Bioresource Technology 320 (2021) 124426

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Citric acid bioproduction and downstream processing: Status,

opportunities, and challenges
Sabrina Mores a, Luciana Porto de Souza Vandenberghe a, *, Antonio Irineudo Magalhães
Júnior a, Júlio César de Carvalho a, Ariane Fátima Murawski de Mello a, Ashok Pandey b,
Carlos Ricardo Soccol a
a
Federal University of Paraná (UFPR). Department of Bioprocess Engineering and Biotechnology. P.O. Box 19011, ZIP Code 81531-990, Curitiba, Paraná, Brazil
b
Centre for Innovation and Translational Research, CSIR-Indian Institute of Toxicology Research, Lucknow, 226 001, India

H I G H L I G H T S

• CA production processes are now turned to the search of new alternative feedstocks.
• Metabolic engineering should be better explored in the bioproduction of CA.
• Adsorption, extraction, and membrane separation have a lower environmental impact.
• Reactive extraction and adsorption have great potential in CA recovery.
• More studies with adsorption and reactive extraction are necessary.

A R T I C L E I N F O A B S T R A C T

Keywords: Citric acid (CA) has been widely used in different industrial sectors, being produced through fermentation of low-
Citric Acid cost feedstock. The development of downstream processes, easier to operate, environmentally friendly, and more
Feedstocks economic than precipitation, is certainly a challenge in CA bioproduction. Large volumes of by-products
Adsorption
generated in precipitation require treatment before disposal. Adsorption, extraction, and membrane separa­
Extraction
tion have been shown to have a lower environmental impact than precipitation, but the technological maturity of
Precipitation
these methods is still limited. However, reactive extraction and adsorption have great potential for industrial
applications. This review shows that there is still much to be explored, both about the factors that are intrinsic to
the techniques, but also in their combination for new processes’ development. This review reports the most
recent advances on CA bioproduction, with significant information about recovery and purification methods
involving this highly industrially demanded organic acid.

1. Introduction feedstocks for CA manufacture.

Citric acid (CA, or 2-hydroxy-propane-1,2,3-tricarboxylic acid),
bioproduction involves a classical technology of industrial-scale
fermentation. CA is one of the most industrially used organic acid
with different applications. The production for 2020 is estimated by 2
million tons (Ciriminna et al., 2017; Technavio, 2019) with a global
market estimated at US$ 3.6 billion in 2025 (ReportLinker, 2020). China
is the largest world producer with annual growth around 4% in 2019. CA
market has the potential to grow at over 5.24% by the year 2025
(ReportLinker, 2020), which justifies the search for new alternatives and
The world population growth along with increasing consumption of
processed food has contributed for higher CA demand. In 2016, the food
and beverage industry accounted for 62.53% of the global CA market,
followed by the pharmaceutical and personal care industry (17.08%),
detergents and cleaners (12.27%), and others (8.12%). Likewise, the
growth projection from 2016 to 2021 is greater for the food and
beverage market (6.03%), followed by pharmaceutical and personal
care (5.84%). There is a trend towards greater consumption of beverages
and processed foods, which require the use of additives, while people
have shown interest in the consumption of products with ingredients of

* Corresponding author.
E-mail address: lvandenberghe@ufpr.br (L.P.S. Vandenberghe).

