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Citric acid production by yeasts: fermentation conditions, process optimization and strain improvement

Authors:
Citric acid production by yeasts: Fermentation conditions, process
optimization and strain improvement
Seda Karasu Yalcin
1
, M. Tijen Bozdemir
2
and Z. Yesim Ozbas
3*
1
Department of Food Engineering, Faculty of Engineering and Architecture, Abant Izzet Baysal University, Golkoy
14280, Bolu, Turkey.
2
Department of Chemical Engineering, Faculty of Engineering, Hacettepe University, Beytepe 06532, Ankara, Turkey.
3
Department of Food Engineering, Faculty of Engineering, Hacettepe University, Beytepe 06532, Ankara, Turkey.
*Corresponding author: e-mail: yesim@hacettepe.edu.tr, Phone: +90 312 297 71 12, Fax: +90 312 299 21 23
Citric acid is the most important organic acid produced by fermentation, widely used in food, pharmaceutical and chemical
industries. Although Aspergillus niger is the traditional producer of citric acid, during the last 30 years the interest of
researchers has been attracted by the use of yeasts for citric acid fermentation processes. Among the yeast species,
Yarrowia lipolytica is known as a potential producer of citric acid. Environmental factors that have been shown to exert an
effect on citric acid production are the type and concentration of carbon source of the fermentation medium, nitrogen and
phosphate limitations, aeration, trace elements, initial pH and temperature. Besides the regulation of product formation by
environmental conditions, strain selection and improvement has become the important factor. The improvement of citric
acid producing yeast strains has been carried out by mutagenesis and selection. Because of annual growths in demand of
citric acid, using alternative processes and strains for its production are in progress.
Keywords citric acid; yeasts; Yarrowia lipolytica; fermentation parameters
1. Citric acid production by yeasts
Citric acid is a commercially valuable organic acid, widely used in food, pharmaceutical and beverage industries
[1]. It
is the main additive used in the food industry. Citric acid is widely used to impart a pleasant, tart flavour to foods and
beverages. It also contributes to the formulation of many foods as an acidulant, antioxidant, emulsifier or preservative
[2, 3]. Among the uses of citric acid, about 70% is used in the food industry, and 10% in the cosmetics and
pharmaceuticals [4]. There is a great worldwide demand for citric acid consumption due to its low toxicity when
compared with other acidulants [3]. It is reported that the supply of natural citric acid is very limited and the demand
can only be satisfied by biotechnological fermentation processes. Citric acid is known as the most important organic
acid produced in tonnage by fermentation
and is the most exploited biochemical product [3, 5]. The annual production
of citric acid was reported as 700 thousand tons in 1993 [6], 1.4 million tons in 2004 [3], and 1.6 million tons in 2008
[7, 8]
.
A large number of microorganisms have been employed for citric acid production, but a few of them can produce
citric acid in industrial scale
[3]. It is reported that Aspergillus niger is almost exclusively used for industrial scale
production of citric acid [5], but during the last 30 years the interest of researchers has been attracted by the use of
yeasts as citric acid producers
[9]. A number of different strains, mostly belonging to the Candida (Yarrowia) genus
have been used for citric acid production, mainly in conventional batch processes, but also in continuous culture, and
with immobilized cells [10]. The yeast species which were reported to produce citric acid are; Candida (Yarrowia)
lipolytica, Candida guilliermondii, Candida oleophila, Candida intermedia, Candida paratropicalis, Candida
zeylanoides, Candida catenulata, Candida parapsilosis, Pichia anomala, and some Rhodotorula species [6, 11].
Among the yeast species, Yarrowia lipolytica is known as a potential producer of citric acid [1, 12]
and has been
developed as a microbial cell factory for citric acid production in recent years [7]. The main advantages of using yeasts
are mentioned as follows: Yeasts are characterized by greater resistance to high substrate concentrations than fungi,
with comparable conversion rates and have greater tolerance to metal ions that allows the use of less refined substrates.
Using yeasts also gives a better process control due to their unicellular nature
[13, 14]. It was reported that citric acid
production by yeast could be in the future an alternative to A. niger one, especially if the yeast biomass became an
additive to animal food and not a by-product [15]. However, the major disadvantage of using yeasts is the simultaneous
production of citric and isocitric acids
[13, 16]. It is reported that the ratio of citric:isocitric acid can vary between 1:1 to
20:1 according to the yeast strain, carbon source and micronutrient concentration
[13]. Selection of a yeast strain with
high citric acid production and giving high citric acid:isocitric acid ratios has been reported as the principal step of a
citric acid production process.
It is reported that more than 90% of the citric acid produced in the world is obtained by fermentation. The industrial
citric acid production can be carried out in three different ways: by submerged fermentation, surface fermentation and
solid-state fermentation or “Koji” process [3]. It is estimated that about 80% of the world citric acid production is
obtained by submerged fermentation in stirred tanks of 40-200 m
3
or larger airlift fermentors of 200-900 m
3
capacity
[17]. Submerged fermentation can be carried out in batch, fed batch or continuous systems, although the batch mode is
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more frequently used [3]. Citric acid production by yeasts is exclusively carried out by submerged cultivation. The
submerged fermentation process is desirable because of its higher efficacy due to higher susceptibility to automatization
[18]. Solid-state fermentation is commonly used for A. niger. However, there have also been reports for yeasts [3].
