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Advances in Citric Acid Fermentation by Aspergillus Niger: Biochemical Aspects, Membrane Transport and Modeling
Advances in Citric Acid Fermentation by Aspergillus Niger: Biochemical Aspects, Membrane Transport and Modeling
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Abstract
Citric acid is regarded as a metabolite of energy metabolism, of which the concentration will rise to appreciable amounts only
under conditions of substantive metabolic imbalances. Citric acid fermentation conditions were established during the 1930s and
1940s, when the effects of various medium components were evaluated. The biochemical mechanism by which Aspergillus niger
accumulates citric acid has continued to attract interest even though its commercial production by fermentation has been established
for decades. Although extensive basic biochemical research has been carried out with A. niger, the understanding of the events
relevant for citric acid accumulation is not completely understood. This review is focused on citric acid fermentation by A. niger.
Emphasis is given to aspects of fermentation biochemistry, membrane transport in A. niger and modeling of the production process.
© 2007 Elsevier Inc. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
2. Fermentation conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
2.1. Submerged fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
2.1.1. Carbon source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
2.1.2. Nitrogen and phosphate limitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
2.1.3. Broth pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
2.1.4. Aeration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
2.1.5. Trace elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
2.1.6. Fungal morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
3. Biochemistry of citric acid formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
3.1. Invertase, hexokinases, and glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
3.2. Phosphofructokinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
3.3. Glycolytic and the pentose phosphate pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
0734-9750/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2007.01.002
M. Papagianni / Biotechnology Advances 25 (2007) 244–263 245
acid, an unwanted byproduct. Selection for mutants with especially so in submerged fermentation processes.
very low aconitase activity has been used in attempts to Other than the early studies by Currie (1917), further
reduce production of isocitric acid. systematic studies relating to medium composition did
Although many microorganisms can be used to produce not occur until the 1940s (Shu and Johnson, 1948a,b).
citric acid, A. niger remains the main industrial producer. Shu and Johnson (1948a,b) developed a medium that
Specific strains that are capable of overproducing citric provided the foundation for subsequent research on
acid have been developed for various types of fermentation citric acid production. The following main factors were
processes. The yield of citric acid from these strains often found to affect citric acid fermentation: type and
exceeds 70% of the theoretical yield on the carbon source. concentration of the carbon source, nitrogen and
Despite a long and successful history of production of citric phosphate limitation, pH, aeration, concentration of
acid, there is still no single explanation for the biochemical trace elements and the morphology of the producer
basis of the process to consistently explain the observed organism. Certain nutrients needed to be in excess (i.e.
behavior of this fermentation. This review discusses the sugar, protons, oxygen), others had to be limiting (i.e.
biochemical factors that contribute to citric acid accumu- nitrogen, phosphate) and some otherwise common feed
lation in A. niger fermentations. components had to remain below defined limits (i.e.
trace metals, especially manganese).
2. Fermentation conditions
2.1.1. Carbon source
The biochemical mechanism by which A. niger The carbon source for the citric acid fermentation has
accumulates citric acid has attracted much interest. been the focus of much study, frequently with a view to
Fermentation conditions were established during the the utilizing polysaccharide sources. In general, only
1930s and 1940s when the effects of various medium sugars that are rapidly taken up by the fungus allow a
components were evaluated. The simple surface culture high final yield of citric acid (Mattey, 1992). Poly-
methods used in the first commercial production plants saccharides, unless hydrolyzed, are generally not a
were labor intensive by modern standards, and although useful raw material for citric acid fermentation because
submerged culture techniques had been developed they are broken down too slowly to match the high rate
before 1940, some old-style surface culture plants of sugar catabolism required for citric acid production.