https://doi.org/10.1016/j.biortech.2020.124426
Received 1 October 2020; Received in revised form 12 November 2020; Accepted 14 November 2020
Available online 20 November 2020
0960-8524/© 2020 Elsevier Ltd. This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/).
S. Mores et al.
natural “status”, as it is the case of CA. Also, CA has low cost, is envi­
ronmentally friendly due to its biodegradability, has the status of
“generally considered safe” (GRAS) by the Food and Drug Administra­
tion (FDA), and enables a wide range of applications, which justifies the
growth of demand for this organic acid.
In the food industry, CA is the main food acidulant used to prevent
oxidative deterioration of the taste and color of products, such as jellies,
sweets, and soft drinks. For application as a food additive, CA must have
a purity of 99.5% (United States Pharmacopeial Convention, 2014). The
antioxidant properties of CA are associated with its ability to chelate
metal ions, such as calcium, iron and copper that are present at signif­
icant levels in several food systems (Allen, 2015; Ciriminna et al., 2017).
In the pharmaceutical industry, sodium citrate is used as an anticoagu­
lant in blood transfusions (Cavallo et al., 2017). CA is also used to mask
unpleasant flavors, improve palatability, and as a crosslinking agent in
films for controlled drug release (Cavallo et al., 2017; Ciriminna et al.,
2017; Ghorpade et al., 2018; Okafor and Okeke, 2017). CA also has
applications in the cosmetic and chemical industry, being used in
astringent lotions, such as aftershave, due to its low pH. The acid, under
neutral or low pH conditions, has a strong tendency to form complexes,
so it is widely used in electroplating, leather tanning, and removing
metal oxides from surfaces. CA is used in detergents replacing phos­
phates, which can cause eutrophication (Okafor and Okeke, 2017;
Soccol et al., 2006). In addition, CA properties have been explored for
use in new applications, such as components of biodegradable pack­
aging, due to its crosslinking capacity, disinfectant agent, extracting
agent, fruit preservative component, and environmental remediation
(Ciriminna et al., 2017). This review reports the most recent advances
about CA microbial production, with significant information about re­
covery and purification methods involving this highly industrially
demanded organic acid.
2. Citric acid production
Since the discovery of CA production by different micro­
organisms>100 years ago, several studies have been conducted to
improve fermentation processes. Several reports indicate physical and
chemical conditions that favor microbial CA production. The nature and
concentration of sugar in the medium are crucial factors for high CA
production (Hossain et al., 1984; Xu et al., 1989). Maltose, sucrose,
glucose, mannose, and fructose in a range of 120 g L-1 to 180 g L-1 has
shown to be appropriate for CA production by A. niger (Hossain et al.,
1984; Ikram-Ul et al., 2004; Sun et al., 2018; Xu et al., 1989; Yin et al.,
2017). Generally, high CA productions occur with the presence of high
sugars’ concentrations, which inhibit the activity of α-ketoglutarate
dehydrogenase, thus interrupting the TCA cycle (Hossain et al., 1984; Xu
et al., 1989).
Nitrogen and phosphate limitations is also an important factor for
biomass production limitation (Max et al., 2010). Usually, the medium is
supplemented with ammonium salts such as urea, ammonium sulfate, or
ammonium nitrate, since these components promote pH reduction,
which is favorable to CA synthesis (Papagianni, 2007). Mostafa and
Alamri (2012) showed that 0.1% of ammonium nitrate and 0.1 g L-1 of
KH2PO4 were optimal concentrations for CA production using date
syrup medium. Ali et al. (2002) reached high CA concentrations when
sugarcane molasses was supplemented with 0.2% of ammonium nitrate.
Zhou et al. (2017) improved CA production from corn stover hydrolysate
with supplementation of 1.0 g L-1 of NH4, but observed a decrease in
production when high concentrations of the nitrogen source were
employed. Also, the addition of KH2PO4 did not affect CA yield.
The presence of heavy metals such as Zn, Mn, Fe, and Cu affect the
CA production especially in liquid cultures. Low concentrations of
manganese ions (<2 µg L-1) must be used. The influence of manganese is
complex, and it is not still completely elucidated. Nevertheless, studies
indicate that these ions cause undesirable morphological changes and
have a negative impact on the activity of some enzymes (Berovic and
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Bioresource Technology 320 (2021) 124426
Legisa, 2007; Clark et al., 1966; Mirminachi et al., 2002; Wallrath et al.,
1992). Iron ions also alter the morphology of fungal pellets and their
concentration must be controlled below 1 mg L-1 (Mirminachi et al.,
2002). Usually, the addition of small amounts of copper and zinc ions is
carried out to produce an antagonistic effect on deleterious action of
iron and manganese ions (Mirminachi et al., 2002; Zhang and Roehr,
2002).
The pH of culture medium must be higher than 5 at the beginning of
fermentation to boost mycelium formation. Biomass growth occurs
during the first 48 h (trophophase), and pH is reduced to about 3 as a
result of nitrogen metabolism. At pH 3 (idiophase), the production of CA
begins (Max et al., 2010). This pH < 3 condition is important to inhibit
the production of by-products, such as oxalic and gluconic acid, and to
prevent contamination (Karaffa and Kubicek, 2019; Roukas and Harvey,
1988). The appropriate temperature for CA production depends on the
employed strain. However, A. niger fermentation is generally carried out
in the range of 30 to 37 ◦ C (Ikram-Ul et al., 2004; Sun et al., 2018; Wang
et al., 2015; Zhou et al., 2017). Another requirement for CA production
is a high dissolved oxygen tension, which must be controlled through the
regulation of agitation intensity and aeration rate during fermentation
(Ikram-Ul et al., 2004; Karaffa and Kubicek, 2019).
Some advances involving the composition of the medium, process
parameters (pH, temperature, aeration, agitation, culture time) and
strains’ improvement led to high yields (>70%) and very close to the
theoretical expected yield (1.12 g of anhydrous CA per g of sucrose), as it
can be seen in Table 1.
Wang et al. (2015) used A. niger CGMCC 5751 strain to produce CA in
liquefied corn medium (184 g L-1 of initial total sugar). The addition of
0.2 mg L-1 of antimycin A at the 24-h time point of fermentation
increased CA production by 19.89%, resulting in 151.67 g L-1 of CA, a
product yield of 0.90 g g− 1 and 1.58 g L-1h− 1 of productivity. Antimycin
A improves glycolysis by suppressing the ATP synthesis that is respon­
sible for feedback inhibition of phosphofructokinase. Ikram-Ul et al.
(2004) obtained A. niger mutants by UV-irradiation followed by MNNG-
mutagenesis. The best mutant (GCMC-7) produced 96.1 g L-1 of CA,
while the parent strain produced 31.1 g L-1 using clarified sugarcane
molasses medium. In a 9-L STR bioreactor, the addition of magnesium
sulfate in 6 h of fermentation increased CA production from 78.2 g L-1 to
113.6 g L-1, resulting in a product yield of 0.8 g g− 1. The transformant
A. niger OG1, which was constructed through the deletion of the
α-glucosidase gene combined with overexpression of the endogenous
glucoamylase gene, produced higher CA titer (185.7 g L-1), product yield
(YCA/S = 1.03 g g− 1), and productivity (2.57 g L-1h− 1) in liquefied corn
medium. In comparison with the parental strain (A. niger CGMCC
10142), the transformant promoted reduction of 88.2% of residual sugar
concentration with a CA production increase of 16.9% (Wang et al.,
2016). Yu et al. (2018) developed a material for A. niger immobilization
that was employed in a repeated-fed batch fermentation system using
liquified cassava as substrate. The authors obtained high CA concen­
trations (162.7 g L-1) with a high productivity (2.26 g L-1h− 1) and
product yield (0.9 g g− 1).
Also, as shown in Table 1, genetically engineered yeast strains have
been employed in CA production. The use of metabolic engineering tools
has enabled high conversion rates (>70%), the use of new substrates,
such as inulin and extract of Jerusalem artichoke tubers, with decreased
by-products formation (Liu et al., 2013; Rakicka et al., 2019; Wang et al.,
2013). These high yields obtained with the use of low-cost raw materials
indicate a high technological maturity of CA fermentation process,
which is unfortunately different from that observed in CA downstream
processes.
2.1. Microorganisms
The most used microorganism in CA industrial production is the
fungus Aspergillus niger (Hu et al., 2019; Max et al., 2010; Soccol et al.,
2006). This is related to A. niger’s ability to generate large amounts of CA
S. Mores et al. Bioresource Technology 320 (2021) 124426

Table 1
Comparison of citric acid production from different carbon sources, microorganisms, and culture mode.
Strain Carbon source (total sugar) Culture Time CA (g YCA/S (g Productivity (g Reference
mode (day) L− 1) g− 1) L− 1h− 1)

A. niger MH 15-15f Sucrose (14%) F 14 53 0.48 0.15 Hossain et al. (1984)

A. niger MH 15-15f Glucose (14%) F 14 31 0.35 0.09 Hossain et al. (1984)
A. niger GCMC-7f Cane molasses (15%) F 7 96.1 1.0 0.57* Ikram-Ul et al. (2004)
A. niger W5f Cane molasses (16%) F 6 98.1 0.61 0.68* Lotfy et al. (2007)
A. niger W5f Beet molasses (16%) F 6 113.5 0.71 0.79* Lotfy et al. (2007)
A. niger J4 Date syrup (15%) F 6 56.7 0.70 0.39 Mostafa and Alamri
(2012)
A. niger L2g Corn starch (12%)a F 7 76 – 0.45 Yin et al. (2017)
A. niger A1g Corn starch (12%)a F 4 117 – 1.22 Yin et al. (2017)
g
A. niger H915-1 Corn starch (12%)a F 4 157 – 1.64 Yin et al. (2017)
A. niger 831f Liquefied cassava (18%) F 4 162.7 0.9 2.26 Yu et al. (2018)
A. niger LPB 21 Cassava bagasse F 6 220c – 1.52d Vandenberghe et al.
(2004)
A. niger B60 Pomegranate peel wastes F 8 351.5c – 1.79d Roukas and Kotzekidou
(2020)
Y. lipolytica NRRL YB-423 Pure glycerol (40%) F 9 21.6 0.54 0.10 Levinson et al. (2007)
Y. lipolytica ACA-DC 50,109 O.M.W.based media (6.5%)b F 15 28.9 0.53 0.08* Papanikolaou et al.
(2008)
Y. lipolytica W29g Glucose (9%) F 5 30.1 0.33* 0.25* Yuzbasheva et al. (2019)
A. niger MH 15–15 Sucrose (14%) B 10 38 0.34 0.15 Hossain et al. (1984)
A. niger MH 15–15 Glucose (14%) B 10 13 0.13 0.05 Hossain et al. (1984)
A. niger GCMC-7 g Cane molasses (15%) B 6 113.6 0.80 0.78 Ikram-Ul et al. (2004)
A. niger CGMCC 5751 g Liquefied corn (18.4%) B 4 151.67 0.90* 1.58* Wang et al. (2015)
A. niger OG1g Liquefied corn (17.98%) B 3 185.7 1.03* 2.57* Wang et al. (2016)
A. niger chsC-3 g Liquefied corn (17.7%) B 3 180.3 1.02 2.50* Sun et al. (2018)
A. niger LPB 21 Cassava bagasse B 6 269c – 1.87d Vandenberghe et al.
(2004)
A. niger NRRL 567 Apple pomace B 5 294.2c – 2.45d Dhillon et al. (2013)
C. lipolytica Y 1095 Glucose (15%) B 4 50 0.61 0.54 Rane and Sims (1993)
Y. lipolytica Wratislavia 1.31f Raw glycerol (55%) B 6 124.5 0.62 0.88 Rywińska et al. (2010)
Y. lipolytica strain 30 g Extract of Jerusalem artichoke B 14 68.3 0.91 0.20* Wang et al. (2013)
tubers (8.43%)
g
Y. lipolytica transformant 30 Inulin (10%) B 9 84 – 0.39* Liu et al. (2013)
Y. lipolytica W29(YlAMPD Glucose (4%) B 7 97.1 0.50 0.58* Yuzbasheva et al. (2019)
YlYHM2)g
Y. lipolytica AWG7 INU 8 g Inuline B 16 203 0.85 0.51 Rakicka et al. (2019)