Fermentative production of citric acid arises from a primary energy metabolism although it is non-growth associated
[19]. Citric acid is a metabolite of energy metabolism, the concentration of which will only rise to appreciable amounts
under conditions of metabolic imbalances [5, 20]. The first stages of citric acid formation in the cell involve the
breakdown of hexoses to pyruvate in glycolysis, followed by its decarboxylation to produce acetyl – CoA. The CO
2
released during this reaction is not lost, but is recycled by pyruvate carboxylase in the anaplerotic formation of
oxaloacetate. Normally, oxaloacetate would largely be supplied through the completion of tricarboxylic acid (TCA)
cycle, allowing recommencement of the cycle by condensing with acetyl CoA to form citrate, catalysed by citrate
synthase. However, in order to accumulate citrate, its onward metabolism must be blocked. This is achieved by
inhibiting aconitase, the enzyme catalysing the next step in the TCA cycle. Inhibition is accomplished by the regulation
of environmental conditions, such as removal of iron, an activator of aconitase. Consequently, during citrate
accumulation, the TCA cycle is largely inoperative beyond citrate formation, hence the importance of the anaplerotic
routes of oxaloacetate formation [17]. It is reported in some studies that citric acid production by yeasts starts only after
the nitrogen source depletes, that is, at the end of the exponential growth phase and with a production medium devoid of
nitrogen source [10, 21]. Citric acid accumulation is a very complex process affected by fermentation conditions, during
which various metabolic and morphological changes take place in a complex form [21].
2. Effects of fermentation conditions on citric acid production by yeasts
It is reported that citric acid production rates and yields are highly dependent on the type of microorganism, the type of
substrate and the culture conditions [10]. Factors that have been shown to exert an effect on citric acid production are
the type and concentration of carbon source of the fermentation medium, nitrogen and phosphate limitations, aeration,
trace elements, initial pH, and temperature [5, 20].
2.1. Effects of carbon sources
Citric acid accumulation is strongly influenced by the type and concentration of carbon source. The type of the carbon
source can be varied according to the microorganism used. Substrate profiles of A. niger and yeasts used for citric acid
production can be extremely different from each other. For A. niger, sucrose is the most favourable substrate among the
easily metabolized pure carbohydrates, followed by glucose, fructose and galactose. Molasses is often used as raw
material for citric acid production by A. niger. However, in citric acid production processes of yeasts; many distinct
substrates could be used besides carbohydrates. Some yeasts are known to be able to produce citric acid from a wider
range of carbon sources than fungi do. The invention of citric acid production by yeasts dates back to investigations on
their abilities to grow on n-alkanes as carbon sources and to produce various valuable substances such as citric acid.
Various fractions of straight-chain paraffins were the preferred substrates according to the studies in 1970s [6].
However, the world oil crisis of 1973/74 almost entirely ended the exploitation of n-alkanes as feedstock for the
industrial production of citric acid. It was then discovered that certain yeasts could also produce citric acid from
carbohydrates, especially from glucose as carbon source. Many yeasts that grow on carbohydrate substrates have the
ability to accumulate high concentrations of citric acid during tricarboxylic acid cycle respiration. However, Y.
lipolytica is the only species known for its capability of maximizing citric acid production
[22]. Y. lipolytica and other
Candida strains are able to produce citric acid from various carbohydrates, whereby glucose has generated increasing
interest
[21]. Other carbon sources such as edible oils, ethanol, molasses, starch hydrolysates and pure or raw glycerol
have been used to produce citric acid from Y. lipolytica [12, 16, 23]. It was also reported that lower isocitric acid
concentations were obtained in media containing glucose than those obtained when n-alkanes were used.
Although fructose assimilation levels of some yeasts are reported as low, molasses or invert sugar mixtures can also
be used for citric acid production by yeasts [24]. In a study performed with C. lipolytica, it was reported that high citric
acid yields were obtained when glucose, fructose and glycerol were used as substrates [25]. In a study carried out by
Karasu-Yalcin et al. [26], growth and citric acid production characteristics of a novel endogenic strain Y. lipolytica 57
were investigated in comparison with a citric acid producer strain, Y. lipolytica NBRC 1658, in glucose and fructose
media in a batch system. The best results for citric acid production were obtained when initial substrate concentrations
were above 100 g/L. The ratio of citric:isocitric acid was changed between 11.20-16.62 in the examined media. It was
also reported that the highest citric acid production rates were obtained with the endogenic strain by using fructose as
substrate. Maximum citric acid concentration was obtained as 65.1 g/L in a medium containing 200 g/L of fructose with
the novel strain. The changes in the maximum specific citric acid production rates and specific growth rates with the
initial fructose concentration of the medium for two Y. lipolytica strains are shown in Table 1. Maximum specific
growth rate was obtained at 50 g/L initial fructose concentration for both strains. Specific citric acid production rate was
maximum at 100 and 150 g/L initial fructose concentrations for the endogenic and NBRC strains, respectively. Table 1
also represents the difference between the production characteristics of the two strains in the same medium and
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conditions. Concentration of carbon source used for citric acid production should be very high (100 to over 200 g/L),
which seems logical when a bulk product is produced economically [18]. High substrate concentration (120-250 g/L) is
given as a factor enhancing microbial citric acid production [10]. It was reported that very low amounts of citric acid
was produced at sugar concentrations below 50 g/L [18], which can also be observed from Table 1. In a study
performed by Antonucci et al.
[10], it was reported that both the specific rate of product formation and rate of substrate
consumption increased at high substrate concentration. With respect to the biochemical basis for the relationship
between citric acid accumulation and sugar concentration, it was shown that a high sugar concentration induces an
additional glucose transport system. It is stated that the increased uptake of glucose under conditions of high sugar
supply will counteract the inhibition of hexokinase by trehalose 6-phosphate [18].
Table 1 Variations of maximum specific citric acid production rate and specific growth rate with initial fructose concentration of
the medium for two Y. lipolytica strains at initial pH 5.2 and 30
o
C [26].
S
Fo
: Initial fructose concentration, υ
m
: Maximum specific citric acid production rate, µ: Specific growth rate
In another study by Karasu-Yalcin et al. [27], mannitol and glycerol were used for citric acid production by two Y.
lipolytica strains and satisfactory results were obtained in both media. Mannitol was determined as a novel potential
polyalcohol for citric acid production, suggesting the use of raw materials containing mannitol. Ethyl alcohol is also
another substrate choice for citric acid production by yeasts in recent years [9, 28, 29]. Arzumanov et al. [30], reported
citric acid biosynthesis by Y. lipolytica repeat-batch culture on ethanol. The highest citric acid concentration (105 g/L)
and product yield (88.3%) were determined with 50% feed every three day, with ethanol concentration not exceeding
1.2 g/L, at pH 4.5 and 28
o
C.
