, total investment costs for the submerged process are about
15% lower than for surface fermentation. Consumption of electrical
energy in the submerged process is about 30% higher than surface fer-
mentation, whereas the labor costs in highly developed countries for
surface fermentation is considerably higher. Similarly, countries where
cooling water temperature exceed 20◦ C, additional expenses for cool-
ing the bioreactors are required for the submerged process. On the
other hand, the submerged fermentation is sensitive to short interrup-
tions or breakdowns in aeration, which results not only in losses of yield
but also in the total breakdown of the respective batch.
13 FUTURE PROSPECTS
An increase in CA productivity with lower cost is a major interest.
There is a need of new technology and innovations not only for biore-
actor designs that could overcome the problems of scale-up in fermen-
tation processes but also the online monitoring and control of sev-
eral parameters. Less expensive substrates such as by-products and
residues of the agro-industry can be used for CA production to reduce
cost and environmental problems. High CA yielding strains need to be
explored through strains improvement and can be achieved by several
high throughput technologies. Recovery and purification of end prod-
ucts are the major challenges that have been stimulating researchers to
thrive hard to find the solutions. The integrated fermentation and prod-
uct recovery method should be used, which we can apply in large-scale
production of CA. The above-mentioned innovative strategies can not
only decrease the cost of production and recovery but also save the
environment from harmful chemicals being used in conventional CA
production.
14 CONCLUSION
Broad aspects of CA are mainly due to its wide range of applications
in different industrial sectors. To meet the increasing global demand
of CA, there is an urgent need of cost-effective industrial produc-
tion process that is highly complex, sensitive, and depends upon sev-
eral parameters such as choices of microorganism, raw materials used,
types of fermentation technique employed, designing of appropriate
bioreactors with precise control over process parameters, biochemical
pathways, factors affecting CA production, quantification techniques,
recovery techniques, strain improvement, and so forth. Utilization of
agricultural wastes for CA production can not only solve the problem
of waste disposal but also save valuable foreign exchange by reducing
BEHERA ET AL.
the CA imported from abroad. Adoption of advanced technology and
metabolic engineering can also help to overcome some critical param-
eter raised during fermentation and significant research efforts are
needed.
ACKNOWLEDGMENTS
The author is grateful to the faculty and staff of SBS, NISER,
Bhubaneswar, for their help and cooperation.
CONFLICT OF INTEREST
The author declares no financial or commercial conflict of interest.
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