F: Flask; B: Bioreactor; a Concentration of reducing sugar; b Olive-mill wastewater-based media; c g/kg dry substrate; d g/kg/h; e repeated-batch culture with
replacement by fresh medium after substrate exhaustion; f Mutant strain obtained with physical or chemical agents; g Genetically engineered strain * Calculated from
the data.

using low-cost raw materials, such as molasses and corn flour liquefied
(Ikram-Ul et al., 2004, 2001; Sun et al., 2018; Wang et al., 2017) .
Furthermore, A. niger is easy to handle and does not produce significant
amounts of by-products, such as isocitric acid, observed in fermentation
using Y. lipolytica yeast (Ozdal and Kurbanoglu, 2019; Soccol et al.,
2006). Despite that, several bacteria, such as Arthrobacter paraffinens,
Bacillus licheniformis, and Corynebacterium ssp, and yeast, such as
Candida tropicalis, C. oleophila, C. guilliermondii, C. citroformans, Hanse­
nula anomala, and Yarrowia lipolytica, can also produce CA.
Mutagenesis with chemical and physical agents have been exten­
sively explored and applied in CA production, which allows obtaining
hyperproductive strains, such as 2-deoxy-d-glucose resistant mutant
strains (Ikram-Ul et al., 2004, 2001; Lotfy et al., 2007). Nowadays, more
sophisticated techniques such as those involving genomic editing have
been explored. In addition to the ability to generate overproducer mu­
tants, metabolic engineering has shown great potential by enabling a
reduction in the generation of by-products, reduction of residual sugar,
use of new substrates, and regulation of fungal morphology (Amato
et al., 2020; Cairns et al., 2018; Tong et al., 2019).
Wang et al. (2016) constructed transformants of A. niger by deletion
of the α- glucosidase-encoding agdA gene in a parent strain (A. niger
CGMCC 10142) genome using Agrobacterium tumefaciens-mediated
transformation. These changes resulted in increased glucoamylase ac­
tivity and decreased α-glucosidase activity in the transformed strains.
This enabled an 88.24% reduction in residual reducing sugars and an
increase of 16.87% in the production of CA when using liquefied corn
starch as substrate. Similarly, Ming et al. (2018) described a reduction in
content of residual sugar by up to 10.26% and an increase in CA yield by
up 12.16% when genetic engineering strains, which were obtained by
knocking out glucosyltransferase genes in A. niger, were employed.
Since the mycelial morphology of A. niger plays an important role in
submerged fermentation for CA production, the use of molecular tech­
niques with focus on the regulation of morphology can be interesting.
Sun et al. (2018) built an RNAi system to silence the chsC gene, which
promoted a decrease in mycelial pellet compaction. These morpholog­
ical changes promoted an increase in CA production and glucose con­
version rate in both shake flask and stirred tank bioreactor. In shaker
flasks, CA production by engineered strains was improved by 42.6%. In
stirred tank bioreactors, CA yield and the conversion rate of glucose
were 180.3 g L-1 and 101.9%, respectively, which represented an in­
crease of 3.6% for both, when compared with the parental strain.
The application of genetic manipulation tools is facilitated in yeasts
since it has a less complex genetic background than filamentous fungi
(Mei et al., 2019). Studies reported the use of genetic tools in Yarrowia
lipolytica to obtain transformed strains with the ability to use inulin, a
renewable substrate widely available. Besides, these genetically modi­
fied strains are capable of producing more citric acid, generating less
isocitric acid, and decreasing in residual sugar content (Liu et al., 2013;
Rakicka et al., 2019).
2.2. Fermentation techniques
Fermentation processes using A. niger can be performed by using
three different techniques: submerged (SmF), surface (SF), and solid-