Some yeasts which assimilate n-alkanes, can also assimilate some hydrophobic substrates such as fatty acids,
triglycerites, oils and fats [31, 32]. Y. lipolytica, C. tropicalis and some Rhodotorula strains can produce citric acid by
using edible oils [33]. For this purpose, sunflower, soybean, olive and rapeseed oil could be used [3]. Kamzolova et al.
[2], reported that maximum citric acid concentration was 135 g/L when rapeseed oil was used in a batch system and its
concentration was kept above 5 g/L. In another study, besides rapeseed oil, sunflower oil was suggested as a potential
substrate for citric acid production by Y. lipolytica [14]. Maximum citric acid concentration obtained by Y. lipolytica
704 was reported as 40 g/L in a medium containing sunflower oil in fed-batch culture at pH 6.0. It was reported that the
hydrolysis of vegetable oil by yeast lipase occured with the production of two types of substrates, glycerol and fatty
acids. Kamzolova et al. [14], demonstrated that Y. lipolytica 704 simultaneously utilize fatty acids and glycerol in the
presence of vegetable oils.
For both increasing production yields and process economy, many natural substrate sources could be used for citric
acid production. Table 2 represents various raw materials as well as pure substrates used for citric acid production by
yeasts. The utilization of relatively impure raw materials such as crude, unfiltered starch hydrolyzates as well as raw
glycerol marks a significant improvement in industrial citric acid production [6, 34]. In recent years, considerable
interest has been developed in utilization of agricultural wastes including date seeds, whey [35-37], molasses, apple
pomace, grape pomace, carob pod [38, 39], ram horn hydrolyzate [40, 41], and olive-mill waste water [42] for citric
acid production. In a study performed by Wojtatowicz et al.
[24], glucose hydrol was used as a substrate and 80 g/L
citric acid was obtained at 100 g/L initial glucose concentration with a mutant strain of Y. lipolytica.
Raw glycerol, by-
product of bio-diesel production process, was also used as carbon source in citric acid production [42]. It was reported
that during the manufacture of 10 kg of biodiesel by esterification of rapeseed oil, 1 kg of raw glycerol was also
produced. In a study performed with acetate mutants of Y. lipolytica, a maximum citric acid concentration of 124.5 g/L
was reached by using raw glycerol at an initial concentration of 200 g/L [22]. Whey, a significant by-product of dairy
industry, can also be used as a natural fermentation medium for citric acid production. Sometimes, whey can be used
after addition of some nutrients such as sugars or nitrogen sources. In a study by Abou-Zeid et al. [35], whey was used
for citric acid production by Y. lipolytica, after addition of glucose, maltose, saccharose and date-seed hydrolysate at
different concentrations. It was suggested that whey supplemented with date seed hydrolysate could be a potential cheap
raw material for citric acid production. In another study, citric acid production characteristics of two Y. lipolytica strains
were investigated in whey supplemented with glucose, fructose and some nitrogen nutrients. Maximum citric acid
S
Fo
(g/L)
υ
m
(g citric acid/g mo.h)
µ
(h
-
)
Y. lipolytica 57 Y. lipolytica
NBRC 1658
Y. lipolytica 57 Y. lipolytica
NBRC 1658
0 0 0 0.003 0.003
20 6.7x10
-
4
7.8x10
-
4
0.012 0.096
50 0.0012 0.0026 0.029 0.122
100 0.0179 0.0042 0.024 0.106
150 0.0158 0.0055 0.025 0.063
200 0.0160 0.0048 0.024 0.063
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concentration was obtained as 49 g/L in whey supplemented by fructose, with the use of a novel endogenic Y. lipolyica
strain [43]. Grape must, naturally containing glucose and fructose as substrates (Table 2) was a potential medium
supporting yeast growth and also citric acid production. The same authors also suggested that grape must could be used
as a novel natural substrate for citric acid production by Y. lipolytica and concluded that enzyme activities related to
substrate consumption of the strains could be affected by the composition of the grape must, indicating the complexity
of natural media [43]. It is well known that the citric acid fermentation is greatly affected by the presence of some trace
metals, so various techniques have been used to remove metallic inhibitory substances from the raw materials. Heavy
metals can inhibit the growth of microorganisms, influence the ionic strength and pH of the medium, and are involved
in the inactivation of enzymes associated with citric acid metablism in the TCA cycle. Deionization or some chemical
pretreatment methods should be applied to raw materials before using them as substrates for citric acid production [44].
2.2. Effects of the other fermentation parameters
The pH of fermentation medium is known as one of the most critical parameter for citric acid production processes by
yeasts. It is reported that initial pH of the fermentation medium must be very well defined and optimized depending on
the microorganism, substrate and production technique. When working with yeasts, initial pH values above 5 should be
used since citric acid production is adversely affected below pH 5 [45]. It is reported that citric acid concentration
decreases below pH 5 due to the accumulation of some polyalcohols like erythritol, arabitol and mannitol, instead of
citric acid [6, 45]. Adverse effect of low pH is also explained by inhibition of citrate production in the cell and transport
of citrate from cell membrane. In a study performed with C. oleophila, effect of pH on citric acid release by specific
active transport system was investigated in a continuous system [46]. It was demonstrated that active citric acid
transport system was a pH-dependent mechanism. In the same study, it was also reported that growth of the yeast,
composition of biomass and citric acid release were directly affected by pH, and maximum citric acid concentration was
obtained at pH 5. Karasu-Yalcin et al. [47], reported that maximum citric acid concentrations and citric acid yields
obtained with Y. lipolytica 57 and Y. lipolytica NBRC 1658 were maximum between the initial pH range of 5.2-7.0 in a
fermentation medium containing glucose. In the same study, maximum specific growth rates were obtained at pH 5.2
and 6.0 for the strain 57 and NBRC 1658, respectively. However, Kamzolova et al. [48], reported that citric acid
production, and ratio of citric acid to isocitric acid depended on pH of the medium, and Y. lipolytica produced almost
equal amounts of citric and isocitric acids at pH 4.5 while predominantly accumulated co-product isocitric acid at pH
6.0. A list of some data in the literature on citric acid production by several yeast strains at various fermentation
conditions were presented in Table 2.