apparently still thrive (Mattey, 1992). The slow hydrolysis of polysaccharides is due to the low
Since 1930s, various explanations have been proposed activity of the hydrolytic enzymes at the low pH that is
for the accumulation of citric acid. Citric acid only necessary for producing citric acid. According to
accumulates when several nutrients are present either in Kubicek and Röhr (1989) sucrose is preferable to
high concentrations (i.e. sugar, acidity, dissolved oxygen) glucose as A. niger has a potent extracellular mycelium-
or at suboptimal levels (i.e. trace metals, nitrogen, bound invertase that is active at low pH and rapidly
phosphate). Many biochemical events likely jointly hydrolyzes sucrose. Superiority of sucrose over glucose
contribute to the observed overproduction. In view of and fructose was demonstrated by Gupta et al. (1976),
the nature of production biochemistry, influence of Hossain et al. (1984) and Xu et al. (1989).
individual factors cannot always be assessed without The carbon source used in industrial fermentations is
influencing the other factors. Moreover, some of the typically beet molasses, although cane molasses, fruit
literature studies have used low- or moderately over- pulp, polysaccharides and sugars are used if local
producing strains, or used conditions that were not conditions permit their economic use. Such low-grade
optimal for accumulation of citric acid. This has made a carbon sources are waste products and generally contam-
comparison of data difficult and adversely affected the inated with high levels of cations from prior processes.
prospects for creating a better overall picture of the Cations are typically made insoluble by precipitation with
commercial citric acid fermentation. Although much potassium ferrocyanide or they are removed by using
basic biochemical research has been carried out with cation exchange resins. Activated carbon, bone char and
A. niger, the fundamental understanding of factors leading bauxite have also been proposed for removing the ions
to citric acid accumulation remains poor. (Prescott and Dunn, 1959). In view of the complexity and
messiness of pretreatment of crude carbon sources, most
2.1. Submerged fermentation of the published research has been done using refined
sugars, generally glucose or sucrose.
Accumulation of citric acid is strongly influenced by Both the type of carbon source and its concentration
the composition of the fermentation medium. This is are critical to citric acid fermentation. The final yield
M. Papagianni / Biotechnology Advances 25 (2007) 244–263 247
of citric acid increases when the initial sugar ing to Shu and Johnson (1948a,b), phosphate does not
concentration is increased and maximal production need to be limiting for citric acid production; however,
rates are usually achieved at 14–22% of sugar (Shu when trace metal levels are not limiting, additional
and Johnson, 1948a,b; Honecker et al., 1989). Xu et al. phosphate results in side reactions and increased
(1989) studied the effect of carbohydrate concentration biomass growth (Papagianni et al., 1999a,b,c). Kubicek
on citric acid yield in submerged fermentation. The and Röhr (1977) showed that citric acid accumulated
sugars maltose, sucrose, glucose, mannose and fruc- whenever phosphate was limited even when nitrogen
tose were tested and highest yields were observed at was not. In contrast, Kristiansen and Sinclair (1979)
sugar concentration of 10% w/v, with the exception of using continuous culture concluded that nitrogen
glucose where 7.5% gave the best results. No citric limitation was essential for citric acid production.
acid was produced in media that contained less than
2.5% sugar. Xu et al. (1989) noted that an increase in 2.1.3. Broth pH
sugar concentration from 1% to 14% increased the The pH of the medium is important at two different
duration of the lag phase of growth from 12 to 18 h times in the fermentation for different reasons. Firstly,
and decreased the growth rate by around 20%. Hossain the spores require a pH N 5 in order to germinate. All
et al. (1984) suggested that high concentrations of fermentations are started from spores even if the culture
appropriate carbon sources lead to repression of α- is “scaled up” through seed fermenters before being
keto-glutarate dehydrogenase, hence explaining the transferred to production fermenters. Secondly, the pH
effect of the sugar concentration and source in terms of for citric acid production needs to be low (pH ≤ 2). A
enzyme repression. low pH reduces the risk of contamination of the
Papagianni (1995) and Papagianni et al. (1999a,b,c) fermentation with other microorganisms. A low pH
studied the influence of glucose concentration on also inhibits the production of unwanted organic acids
growth and citric acid production by A. niger in batch (gluconic acid, oxalic acid) and this makes the recovery
and fed-batch cultures in which the concentration of of citric acid from the broth simpler. The uptake of
glucose was maintained stable (glucostat). The level of ammonia by the germinating spores causes a
glucose had a marked effect on citric acid production corresponding release of protons that lower the pH to
rate. The specific rate of citric acid production increased approximately the right level after spore germination has
with increasing initial glucose concentration (batch occurred. Increasing the pH to 4.5 during the production
culture) or increasing constant glucose level (glucostat phase reduces the final yield of citric acid by up to 80%
culture). In both culture methods, the specific growth (Papagianni, 1995).