3
S. Mores et al.
state fermentation (SSF). All methods have their advantages and dis­
advantages, as shown in Fig. 1. The SSF method enables the use of
different inexpensive wastes as substrate, resulting in lower energy,
water consumption, and, consequently, lower wastewater generation
(Soccol et al., 2017). However, the technological maturity of the SSF
method is not yet industrially adequate. SSF requires intensive labor in
the loading, unloading, and cleaning stages, resulting in longer overall
process time and high operational cost, a problem that is also associated
with SF (Vandenberghe et al., 2016). SmF involves more complex
equipment that results in higher installation investments. Despite that,
the SmF automatization allows the standardization of the process,
higher yields, reduced labor and space, and less risk of contamination.
Usually, beet and sugarcane molasses are used as substrates.
Nevertheless, it is possible to use other substrates composed of mono-
and disaccharides. Due to the various advantages of SmF compared to
other methods, about 80% of the world production of CA is carried out
through this fermentation method (Amato et al., 2020; Roukas and
Kotzekidou, 2020; Tong et al., 2019). Some small and medium-sized
industries that started the production of CA commercially using the
surface technique remain in operation, which contributes to the fact that
currently about 99% of the acid is produced through fermentative pro­
cesses (Cavallo et al., 2017; Max et al., 2010).
Recently published reviews indicate that innovations in CA produc­
tion processes include the use of the fungal immobilization method,
multiple step processes, and the substitution of traditional glucose by
food and agriculture waste. Different kinds of agro-industrial wastes as
corn, fruits, vegetables, liquefied starchy materials, lignocellulosic
biomass, and molasses have been researched. Some of them are
employed industrially for CA production but the most part still faces
limitations due to the need for pre-treatment or difficulty in scale-up in
the case of solid state fermentation (Amato et al., 2020; Hu et al., 2019).
Fig. 1. Main characteristics of the three fermentation methods in citric acid
production: Solid-state fermentation; Submerged fermentation; and Surface
fermentation.
4
Bioresource Technology 320 (2021) 124426
3. Citric acid recovery
In industrial CA production, the downstream process represents
30–40% of the total production cost (Straathof, 2011). Traditionally, CA
separation from fermentation broths is carried out by precipitation using
calcium salt. A recovery step with sulfuric acid is then required. This
technique involves several and laborious stages, requires significant
amounts of reagents, and generates huge amounts of waste whose
disposal can represent up to 85% of the overall environmental cost
(Harrison et al., 2015). Because of that and due to the growing demand
for high purity acid it is necessary and there is a great interest in research
and development of more sustainable downstream methods and pro­
cesses. Thus, this overview provides a comprehensive knowledge of the
most recent developments of CA recovery and purification.
3.1. First step of citric acid recovery
In the CA recovery obtained by SF, after draining the liquid from the
trays, the mycelia are washed with water to extract the retained acid. In
SSF, recovery begins with the extraction of the acid adhered to the solid
material using hot water. In SmF, CA is already dissolved in the
fermentation broth, so the solubilization steps are not necessary. Despite
that, the fermentation broth can be heated to obtain a partial protein
coagulation to facilitate the subsequent stage of mycelium removal. The
removal of biomass from the aqueous solutions is carried out by
centrifugation or filtration. Then, several unit operations can be used for
CA separation and purification (Berovic and Legisa, 2007; Felman et al.,
1998; Max et al., 2010). At the end of an industrial SmF process by
A. niger, CA concentration is about 150 g L-1 in the fermentation broth
(Table 1) and a low pH of 2 (Max et al., 2010). The presence of other
compounds varies according to the sources of carbon, nitrogen, metals,
and other added compounds or medium components. Agricultural and
food industry wastes are universally available and their use in CA pro­
duction promotes cost reduction that is why they are mostly used as raw
material for fermentation (Amato et al., 2020; Show et al., 2015).
However, these materials are complex and composed of several com­
ponents, which require separation after fermentation, implying in a
complex downstream step to obtain a high purity product (Hu et al.,
2017; Pais et al., 2016). Residual sugars, proteins, and inorganic salts are
the main components of the fermentation broth that need to be sepa­
rated (Wu et al., 2009).
3.2. Precipitation
The classical precipitation technique remains one of the most widely
used recovery methods in the production of CA (Sun et al., 2017;
Widiasa et al., 2004). This technique (Fig. 2) consists of using lime at
high temperatures (85 to 90 ◦ C) to precipitate CA, present in the
fermentation broth, in the form of citrate (calcium, potassium, or so­
dium). Then, the insoluble citrate that is formed is separated from the
liquid phase by filtration and then it is washed several times to remove
impurities, such as sugars and other fermentation media components.
The aqueous suspension is then treated with sulfuric acid. The CA is
separated from the calcium sulfate by a new filtration step (López-
Garzón and Straathof, 2014; Sun et al., 2017). To obtain a product of
greater purity, subsequent steps are necessary, such as adsorption using
resins and activated charcoal, evaporation, crystallization, and drying
(Feng et al., 2016; Max et al., 2010).
The first step in precipitation is the conversion of CA (soluble) to
calcium citrate (insoluble) by the addition of calcium hydroxide
(Equation (1)):
2C6 H8 O7 + 3Ca(OH)2 →(C6 H5 O7 )2 Ca3 .4H2 O + 2H2 O (1)
In the second stage (Equation (2)), the reaction of sulfuric acid with
calcium citrate (insoluble) results in the production of CA (soluble) and
calcium sulfate (insoluble):
S. Mores et al.
Fig. 2. Schematic diagram for citric acid recovery by precipitation.
(C6 H5 O7 )2 Ca3 .4H2 O + 3H2 SO4 →2C6 H8 O7 + 3CaSO4 .2H2 O + 2H2 O (2)
For each 1 kg of CA processed, 0.58 kg of calcium hydroxide (Ca
(OH)2) and 0.76 kg of sulfuric acid (H2SO4) are required. At the end, 1.3
kg of gypsum are generated and approximately 18 L of water are
consumed. To decrease these quantities, another recovery method is
required. Although gypsum (calcium sulfate) can be treated for use in
the construction industry, treatment costs are too high considering the
value of the final product. For this reason, gypsum is usually discarded in
landfills, causing environmental impacts (Kristiansen et al., 1998). Ac­
cording to Harrison et al. (2015), chemicals such as sulfuric acid and
calcium sulfate, which are used in precipitation, account for 11.4% of
overall operating cost of CA production. Also, the disposal of the
generated gypsum (calcium sulfate) after precipitation represents 85%
of environmental cost.
Processing factors, such as temperature, pH, and reaction time,
interfere in the precipitation. Heding & Gupta (1975) performed the
precipitation of CA with calcium carbonate at pH 5 and different tem­
peratures. The authors observed that at high temperatures (80 ◦ C and
100 ◦ C) it was possible to recover almost all CA and the reaction
occurred instantly, however, the precipitate was difficult to filter and
presented in the form of clusters after drying, which made it difficult to
handle in subsequent processes. At temperatures below 50 ◦ C, the au­
thors observed a significant increase in reaction time and a decrease in
the rate of CA recovery. On the other hand, the precipitation conducted
at 50 ◦ C had a reaction time of about 20 min, with recovery of about all
CA, resulting in a product with interesting characteristics for the sub­
sequent purification steps. The temperature control during the precipi­
tation process is especially important because it determines the
characteristics of citrate. It is desirable the formation of small particles
in the precipitate, which show a larger surface area that is available for
reaction with sulfuric acid, increasing its efficiency. Wang et al. (2013)
recovered CA from the fermentation broth using steps of centrifugation,
precipitation (CaCO3 at 85 ◦ C), solubilization (H2SO4 10% v/v), and
crystallization (60 and 80 ◦ C). The yield of the downstream process was
67.2% with a purity of 96% CA. A patent, assigned to Bayer Corp and
Tate and Lyle Citric Acid Inc, describes a precipitation process with the
aim of reducing waste formation and operating costs as well as
increasing productivity and final product quality (Felman et al., 1998).
The process consists of slow addition of a solution of strong acid or salt,
such as sulfuric acid, nitric acid, hydrochloric acid, sodium chloride, or
sodium sulfate, to the fermentation broth. CA crystallizes rapidly in the
mixture, which is stirred and gradually cooled to 30 ◦ C and filtered. With
5
Bioresource Technology 320 (2021) 124426
this process, it is possible to recover at least 80% of the CA and reduce
impurities by 90%.
The classical method of precipitation involves high capital in­
vestments and operating costs due to the kind and number of operations
required for product recovery as shown in Fig. 2. These operations
employ high amounts of labor, reagents, energy, water, and generate
large amounts of waste (liquid and gypsum) that need treatment before
disposal (Delgado Dobladez et al., 2019; Shishikura et al., 1992; Wang
et al., 2020, 2015). Despite the various disadvantages, this technique
remains widely used for CA recovery because it does not require com­