It is known that optimum temperatures for growth of cells and product formation may be different in some
fermentation processes [49]. It is also quitely deliberated that determination of the optimum temperature for a batch
process is necessary before scaling-up the process [50, 51]. Optimum temperature for citric acid production may change
relative to the used strain and medium conditions. Effects of temperature on growth and citric acid productions of
different strains were submitted in a number of studies. Temperature reported in various researches on citric acid
production by yeasts was between 22-35
o
C. The optimal temperature range for both citric acid production and biomass
models of C. lipolytica was stated as 26-30
o
C [50]. Rane and Sims [13], determined the optimal temperature for growth
of C. lipolytica Y 1095 as 27
o
C. In another study, growth and citrate production of C. lipolytica were reported even at
35
o
C by using a medium containing glucose [52]. Karasu-Yalcin et al. [47], obtained that citric acid productions of Y.
lipolytica NBRC 1658 and Y. lipolyica 57 both decreased when temperature was increased from 30 to 35
o
C. Although
the maxima of cell dry mass were reported at 20
o
C for both strains, the best results for citric acid production were
observed at 30
o
C in the same study.
Citric acid production is directly influenced by the concentration and nature of the nitrogen source in the
fermentation medium [3, 54]. Corn-steep liquor, urea and ammonium salts have all been used as nitrogen sources in
citric acid production by yeasts. The most suitable organic and inorganic nitrogen sources for citric acid production by
Y. lipolytica, C. paratropicalis and C. guilliermondii have been reported to be yeast extract and ammonium chloride,
respectively [19]. It was reported that citric acid production began after depletion of nitrogen source in the medium
[55]. For this reason, the source of nitrogen and its concentration has a primary role on citric acid production, and high
concentrations of nitrogen compounds may have negative effects on citric acid production rate. In a research performed
by C. lipolytica, effects of different nitrogen sources on citric acid production were investigated and the best results
were obtained with ammonium chloride [25]. In the same study, it was reported that the experiments were carried out
with using 0 to 4 g/L of ammonium chloride, and the highest citric acid production was obtained when ammonium
chloride concentration was 1.5 g/L. Imandi et al. [12], investigated citric acid production of Y. lipolytica from raw
glycerol by using yeast extract as a nitrogen source with an optimum concentration of 0.2682 g/L. Karasu-Yalcin et al.
[47], investigated the effect of ammonium chloride concentration on citric acid production by Y. lipolytica strains in the
range of 0-6 g/L. It was reported that citric acid production increased by increasing ammonium chloride concentration
from 0 to 2 g/L in a defined fermentation medium containing glucose. However, addition of this nitrogen source to
whey at a concentration of 2 g/L extremely decreased citric acid production by approximately 50% [43]. It should be
noted that the effect of nitrogen sources is mainly observed in chemically defined media, as no further nitrogen is
necessary when some raw materials are used as carbon source [18].
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Table 2 Citric acid production by several yeast strains at different fermentation conditions
S
o
: Initial substrate concentration, S
Fo
: Initial fructose concentration of the grape must, S
Go
: Initial glucose concentration
of the grape must, T: Temperature, C
c m
: Maximum citric acid concentration, Y
P/S
: Citric acid yield based on consumed
substrate, n: Not indicated, OMW: Olive-mill waste water
The effects of trace metal ions have been known for a long time and had been the key to the establishment of
successful fermentation processes, although the effect is much more pronounced in the submerged fermentation [18].
Different explanations have been offered regarding the biochemical mechanism of action of trace metals [6]. Divalent
metal ions such as zinc, manganese, iron, copper and magnesium have been found to affect citric acid production [3]. It
Yeast strain Fermentation
type
Substrate S
o
pH & T C
c m
Y
P/S
(g/g)
Ref
Y. lipolytica
N1
Submerged,
continuous
Ethanol 0.01-1.0 g/L pH 4.5
28
o
C
14.4-19.2 g/L n [28]
Y. lipolytica
NCIM 3589
Batch Raw
glycerol
54.4 g/L pH n
30
o
C
77.39 g/L n [12]
C. lipolytica
Y 1095
Submerged,
batch
Glucose 50-150 g/L pH 5.5
27
o
C
13.6-78.5 g/L 0.50-
0.79
[13]
Y. lipolytica
A-101
Repeated
batch
Glucose 92 g/L pH 5.5
29
o
C
34.3 g/L 0.84 [53]
C. lipolytica
Y 1095
Batch n-paraffin 100-150 g/L pH n
26-30
o
C
9.8 g/L n [50]
Y. lipolytica
NCIM 3589
Solid state Pineapple
waste
n pH n
30
o
C
202.35 g/kg n [1]
C. oleophila
ATCC 20177
Submerged,
continuous
Glucose 250 g/L pH 5.0
30
o
C
57.8 g/L n [46]
Y. lipolytica
A 101-1.14
Submerged,
batch
Glucose
hydrol
(39.9%
glucose)
400 mL/L pH 5.5
30
o
C
> 80 g/L 0.93 [24]
Y. lipolytica
UOFS Y-
1701
Batch Sunflower
oil
30 g/L pH 5.8
26
o
C
18.7 g/L n [33]
Y. lipolytica
1.31
Submerged,
batch
Raw
glycerol
200 g/L pH 5.5
30
o
C
124.5 g/L 0.62 [22]
Y. lipolytica
187/1
Submerged,
batch
Rapeseed oil > 5 g/L pH 5.0
28
o
C
135 g/L 1.55 [2]
Y. lipolytica
ACA-DC
50109
Batch Glucose in
OMW-based
medium
65 g/L pH 5.0-
6.0
28
o
C
28.9 g/L 0.82 [42]
Y. lipolytica
NRRL YB-
423
Batch Pure
glycerol
40 g/L pH 6.0
28
o
C
21.6 g/L 0.54 [16]
Y. lipolytica
LGAM S(7)1
Batch Raw
glycerol
80-120 g/L pH>5
28
o
C
33-35 g/L 0.42-
0.44
[23]
Y. lipolytica
NBRC 1658
Batch Mannitol 120 g/L pH 5.2
30
o
C
20.25 g/L 0.17 [27]
Y.lipolytica
57
Batch Pure
glycerol
160 g/L pH 5.2
30
o
C
32.80 g/L 0.21 [27]
Y. lipolytica
NBRC 1658
Batch Glucose in
whey-based
medium
100 g/L pH 5.2
30
o
C
38.88 g/L 0.38 [43]
Y. lipolytica
57
Batch Fructose in
whey-based
medium
150 g/L pH 5.2
30
o
C
49.23 g/L 0.33 [43]
Y. lipolytica
57
Batch Grape must
S
Go
= 78.30 g/L
and
S
Fo
= 85.16 g/L
pH 5.2
30
o
C
32.09 g/L 0.48 [43]
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was reported that fermentation medium should contain metal ions especially manganese, iron and zinc at required
amounts for inducing cell growth to obtain high citric acid yields [18]. 20% decrease in citric acid accumulation was
reported when concentration of manganese exceeded 2 µg/L. The concentrations of metal ions below which citric acid
is accumulated in high amounts are not absolute, and they depend on their relative proportion to other nutrients,
particularly phosphate. It was reported that some researchers have claimed a particularly strong influence of iron on
citric acid accumulation, which is, however, not supported by others. Iron limitation has been claimed to lead to an
inactivation of aconitase, the enzyme catalysing further degredation of citric acid within the tricarboxylic acid cycle
[18]. In a study performed by Anastassiadis and Rehm [46], the addition of iron has been found to enhance biomass
formation and to affect continuous citric acid production significantly, for yeast C. oleophila growing on glucose.
Finogenova et al. [28], reported that the main factor determining citric acid production in Y. lipolytica was growth
limitation by nitrogen, whereas zinc or iron limitation conditions resulted in insignificant cell growth without citric acid
production. Addition of zinc to the medium alleviated the zinc deficiency symptoms and increased the production of
citric acid. In the same study, it was also indicated that the intensive citric acid production in Y. lipolytica required high
intracellular iron amounts in the range of 0.2-2.5 mg/g, in conditions of nitrogen limitation growth of cells, grown on
ethanol. At an intracellular iron content of 7.0 mg/g, citric acid production was completely inhibited. Karasu-Yalcin et
al. [47], reported the effects of different mineral salts on citric acid production by two Y. lipolytica strains. It was found
that iron and copper inhibited citric acid production for the used concentrations, while cell growth was positively
affected by the addition of these minerals. By the addition of zinc sulphate into the fermentation medium, citric acid
production of the two yeast strains were differently affected. When compared with the medium without the zinc
sulphate, maximum citric acid concentration obtained by Y. lipolytica NBRC 1658 was decreased in the media
containing the salt. The highest value of the maximum citric acid concentration analyzed was 41.63 g/L for the
domestic Y. lipolytica strain 57 in the medium containing 0.008 g/L
of the same zinc salt.
Since citric acid production is an aerobic process, accumulation of high amounts of citric acid is also dependent on
strong aeration [18, 56]. The role of oxygen as a factor influencing citric acid production has been investigated with
yeasts as well as with A. niger grown on various substrates [57, 58]. The necessity of high oxygen uptake is evident
from the metabolic balance of citrate formation and the high sugar concentration used in the fermentation media [6].
Dissolved oxygen concentration influences the citric acid formation directly. The high demand of oxygen is reached by
constructing appropriate aeration devices, which is also dependent on the viscosity of the fermentation medium [3]. In
hydrocarbon fermentations, both aeration and agitation affect the extent of dissolved oxygen and available substrate in
the medium [59, 60]. It is known that agitation increases the area available for oxygen transfer by dispersing air and
insoluble substrate in the culture fluid in the form of fine bubbles. However, increased agitation can impose shear stress
on cell walls as well as the cell-insoluble substrate interface. Therefore, an optimum agitation speed is required to
maximize product production [59]. It was reported that the concentration of citric acid produced by C. tropicalis
increased with an increase in dissolved oxygen concentration, whereas the isocitric acid concentration decreased at the
same time. In another study, it was determined that citric acid production of Y. lipolytica 704 was completely inhibited
by decreasing dissolved oxygen concentration from 60-95% to 28-30% saturation [58]. However, Kamzolova et al.
[58], reported that oxygen requirements of Y. lipolytica N1 for growth and citric acid synthesis depended on the iron
concentration in the medium. At relatively low dissolved oxygen concentrations and a high iron concentration, citric
acid accumulation was as high as 120 g/L in continuous culture.
The optimization of fermentation conditions are of primary importance in the development of any fermentation
process owing to their impact on the economy and practicability of the process
[5]. Work to improve fermentative
production of citric acid is continually in progress. Different techniques of production are continuously studied showing
new perspectives for the production of this organic acid [3].
3. Strain selection and improvement for overproduction
By using conventional techniques of fermentation in stirred vessels and modifying fermentation parameters, citric acid
productivity can be achieved up to certain level [4]. It is known that citric acid production characteristics of yeasts are
highly dependent on strain diversity. This phenomenon was demonstrated in several strain-screening studies [2, 16, 61].
Basically, selection of strains begins with isolation from natural habitats according to common microbiological
methods, followed by screening for their citric acid production capabilities. Several of such strains have been
incorporated in governmental or industrial culture collections [6]. Besides, finding novel potential yeast strains to be
used in citric acid production processes is interest of researchers in recent years.