plex technologies and equipment, and the characteristics of the process
are well known, which facilitates its implementation and use.
3.3. Solvent extraction
Unlike the precipitation method, in solvent extraction, there is no
change in the aggregate state of the product, so steps of the separation
phase and the repeated dissolution are not necessary, and gypsum is not
produced. Therefore, solvent extraction is characterized by high effi­
ciency, low consumption, and in situ or continuous recovery (Cheng
et al., 2012; Feng et al., 2016; Wennersten, 1983). Although various
compounds can be used as solvents in the extraction, some character­
istics are necessary for process efficiency. The ideal solvent for appli­
cation in the recovery of organic acids should extract the maximum
amount of the product of interest and minimum of impurities. Each
solvent is characterized by an equilibrium distribution coefficient,
which is defined as the ratio between the concentration of acid in the
extract and the concentration of acid in the aqueous phase at equilib­
rium (Kristiansen et al., 1998; Wennersten, 1983). Conventional organic
solvents such as long-chain alcohols and ketones give low distribution
coefficients, because of the high affinity of acid for water. However,
some solvents such as butanol have a high coefficient of distribution, but
they are miscible with water and require an extra recovery stage. In low
concentrations of CA, such as fermentation broths < 15%, the distri­
bution coefficient strongly depends on the type of solvent, and an
additional concentration step is needed before extraction (Apelblat,
2014; Kristiansen et al., 1998; Wennersten, 1983). Thus, the low dis­
tribution coefficients of the components imply the need for large
amounts of solvent in the feed and an increase in the number of stages
required for CA separation.
Due to the low efficiency of liquid–liquid extraction with conven­
tional solvents, alternative techniques that are economically viable for
the recovery of organic acids from fermentation broths have been
S. Mores et al.
explored, such as reactive extraction (Kristiansen et al., 1998; Rani et al.,
2010; Thakre et al., 2016). In reactive extraction, chemical (reaction
between the solute and extractant) and physical phenomena (diffusion
and solubilization of the formed compound) occur (Rani et al., 2010).
This technique consists of two main steps: extraction and back extrac­
tion. In the first stage, the fermentation broth is mixed with the
extractant/diluent, promoting the complexation between the extractant
and the carboxylic acid and the formation of an aqueous raffinate with a
low concentration of carboxylic acid. Then, the extraction of the
carboxylate present in the organic complex is carried out to an aqueous
phase while the extractant is regenerated and recycled to be used again
in the extraction process. Back extraction can be conducted using
different methods, such as changing temperature, pH, diluent compo­
sition, among others (Datta et al., 2015b; Hong et al., 2001; López-
Garzón and Straathof, 2014). Extractant recovery is possible because the
complexation reaction is reversible. Extractants should be chosen so as
to provide a complexation capable of overcoming the low activity of the
solute in the aqueous phase, but which is not so strong as to make
regeneration difficult or impossible (Hong et al., 2001; Magalhães et al.,
2017).
The various extractants that can be used in reactive extraction are
divided into three categories: carbon-bonded oxygen-bearing,
phosphorus-bonded oxygen-bearing and aliphatic amines. Among these,
the aliphatic amines, mainly tertiary, have been outstanding in the re­
covery of CA from dilute aqueous solutions (Baniel and Gonen, 1991;
Bízek et al., 1993; Datta et al., 2015a; Verser et al., 2014; Wennersten,
1983). Usually, combinations of extractants are used, or the extractants
are mixed with solvents to reduce costs and modify physical–chemical
properties (Thakre et al., 2016). Tertiary amines, for instance, have high
viscosity, corrosivity, and surface tension that are undesirable properties
in the extraction process, therefore they are used in solution with
organic solvents (Djas and Henczka, 2018; Hong et al., 2001). Diluents
are divided into two categories: inactive and active. The active diluents
are polar with several functional groups that increase the solubility of
the extractant-solute complexes, such as chlorinated hydrocarbons,
vegetable oils, and kerosene, and the inactive diluents, which include
alkanes, benzenes, and aromatics substituted with alkyl, are nonpolar
and provide a very low solubility of the complex (Djas and Henczka,
2018; Thakre et al., 2016). The number of impurities, the concentration
of the acid, the extractant, and the employed diluent, as well as their
physical–chemical properties, are some of the factors that influence the
extraction efficiency (Araújo et al., 2017; Rani et al., 2010). Studies
conducted by Thakre et al. (2016) evaluated the reactive extraction of
CA from dilute aqueous solutions. The tri-n-butyl phosphate (TBP), tri-n-
octylamine (TOA), and Aliquat 336 (A336) extractants dissolved into
three different diluents: butyl acetate, decanol, and benzene were tested.
The maximum degree of extraction (95.5%) was obtained with the 20%
(v/v) TOA + butyl acetate system resulting in a distribution coefficient
of 21.25.
The choice of the solvent is an extremely important factor to obtain a
viable extraction process. Many solvents are toxic, which makes it
difficult to apply the technique in situ since they can inhibit enzymes,
modify the permeability of cell membranes and hinder the diffusion of
nutrients (Keshav et al., 2012). Furthermore, industrially, the use of
potentially toxic solvents may represent an increase in costs, due to the
need for greater process safety and environmentally correct disposal
(Choi and Verpoorte, 2019). To obtain food grade CA, the solvent op­
tions are even more restricted. In the search of a more environmentally
friendly alternative, Djas & Henczka (2016) investigated the reactive
extraction of CA aqueous solution using the supercritical carbon dioxide
and tri-n-octylamine (TOA). The authors obtained a high efficiency
(96.9%) using a flow of supercritical carbon dioxide saturated with TOA
at high pressure (16 MPa) and low temperature (35 ◦ C) and concluded
that this type of extraction is efficient for the recovery of carboxylic
acids from aqueous solution. Although, in recent years, the supercritical
extraction technology have shown reduced operating costs and use of
6
Bioresource Technology 320 (2021) 124426
equipment , this method remains limited to products with greatest
commercial value (Djas and Henczka, 2018).
Keshav et al. (2012) tested the use of different non-toxic natural
diluents (rice bran oil, sunflower oil, soy oil, and sesame oil) associated
with a tertiary amine (tri-n-octylamine, TOA) to recover CA from solu­
tions aqueous. The authors found a decrease in the distribution coeffi­
cient with increasing acid concentration, suggesting that reactive
extractions using TOA may be interesting in removing CA from diluted
solutions. The authors obtained a high degree of extraction when 30%
(v/v) TOA was used with these diluents. The diluents had a distribution
coefficient>12, with diluents in decreasing order of degree of extrac­
tion: rice bran oil (95%), soybean oil (94%), and sesame oil (94%), and
sunflower oil (93%). Liu et al. (2020) proposed a new way to separate
CA from the fermentation broth using ten different dialkyl amides as
extractants and isostearyl alcohol as a diluent. This method investigated
the use of amides as hydrogen bonds acceptors and CA as the hydrogen
bond donor for the formation of deep hydrophobic eutectic solvents.
With a contact time of 20 min at 30 ◦ C, the authors extracted 75% of CA
with a high degree of purity (99.8%). Besides, it was possible to
regenerate the extractant quickly and easily with an increase in tem­
perature (75 ◦ C). Extractant recycling was possible without significant
loss of capacity for at least 5 cycles.
Another process for CA recovery using reactive extraction was
described by Rongjie et al. (2017). The method proposes the mixing of
CA solution (fermentation broth, for example) with an organic solvent.
Then, the mixture is subjected to vacuum evaporation. As a result, the
organic acid is concentrated in the organic phase, while most inorganic
salts and other impurities remain in the aqueous phase. Then, water is
added to the organic solvent so as CA re-enters the aqueous phase to
obtain an aqueous solution composed of the organic acid. The authors
treated a solution containing 50% (mass percentage) of CA with an
organic solvent mixture composed of trioctyl ammonium, octadecanol,
and kerosene. The CA yield and purity were both 99.0%.
Compared to the classic precipitation technique, reactive extraction
implies lower environmental impacts due to the possibility of reusing
reagents, reducing the generation of solid waste, reducing energy
expenditure, and the possibility of using natural and non-toxic diluents.
Despite that, the technique is still limited by the costs of extractants and
diluents. There is still much to be explored, mainly in the search for new
extractants for the development of processes that are efficient and
economically viable.
3.4. Adsorption
Adsorption is a unitary operation that can be employed at large scale
in the recovery and purification of biomolecules. The properties of the
different adsorbents are exploited by the ability to preferentially interact
with the compound of interest or with the impurities that are also pre­
sent in the broth. Depending on the physicochemical properties such as
charge and polarity, the adsorbent material can interact strongly with
the target biomolecule and adsorb it. Then, the adsorbed compound is