To improve the process productivity and yield of citric acid, either physical or biological parameters require
modification. In this respect, strain improvement has become the important activity [4]. The improvement of citric acid
producing strains has been carried out by mutagenesis and selection. The most employed technique has been by
inducing mutations in parental strains using mutagens. Among mutagens; γ-irradiation, UV irradiation and chemical
mutagens are often used. It is reported that, UV treatment can frequently be combined with some chemical mutagens
[3]. Successive treatments with physical and chemical mutagens followed by testing a large number of colonies will be
necessary before strains with improved performance can be isolated. In selecting strains or mutants for large-scale
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A. Méndez-Vilas (Ed.)
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1379
production, several important factors need consideration. These include stability of strains without undergoing
physiological or biochemical degeneration upon subculture for mass propagation, non-utilization of the acid formed and
non-formation of the other metabolic acids like gluconic, oxalic, and malic acid [4]. In a reported study, C. lipolytica Y-
1095 was treated with two different mutagens, UV-irradiation and N-methyl-NT-nitro-N-nitrosoguanidine (NTG). It
was determined that the UV-irradiation was better in inducing more productive isolates. Four mutants, each of them
giving a yield of about 75-80% of citric acid more than the original parent, were selected [62]. In a study carried out
with four commercial strains and two mutants of Y. lipolytica, a UV-induced mutant was found to be the most suitable
for citric acid production from glucose hydrol [24]. In another study by Finogenova et al. [63], mutants of Y. lipolytica
VKM Y-2373 with increased ability to synthesize citric acid were obtained by using UV- irradiation and NTG. It was
reported that three mutants that displayed higher biosynthetic ability as compared with the initial strain, were selected
after the treatments of UV irradiation or NTG. Additionally, three mutants were generated from the combined action of
UV and NTG, and their biosynthetic activity exceeded that of the initial strain by 43.9%. In another research, a citrate
nonutilizing strain which had an improved (by twofold) citric to isocitric acid ratio was isolated after mutagenesis of
Saccharomycopsis lipolytica ATCC 20228 with NTG [64]. It was reported that a combination of mutagenesis and
medium development had been shown to be complementary methods for improvement of citric acid production from
canola oil by S. lipolytica.
Most strains of Y. lipolytica grow very efficiently on acetate as sole carbon source. Several mutants have been
isolated and characterized, which were blocked in the utilization of acetate. It was reported that utilization of acetate is
related to induction of glyoxylate pathway, which has a major role in citric acid metabolism. Mutants blocked in the
activity of acetyl-coenzyme A synthetase were characterized. Acetyl-coenzyme A is needed for the induction of the
glyoxylate cycle, which is not induced in acetyl–coenzyme A deficient mutants [65]. Rymowicz et al. [22], used three
acetate-negative mutants of Y. lipolytica for citric acid production from raw glycerol and maximum citric acid
concentration was obtained as 124.5 g/L with the strain 1.31.
For improving citric acid production characteristics of Y. lipolytica, some genetic engineering studies other than
mutation were also reported. To extend the substrate spectrum of Y. lipolytica to sucrose-containing mixtures like
molasses, the S. cerevisiae SUC2 gene, expressed with the XPR2 promoter and signal sequence, was introduced in
different strains. Such Suc
+
-transformants were able to grow on sucrose and citric acid could be produced from
molasses [31].
4. Conclusions
Today, yeasts are potential producers of citric acid and strains of Y. lipolytica are used in the industrial productions [22].
It was reported that the consumption of citric acid has an increase of 5% per year [2]. Because of the large and ever-
increasing demand for citric acid, alternative cultivation processes involving yeast strains are used for its production
[22]. In this respect, utilization of some renewable substrates such as agricultural wastes has been the interest of
researchers in recent years [1]. Besides process optimization, there is a great interest in the possibility of obtaining
different strains of yeasts giving high yields of citric acid. Improvement of citric acid producing strains can be achieved
by mutation and selection studies. Strains with superior characters, such as enhanced citric acid production and
increased rate of fermentation can be selected after subjecting the genetic material to physical or chemical mutagenic
agents [66]. In order to make citric acid production by yeasts industrially more feasible, further investigations should be
devoted to selecting a high citrate-producing strain and to optimizing its metabolic pathways and the operating
conditions [10].
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... For A. niger, sucrose is the most favorable substrate among the easily metabolized pure carbohydrates; followed by glucose, fructose and galactose (Yalcin et al., 2010;Wang et al., 2020). Molasses is often used as raw materials for citric acid production by A. niger. ...
... The presence of nitrogen in form of ammonium salt will inhibit iron utilization as stimulant and does not have obvious metabolic effect on the process. But when nitrogen is present as nitrates, iron has an obvious effect as stimulating agent, it obviously increase the mycelium biomass (Yalcin et al., 2010;Show et al., 2015;Almousa et al. 2018). ...
... It is reported that, UV treatment can frequently be combined with some chemical mutagens (Adeoye et al., 2015;Mores et al., 2020). Successive treatments with physical and chemical mutagens followed by testing a large number of colonies will be necessary before strains with improved performance can be isolated (Yalcin et al., 2010). ...
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Agro-wastes have been found useful for the production of different important metabolites through fermentation process. Notable metabolites of industrial importance is citric acid, which has wide applications in many areas of human activities. The huge amount of waste generation from agricultural and industrial activities caused pollution and discomfort. Hence, it poses colossal threat to our health due to indiscriminate disposals. Valorization of these wastes will invariably amount to converting waste to wealth, which is in line with sustainable development goals of united nation. Citric acid is a good natural preservative; it serves as acidulant in many processes because of its low pH and less toxicity compared to other known acidulants. Citric acid is suitably applied in many areas of human activities; both domestics and industries, such applications are in cosmetics, pharmaceuticals, chemicals, textiles and electroplating industries. As an antimicrobial, preservative agent, it found application in many areas of food processing and preservation to inhibit the growth of spoilage organisms. The demand for citric acid is increasing ditto the cost of production due to increase in energy cost and raw materials. Hence, there is need for cheaper means of production. This review will give insight into the innovative raw materials, application of strain improvement, and optimization technique that have been of tremendous benefit.
... Biomass (g/L) was calculated as the dry cell weight produced in liters of liquid medium (Papanikolaou and Aggelis 2002). When CaCO 3 was used to control the pH of the fermentation medium, 6 N HCl was used to dissolve residual CaCO 3 in the fermentation medium and biomass concentration was determined (Karasu- Yalcin et al. 2010Yalcin et al. et al. 2010. The pH value of fermentation medium was measured with a pH meter (Mettler Toledo Ion S220, Switzerland). ...
... Biomass (g/L) was calculated as the dry cell weight produced in liters of liquid medium (Papanikolaou and Aggelis 2002). When CaCO 3 was used to control the pH of the fermentation medium, 6 N HCl was used to dissolve residual CaCO 3 in the fermentation medium and biomass concentration was determined (Karasu- Yalcin et al. 2010Yalcin et al. et al. 2010. The pH value of fermentation medium was measured with a pH meter (Mettler Toledo Ion S220, Switzerland). ...
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In this study, citric acid (CA) production by autochthonous Candida zeylanoides 7.12 was investigated and optimized. Response surface methodology (RSM) was used for the analysis of simultaneous effects of the chosen factors and 2 experiment designs were applied. In the first experimental design, the effects of initial pH value (5.5, 6.0 and 6.5), fermentation time (4, 5 and 6 days) and initial glucose concentration (125, 150 and 175 g/L) on CA production were investigated. Initial pH value was adjusted periodically with NaOH. Results of the statistical analysis showed that the model was found to be not applicable sufficiently to the chosen data. A second experimental design was employed at the same levels of glucose concentration and fermentation time by disabling the pH factor. pH level was kept at 6.5 with CaCO3. Results of the statistical analysis showed that the fit of the model was good and the lack of fit was not significant (P > 0.05). The highest CA concentration of 11.36 g/L was obtained after 6 days of fermentation with an initial glucose concentration of 125 g/L. The results indicated that initial glucose concentration and fermentation time were important parameters for CA production by C. zeylanoides 7.12 and this strain could be used for future studies.
... Additionally, acids are produced by the yeasts as by-products of their metabolic pathways (Whiting, 1976). Selected yeast species are known for the production of citric acid or pyruvic acid during fermentation (Yalcin et al., 2010;Chidi et al., 2015;Afolabi et al., 2018), while Lachancea thermotolerans, besides other yeast species, possess the ability to form high amounts of lactic acid at the expense of ethanol during fermentation (Sgouros et al., 2020;Rodríguez Madrera et al., 2021). Due to the low pH values in the beers, there is a possibility that S. fib SF4 partly converted carbohydrates to organic acids instead of ethanol. ...
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There is a growing trend for beers with novel flavor profiles, as consumers demand a more diversified product range. Such beers can be produced by using non- Saccharomyces yeasts. The yeast species Saccharomycopsis fibuligera is known to produce exceptionally pleasant plum and berry flavors during brewer’s wort fermentation while its mycelia growth is most likely a technological challenge in industrial-scale brewing. To better understand and optimize the physiological properties of this yeast species during the brewing process, maltose and maltotriose uptake activity trials were performed. These revealed the existence of active transmembrane transporters for maltose in addition to the known extracellular amylase system. Furthermore, a single cell isolate of S. fibuligera was cultured, which showed significantly less mycelial growth during propagation and fermentation compared to the mother culture and would therefore be much more suitable for application on an industrial scale due to its better flocculation and clarification properties. Genetic differences between the two cultures could not be detected in a (GTG) 5 rep-PCR fingerprint and there was hardly any difference in the fermentation process, sugar utilization and flavor profiles of the beers. Accordingly, the characteristic plum and berry flavor could also be perceived by using the culture from the single cell isolate, which was complemented by a dried fruit flavor. A fermentation temperature of 20°C at an original gravity of 10 °P proved to be optimal for producing a low-alcohol beer at around 0.8% ( v / v ) by applying the S. fibuligera yeast culture from the single cell isolate.
... Considering that the strains can grow even at high sugar concentrations and have acid production capabilities, it is thought that they can be used in microbial organic acid production. For instance, it is known that citric acid production needs a high substrate concentration (120-250 g/L) in the fermentation medium (Yalcin et al., 2010). ...
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The aim of this study was to investigate some of the technological and functional properties of 16 autochthonous Candida zeylanoides strains isolated and identified from pastırma, a traditional dry-cured meat product. Consequently, it was determined that some strains could grow at high sugar concentrations (45%) while all strains were resistant to 10% NaCl concentration and most strains were tolerant to 10% ethanol and 0.5% bile salt levels. Furthermore, the certain strains showed good growth at pH 3.0 and only 6 strains were able to grow at 42°C. All strains showed catalase activity. It was detected that the strains did not produce hydrogen sulfide (H2S) and also had no DNase, nitrate reductase, proteolytic, and lipolytic activities. It was found that some strains exhibited urease activity and all strains that could grow at 37°C had β-hemolytic activity and formed biofilm. Moreover, C. zeylanoides strains showed sensitivity to nystatin, fluconazole, voriconazole, and ketoconazole.
... This caused a change in the metabolic pathway by producing 1.46 g/L of CA and 16.79 g/L of isocitric acid [85]. The addition of extra nitrogen sources along with whey decreased CA production [86]. ...
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The citric acid (CA) is an organic acid with a global market value of USD 3.6 billion. Due to its non-toxic and biodegradable nature, it is widely used in the food industry and various other industries such as pharmaceutical, biomedical, textile, and leather industries. CA is predominantly produced by Aspergillus niger due to its higher yield and ability to ferment various low-cost feedstock. For a sustainable environment and economic benefit of industries, the use of agricultural and industrial residues as substrates for CA production is the current interest for researchers. This review reports the bio production of CA using different substrates, microorganisms, and fermentation techniques. It also gives significant information on the recovery process, challenges faced during microbial fermentation, and various industrial applications CA.