desorbed by passing a desorbent (eluent) to obtain a solution of the
biomolecule with fewer impurities concentration. The adsorbent is a
solid particulate material that can be disposed in agitated tanks, com­
pacted in columns, or used in moving bed systems (de Carvalho et al.,
2017). The adsorption technique can be performed continuously, using
fixed bed columns coupled to the bioreactors. As the columns is packed
with the adsorbent, there is a greater ease of operation and less loss of
solvent compared to the extraction technique. Nevertheless, there may
be problems with fouling and shortening the useful life of the adsorbents
(López-Garzón and Straathof, 2014). Several studies indicate the use of
adsorption for CA recovery after fermentation step using different ad­
sorbents, mainly those synthesized with polymeric materials. Among the
resins, those of basic anionic exchange impregnated with tertiary and
quaternary amines, or pyridine, stand out for their affinity and selec­
tivity about the CA (Delgado Dobladez et al., 2019; Gluszcz et al., 2004;
S. Mores et al.
Kulprathipanja, 1989; Kulprathipanja and Oroskar, 1991; Martin et al.,
2020; Van den Bergh et al., 2017; Wu et al., 2009).
The high capacity and selectivity of the adsorption to the target
biomolecules are important factors necessary for a greater efficiency of
the process. Besides that, being easily regenerated with the green sol­
vents, such as water and ethanol, is a desirable feature (Van den Bergh
et al., 2017). Van den Bergh et al. (2017) compared the performance of
three resins (PVP, IRA-900, and IRA-67) in CA recovery. The PVP resin
has a high capacity for the adsorption (0.9 g g− 1) and selectivity for the
CA at low pH, as well as in moderate pH solutions with sulfates.
Therefore, the use of PVP resin proved to be a viable alternative for the
direct recovery of CA from fermentation broth (low pH). Another
application is after the precipitation process, which has a solution rich in
sulfate and moderate pH. PVP resin can be regenerated efficiently with
methanol or ethanol at a high temperature, avoiding the generation of
low-value waste, which happens when acids or bases are used in
regeneration.
In the 1980 s, the UOP company applied the first patent for CA re­
covery from fermentation broth with the simulated moving bed (SMB)
technology. In this process, the pH of the fermented broth is adjusted so
that it is below the first CA ionization constant (pKa1). This standardi­
zation is necessary to maintain the selectivity of the adsorbent. Ketone
or inorganic acid are used as eluents (desorbents) (Kulprathipanja,
1989, 1988; Kulprathipanja et al., 1989; Kulprathipanja and Oroskar,
1991). Wu et al. (2009) proposed a process for CA recovery from
fermentation broth using SMB technology with a tertiary poly (4-
vinylpyridine) resin (PVP) as a stationary (adsorbent) phase. The au­
thors performed the tests at pilot scale (flow rate up to 150 mL/min) and
simulated processes with separation requirements defined as at least
99.8% of purity and 90% of CA recovery in the extraction stream. With
the process parameters defined, the authors reached 97.2% of CA re­
covery and a product with 99.8% of purity and the concentration of CA
in the extract being half of its concentration in the pre-treated fermen­
tation broth. This process is environmentally interesting due to the
minimal waste generation, as only water is added to the system and the
mother liquor after separation of the CA can be recycled for the
fermentation step. Using the same SMB technology having a commercial
resin (Reillex® 425) as adsorbent and methanol as eluent, Delgado
Dobladez et al. (2019) developed a theoretical model to describe the
adsorption and desorption of CA from aqueous solution (10% w/w). A
CA purity above 99% and recovery above 95% was obtained with resin
adsorption capacity of 0.57 g g− 1. An evaporation-crystallization
method is designed for the recovery of CA and methanol from the
extract. At the end of the process, the exhausted stream (raffinate
product) can be recycled avoiding the loss of CA in this stream and
allowing the reuse of water and the soluble components in the fermen­
tation process. The recovery of CA and methanol from the process
extract was proposed by evaporation-crystallization at 60 ◦ C.
Wang et al. (2020) compared the economic and environmental
impact of CA recovery through three different processes that employed
precipitation, reactive extraction, or adsorption. The authors concluded
that the adsorption technique is the most environmentally friendly with
the lowest acidification, eutrophication, ecotoxicity, and global warm­
ing potential. They also estimated a higher annual operating cost of
adsorption ($61 million) than solvent extraction ($54 million) and lower
than precipitation ($70 million) as well as total project investments
which were $35.1, $32.6 and $41.5 million, respectively, for a com­
mercial scale of 6400 kg/h CA production rate. Although the adsorption
method reduces the energy and reagents consumption, there are ex­
penses with regeneration and periodic exchange of resins due to the
limited useful life.
3.5. Electrodialysis
Membrane separation techniques, such as ultrafiltration, electrodi­
alysis and electrodeionization, and pertraction, have also been tested for
7
Bioresource Technology 320 (2021) 124426
the recovery of CA from aqueous solutions or fermentation broths
(Angumeenal and Venkappayya, 2013; Basu and Sirkar, 1991; Chandra
et al., 2018; Datta and Bergemann, 1996; Geanta et al., 2013; Luo et al.,
2017; Sun et al., 2017; Tongwen and Weihua, 2002; Widiasa et al., 2004;
Yordanov and Boyadzhiev, 2004). The study of processes involving the
electro membrane has predominated in CA recovery, mainly electrodi­
alysis with bipolar membranes. Electrodialysis processes allow impu­
rities separation and organic acid concentration from an aqueous
solution based on the electro-migration of ions through ion exchange
membranes. Bipolar membranes consist of a layer of an anion exchanger
and a layer of a cation exchanger. At the interface of these bipolar
membranes, the water dissociates to produce acids and bases from salts.
Therefore, from a solution containing sodium citrate, it is possible to
obtain CA and a free base that can be reused in the process (Handojo
et al., 2019; Sun et al., 2017; Tongwen and Weihua, 2002).
The concentration and composition of the feed directly affect the
efficiency of the electrodialysis process. CA recovery is reduced when in
complex media, with the presence of several substances, as shown in
Table 2. Chandra et al. (2018) observed a drop of up to 40% in the ef­
ficiency of CA recovery, when the feeding solution of the electrodialysis
process is composed of strong electrolytes, such as NaCl, CaCl2, or FeCl3.
The adsorption of these metals and organic molecules in the membranes,
which is due to concentration polarization and fouling phenomena, re­
sults in higher operating costs due to the increase in energy demand and
the decrease in the useful life of the membranes. Thus, an adequate
treatment of the fermented broth for the removal of suspended solids
and metallic ions is necessary, such as microfiltration, adsorption with
activated carbon, or resins (Brichant et al., 2018; Handojo et al., 2019;
Hb̌ová et al., 2004; Widiasa et al., 2004). The performance of electro­
dialysis is also influenced by the pH of the feed solution, with greater
efficiency at high pH (above 7). In this pH range, there is a rapid CA
dissociation, since it is superior to the three dissociation constants of the
molecule: 3.1, 4.7, and 6.4 (Nikbakht et al., 2007). The increase in pH
results in a decrease in the electrical resistance of the solution and an
improvement in the separation efficiency. This condition uses low CA
concentrations in the feed, which is not the case with fermentation
broths, or implies the need to correct the pH of the broth before
electrodialysis.
Brichant et al. (2018) developed a CA purification process consisting
of a chromatographic separation step followed by an electrodialysis
treatment. In this process, the pre-treated fermentation broth first passes
through the chromatographic separation unit, where it is retained.
Elution with a sulfuric acid solution generated the refined and extract
streams. The extract containing most of the CA undergoes more purifi­
cation steps to generate a higher purity product. The refined product,
containing CA, sulfuric acid, and impurities that were not been recov­
ered in the extract, is fed to an electrodialysis unit. The authors reported
a recovery of at least 99% of the sulfuric acid and between 60 and 80% of
CA in the concentrate compartment. The solution can then be inserted in
the fermenter, in the adsorption unit or as a cleaning stream for the
filtration membranes, thus reducing the generation of waste and the
consumption of reagents.
Moresi et al. (1998) evaluated the recovery of CA from aqueous so­
lutions using the electrodialysis and operating costs of the technique
were higher (about 50% higher) than the costs of the conventional CA
recovery process. Although there are still no studies comparing CA
downstream methods, Magalhães et al. (2019) simulated the use of
different recovery process of itaconic acid, an organic acid produced by
Aspergillus terreus fungus, from fermentation broth. The authors
compared crystallization, a method traditionally used in the industry to
recover itaconic acid, with adsorption, reactive extraction, and elec­
trodialysis. Only electrodialysis was not able to compete with crystalli­
zation. The economic infeasibly was attributed to the low recovery yield,
membrane durability, and high energy demand. However, the advan­
tages of using the electrodialysis technique include: high purity of the
recovered products, short process time, reduced generation of by-
S. Mores et al. Bioresource Technology 320 (2021) 124426