... While the addition of KH 2 PO 4 had minör effect on the production and mycelial growth, methanol addition (2-4%) markedly increased the production (Hang et al., 1977). The reason for this is due to the shift of production towards cell growth because CA occurs in the exponential growth phase and starts to accumulate after the nitrogen in the environment is depleted (Yalcin et al., 2010). Also, it has been reported that lower alcohols such as methanol, ethanol, n-propanol, isopropanol, or methyl acetate neutralized the negative effect of the metals in CA production when used at about 1 to 5% (Soccol et al., 2006). ...
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With the increasing population, developing technology, and industry, the importance given to waste control/effective assessment studies continue with increasing momentum. The use of wastes in the production of biotechnological products is preferred due to its advantages in reducing environmental pollution, preventing nutrient and biomass losses, recycling, and decreasing costs. Citric acid (CA) is an intermediate product formed by the oxidation of carbohydrates to carbon dioxide in the Krebs cycle. This organic acid is used in many industrial areas such as pharmaceuticals and cosmetics. It is also an important organic acid in the food industry and is used as an acidifier, a stabilizer, an antioxidant, a flavor enhancer, and a preservative. Today, CA production is produced by microorganisms through fermentation. In addition, some wastes, such as molasses, glycerol, whey, olive mill wastewater, and various fruit wastes can be evaluated for use in the production of CA. This study reviewed the microbial production of CA using various wastes and some factors affecting the production.
... Later, it was determined that other Aspergillus species can also produce citric acid. However, studies have indicated that the most suitable mold for the yield of citric acid is A. niger (Yalcin et al., 2010). Mainly citric acid obtained from molds including A. niger is affected from different conditions including carbon source, substrate, pH, nitrogen amount, temperature, trace elements, thiamine amount, aeration. ...
... Yeast initiates the formation of pyruvate from hexoses and arrives to TCA cycle, primarily converted to citrate from oxaloacetic acid (Gancedo, 1986). Citrate assimilation will ensue after the nitrogen source descents encouraging stationary growth of yeast (Yalcin et al., 2010). In this phase, LAB utilizes citrate to convert into lactic acid and acetic acid. ...
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Fermentative production of organic acids by using current research can promote the economy as many countries still depend on importing these multiuse product to meet their needs. Organic acids have various applications in the food, pharmaceuticals, cosmetics, and textile industries. Besides their traditional applications, they are also used for manufacturing bioplastics. Organic acids can be produced commercially by either chemical or biological route, where the latter employs fermentation strategies. Production of organic acids by microbial processes is the preferred method due to its high productivity and environment-friendly procedure. Organic acids are low-value and high-volume products, and therefore the use of economical substrates can boost their commercialization. The application of strategies such as recombinant DNA technology (RDT) and metabolic engineering have the capability to develop strains with high organic acid productivity. In this chapter, we will discuss industrially important organic acids (such as lactic acid, citric acid, acetic acid, kojic acid, and itaconic acid), their market potential, biosynthetic pathways, suitable substrates and microorganisms, fermentation and purification strategies, and associated challenges.KeywordsLactic acid (LA)Citric acid (CA)Acetic acid (AA)Kojic acid (KA)Itaconic acid (IA)
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In this study, Y. lipolytica NBRC 1658 and a novel domestic strain Y. lipolytica 57 were used for citric acid production from two different substrates; glycerol and mannitol, in a batch system. In each substrate media, the growth was defined by the non-competitive substrate inhibition model for both of the strains. Maximum citric acid concentration obtained by the domestic strain was higher than that of NBRC 1658 in glycerol medium. The best results for productivity and maximum specific citric acid production rate were obtained at 120 g/L initial glycerol concentration for NBRC 1658, and at 160 g/L initial glycerol concentration for the domestic strain. In the medium containing mannitol, maximum citric acid concentration and productivity were obtained at 120 g/L initial mannitol concentration for both of the strains. The study gave promising data for gaining a novel citric acid producer, Y. lipolytica 57, determining substrate profiles of the used strains for citric acid production.
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In this study, utilization of whey and grape must were investigated for citric acid production using Yarrowia lipolytica NBRC 1658 and a domestic strain Y. lipolytica 57. In addition to its use as a sole nutrient source, whey was also fortified with glucose or fructose as well as other nutrients. The best results for citric acid production were obtained in the medium containing whey supplemented with fructose. Maximum citric acid concentrations in this medium were 49.23 and 32.65 g/L for the domestic and NBRC 1658 strains, respectively. In grape must, maximum citric acid concentrations obtained using domestic and NBRC 1658 strains were 32.09 and 10.39 g/L, respectively. Both of the natural nutrient sources were found to be promising for utilization in citric acid production process. A domestic Turkish yeast strain was confirmed to be superior for citric acid production for the first time. This can be targeted for enhancing citric acid production efficiencies from locally available substrates such as whey or grape must.
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Microbial production of citric acid was performed by Yarrowia lipolytica NBRC 1658 and a domestic strain in the media containing glucose or fructose as substrates. The study was carried out in a batch system. The non-competitive substrate inhibition model was proposed for growth of the yeasts in both substrate media. Higher dry mass and lower Ks values were obtained in glucose media when compared to fructose media. The highest citric acid concentration (65.1 g/L) was obtained with the domestic strain in the medium containing 200 g/L fructose. In the media containing fructose, maximum citric acid concentration and productivity values determined with the domestic strain were aproximately two-fold greater than those obtained with NBRC 1658 strain. The required initial concentration of glucose or fructose at which best citric acid production properties were observed changed between 100-200 g/L for both of the strains. The ratio of citric acid to isocitric acid was found to change between 11.70-16.62.
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