Table 2
Methods of citric acid recovery.
Method CA solution (%) CA recovery Purity Temperature Reaction time Processing time Reference
(%) (%) (◦ C) (min) (min)

Precipitation + Crystallization Fermentation broth >75 – 60 to 130 105 – Annadurai et al.

(5%) (1996)
Precipitation + Crystallization Fermentation brotha >80 >90 30 – 120 Felman et al. (1998)
Precipitation + Crystallization Fermentation broth 67.2 96 60 to 85 105 205 Wang et al. (2013)
(6.8%)
Extraction + Precipitation with Dry fermented grainb 96.2 to 99.3 99.1 to 40 30 120 to 180 Shishikura et al.
high pressure 99.9 (1992)
Extraction + Precipitation with Aqueous solution 92.9 to 98.2 97.6 to 40 – – Shishikura et al.
high pressure (56.8%) 99.8 (1992)
Extraction + Precipitation with Fermentation broth 92.4 99.8 35 – – Shishikura et al.
high pressure (56.4%) (1992)
Reactive extraction Fermentation brothc 90f – 50 to 65 – Baniel and Gonen
(1991)
Reactive extractiong Aqueous solution 93f – RT 240 – Keshav et al. (2012)
(4.2%)
h f
Reactive extraction Aqueous solution 94 – RT 240 – Keshav et al. (2012)
(4.2%)
Reactive extractioni Aqueous solution 95f – RT 240 – Keshav et al. (2012)
(4.2%)
Reactive extraction Aqueous solution 98f – 25 15 – (Araújo et al., 2017)
(10%)
Reactive extraction Aqueous solution 99 99 80 – – Rongjie et al. (2017)
(50%)
f
Reactive extraction Fermentation broth 75.3 99.8 30 20 35 Liu et al. (2020)
(12%)
Adsorption Fermentation broth 97.2 99.8 80 25j – Wu et al. (2009)
(70%)
Adsorption Aqueous solution 99 – 60 120 k 240 Van den Bergh et al.
(0.5%) (2017)
j
Adsorption Aqueous solution 95.7 99.4 25 30 – Delgado Dobladez
(1%) et al. (2019)
Electrodialysis Aqueous solution 97 – 33 – – Moresi and Sappino
(5%) (1998)
Electrodialysis Aqueous solution 97.1 – RT – 140 Sun et al. (2017)
(3.3%)
Electrodialysis Aqueous solutiond 8.8 to 23.4 – 25 – 250 Chandra et al. (2018)
(1.92%)
Electrodialysis Aqueous solution 50.4 – 25 – 250 Chandra et al. (2018)
(1.92%)
Electrodialysis Fermentation brothe 60 to 80 – 25 – – Brichant et al. (2018)
Electrodeionization Fermentation broth 50 – RT – – Widiasa et al. (2004)
(0.2%)
a
Fermentation broth concentrated by evaporation to within about 10% of the saturation point of CA and most preferably to about the saturation point of CA; b 36.3%
CA and 7.29% water content; c Fermentation broth concentrated by evaporation with a citric acid concentration of at least 80% of the saturation value at the
evaporation temperature; d Containing 0.1 mol⋅L− 1 NaCl, CaCl2 or FeCl3 e Raffinate of fermentation broth subjected to a step of chromatographic separation, using
sulfuric acid solution as eluent; f Degree of extraction g TOA + sunflower oil; hTOA + soybean oil or sesame oil; i TOA + rice bran oil; j Switching time; kAdsorption time.

products, low space requirement, ease and operational flexibility, and
the possibility of continuously operate (Chandra et al., 2018; Handojo
et al., 2019; Luo et al., 2017; Sun et al., 2017). The use of membranes
allows a longer uninterrupted operation, since the regeneration steps,
such as adsorption with resins, are not necessary. Also, although the
yield is lower than other separation techniques, the material recovered
has high purity, which reduces costs in subsequent purification
operations.
4. Recent patents and innovation on citric acid production and
recovery
Knowledge and innovation are strictly related since one of the most
disseminated definition of innovation is the diffusion of a new knowl­
edge or technology to society through commercialization, which in­
creases the business value (Roper and Hewitt-Dundas, 2015). One way
to measure innovation is the analysis of patents. Although, these docu­
ments may not reveal all the value of an innovation, they can be great
indicators of new products and processes creation and diffusion (Acs
et al., 2002). The field of biotechnology is highly innovative since re­
searchers are constantly developing new fermentative processes to
increase yield and productivity, finding new applications for the
developed products. According to the World Intellectual Property Or­
ganization (WIPO), the number of published patents applications with
biotechnology focus increased 6.3% from 2007 to 2017 (WIPO, 2019).
Therefore, a patents search was conducted for revealing the state of
knowledge and recent innovations in citric acid production and recov­
ery. Lens.org and Latipat databases were employed and the desired
terms were chosen and combined with boolean language in each data­
base. The period of analysis was set from 2000 to September 2020. After
manual revision and sorting, 481 documents were analyzed with
Microsoft Excel software and Python programming language with
Anaconda3 software.
In Fig. 3A, it is shown the evolution of applied documents over the
years, which is following a crescent tendency with some natural fluc­
tuations. Technologies usually pass through 5 phases: Incoming,
Emerging, Key, Maturing and Declining (Sossa et al., 2016). Since
biotechnology is constantly growing as a technology field, the developed
products are in emerging or key phases, with great potential to grow and
change market dynamics. The CA market is also expanding and was
evaluated at USD 2.5 billion in 2016 and has a growth projection of
5.24% in the period of 2020 to 2025 (Grand View Research, 2018;

8
S. Mores et al.
Fig. 3. A) Evolution of applied patents over the years; B) Comparison between applied patents by China and other countries; C) Number of applied patents
by countries.
ReportLinker, 2020). Furthermore, the ADM company - one of the main
players of the CA market - built a food ingredient facility in China in
2009, which explains the increase in the number of documents starting
in 2010. Besides, FoodChem, another major player, was founded in 2006
in China and established an office in Germany in 2011.
According to WIPO, China is the country that has more applied
patents and other scientific and technological publications in the world.
Besides, China is only behind the USA in terms of investment in R&D. In
2016, almost USD 400 million were invested in R&D in China - and yet,
is the country that has more researchers in its population (Cornell
University et al., 2018). This fact has a direct impact on the presented
data about CA patents since China has 86.1% of the applied documents
(Fig. 3B). As mentioned, ADM and FoodChem are great producers of CA
and are established in China, as well as Cofco International. Europe is an
important region in the CA market, being the second region with more
applied documents behind Asia, which is represented by Russia, Poland
and Germany as countries that have a significant number of applied
patents (Fig. 3C). Brazil also appears as a relevant player due to the great
agricultural production in the country, which generates alternative
substrates that can be applied to CA production, reducing processes’
costs.
The most cited patents related to CA production and recovery, were
selected showing the focus of recent innovation in CA bioproduction.
The most cited patents in the last ten years involve SmF (Guangzhi et al.,
2011; Guiyang et al., 2011; Yongsheng et al., 2009; Zhenqiang et al.,
2010). This confirms the predominance of CA production through SmF
at industrial scale. Besides, these patents propose new carbon sources,
which evidences the growing search for alternative raw materials to
9
Bioresource Technology 320 (2021) 124426
meet the growing demand for CA. It is also noteworthy that CA recovery
patents propose new alternative methods to replace traditional precip­
itation techniques (Kulprathipanja, 2007; Qijun and Zheng, 2002; Song
et al., 2010). In all cases, both the academy and the industry are
searching for new economic and environmentally friendly technologies
for CA production with the reuse of different alternatives subproducts as
substrate and reduced use of chemicals.
5. Final remarks
Although the literature indicates high technological maturity of CA
fermentation process, the growing trend in the number of applied doc­
uments per year (Fig. 3A) indicates that there is still room for and in­
terest in improvements.
The data available in the literature indicate that new developed
strategies have shown their potential for CA recovery and purification.
Some employed chemical products, advantages, disadvantages, and
environmental impact of CA recovery methods are shown in Table 3.
Generally, the downstream involves the combination of different
methodologies to achieve a product with the desired degree of purity.
The techniques of solvent extraction, adsorption, and membranes sep­
aration are considered as the most attractive and interesting alternatives
to replace the precipitation technique in the CA recovery. The main
advantages are low reagents consumption, low wastes generation and
reduced processing time. Nonetheless, economic issues and technolog­
ical maturity limit the application of alternative recovery techniques.
The membrane separation technique is still an unfeasible alternative due
to the high energy consumption and equipment costs compared to the
S. Mores et al.
Table 3
Comparison of citric acid recovery methods.
Recovery method Chemical products Advantages
Precipitation Lime and sulfuric acid Process simplicityLow equipment cost
(conventional)
Reactive Extractants (aliphatic Reagents can be reusedLow energy
extraction amines) and diluents consumptionNo prior broth concentration is
(organic solvents, required
vegetables oil)
Adsorption ResinsEluents Reagents can be reusedLow energy
consumptionHigh recovery yieldHigh
selectivityHigh purityNo prior broth
concentration is required
Electrodialysis Cleaning solutions High puritySimultaneous separation and CA
concentrationNo need of regeneration
stepsBases can be recycled
commercialization price of CA. The techniques of reactive extraction
and adsorption enable continuous recovery, increasing productivity.
Environmentally, both techniques are interesting, due to the lower
consumption of reagents, the possibility of reusing them, and the gen­
eration of small amounts of waste. The techniques are also very flexible
since it is possible to combine several extractants, adsorbents, diluents,
and solvents. This makes it possible to develop new processes through
new combinations of compounds or by discovering others. The oper­
ating conditions of adsorption reduce losses and facilitate solvent re­
covery. The technique also allows the efficient use of water and “green
solvents” as eluents and acid recovery in a few steps.
However, if CA separation methods are so efficient in the laboratory,
what could be done to overcome the drawbacks and apply one or more
of these new techniques at industrial scale? Economic issues and tech­
nological maturity limit the industrial application of alternative recov­
ery techniques. As it can be seen in Table 2, many studies evaluated the
efficiency of CA recovery methods using aqueous solutions rather than
fermentation broth. So, it is difficult to accurately assess CA downstream
methods because after fermentation the organic acid is dissolved in a
complex mixture of many components that affect the efficiency of
product recovery (Chandra et al., 2018; Van den Bergh et al., 2017).
Therefore, further studies involving the use of CA fermentation broth, at
pilot scale, and cost analysis of downstream processes are required for
more efficient and economic viable processes. Also, the development of
low-cost and resistant resins, increasing solvent recycling when using
adsorption and solvent extraction methods, are necessary to turn these
techniques feasible alternatives for industrial applications.
Metabolic engineering is another alternative with great potential and
that should be better explored in the bioproduction of CA. As displayed
in this review, the use of genetically modified strains can increase the CA
yields and enable the use of new substrates (Liu et al., 2013; Rakicka
et al., 2019; Sun et al., 2018; Wang et al., 2016). Furthermore, metabolic
engineering tools can be combined in the development of more efficient
downstream processes, since broths with higher concentrations of CA
and fewer impurities (lower concentrations of by-products and sugars)
can be obtained from fermentation with modified strains (Ming et al.,
2018; Ruijter et al., 1999; Sun et al., 2018; Wang et al., 2016). The
problems of high formation of by-products by yeast can be mitigated by
the use of mutant strains, which can be an interesting alternative to
increase the industrial production of CA using these microorganisms and
employing new substrates (Cavallo et al., 2017; Rakicka et al., 2019;
Wang et al., 2013).
6. Conclusions
The well-established CA production processes are now turned to the
10
Bioresource Technology 320 (2021) 124426
Disadvantages Environmental
impact
Use of large amount of water and chemicalsReagents High
cannot be reusedGenerate large amount of wastewater and
gypsum (CaSO4)High energy consumptionLow yieldNeed
subsequent purification stepsDecreased CA
qualityInability to operate continuously
High costs of extractants and diluentsNeed subsequent Medium
purification stepsRelatively complexSome solvents cause
high environmental impact
Need subsequent steps for eluent separation and resin Low
regenerationGeneration of waste streams for resin
regeneration
Concentration polarizationHigh equipment costMembrane Medium
foulingPretreatment of the broth is necessaryHigh energy
consumption
search of new feedstocks and the use of genetically modified strains
together with metabolic engineering methods. Solvent extraction and
adsorption are potential alternatives to the traditional method of CA
recovery. The processing time and environmental impact of using these
techniques are significantly reduced, however, the low level of tech­
nology maturity remains the major obstacle for their application.
Therefore, further studies involving the use of CA fermentation broth, at
pilot scale, and cost analysis of downstream processes are required for
more efficient and economic viable methods for industrial applications.
CRediT authorship contribution statement
Sabrina Mores: Conceptualization, Methodology, Formal analysis,
Investigation, Writing - original draft. Luciana Porto de Souza Van­
denberghe: Conceptualization, Methodology, Supervision, Project
administration, Writing - review & editing. Antonio Irineudo Mag­
alhães Júnior: Conceptualization, Writing - review & editing. Júlio
César de Carvalho: Writing - review & editing. Ariane Fátima Mur­
awski de Mello: Formal analysis, Investigation. Ashok Pandey:
Writing - review & editing. Carlos Ricardo Soccol: Writing - review &
editing.
Declaration of Competing Interest
Ashok Pandey, a co-author on this paper, is the Editor of Bioresource
Technology. He was blinded to this paper during review, and the paper
was independently handled by Huu Hao Ngo as editor. The other authors
declare that they have no known competing financial interests or per­
sonal relationships that could have appeared to influence the work re­
ported in this paper.
Acknowledgements
Authors want to thank Ajinomoto do Brasil Ltda., CNPq, the Brazilian
National Council for Scientific and Technological Development, and
CAPES, the Coordination of Improvement of Higher Education
Personnel for the financial support.
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