rate increased with decreasing glucose concentration for
the first 48 h of fermentation. The size of mycelial 2.1.4. Aeration
clumps was reduced at low glucose levels and their Industrial producers of citric acid have known for a
shape was also affected as the ratio between the long time that variations in the aeration rate can have a
perimeter of the clump and the perimeter of its core detrimental effect on the yield of a batch. If the aeration
increased as glucose concentration decreased. rate is too high (a condition that occurs only at laboratory
scales), the partial pressure of dissolved CO2 in the
2.1.2. Nitrogen and phosphate limitation medium can become too low. Carbon dioxide is important
Complex media such as molasses are rich in as a substrate for pyruvate carboxylase which replenishes
nitrogen-containing compounds and rarely need to be the supply of oxaloacetate for citrate synthase. Sufficient
supplemented with a nitrogen source. The high purity CO2 is produced by the pyruvate decarboxylase reaction
media that are used mainly in research laboratories are to satisfy the stoichiometric demand of the pyruvate
generally supplemented with ammonium salts, particu- carboxylase reaction, but excessive aeration results in
larly ammonium nitrate and ammonium sulfate, to some loss. McIntyre and McNeil (1997) showed that
provide the necessary nitrogen. An advantage of using elevated levels of CO2 in the sparged gas actually had a
ammonium salts is that the pH declines as the salts are detrimental effect upon the final citrate concentration and
consumed and a low pH is a requirement of citric acid final biomass concentration.
fermentation (Mattey, 1992). Other sources of nitrogen The effect of dissolved oxygen has been studied
that have been used include urea and yeast/malt extract in some detail. Even short periods of decreased dis-
(Xu et al., 1989). solved oxygen tension (DOT) cause irreversible changes
Reports concerning the effects of nitrogen and in therate of citric acid production (Kubicek et al.,
phosphate limitations have been contradictory. Accord- 1980). A. niger is known to use two different respiratory
248 M. Papagianni / Biotechnology Advances 25 (2007) 244–263
pathways during citric acid production. Synthesis of citric acid within the TCA cycle. Thus, citric acid is
citric acid is dependent on the cyanide sensitive pathway regarded as an “overflow end product” due to high flux
during the final phase of the fermentation. Both growth rates upstream and reduced flux rates downstream of the
and the start of citric acid production are dependent on accumulation point (Fig. 1).
salicylhydroxamic acid (SHAM)-sensitive respiration. Manganese has also been shown to be important in
A SHAM-sensitive bypass is produced late in tropho- many other cell functions, most notably cell wall
phase, and an alternative ubiquinol oxidase component synthesis, sporulation and production of secondary
of this (Ruijter and Visser, 1999) has been discovered. metabolites (Shu and Johnson, 1948b). Care must
The presence of these bypasses and their importance to therefore be taken when choosing broth ingredients
citric acid production is not coincidental. The produc- and even the materials of construction of the bioreactor
tion of ATP at substrate level via glycolysis is probably vessels, to ensure that traces of manganese do not reduce
sufficient for the cell's energy requirements, and so the yield from the fermentation.
there is little need for much more ATP to be generated
via oxidative phosphorylation of NADH. A high 2.1.6. Fungal morphology
internal concentration of ATP would have an inhibitory The relationship between fungal morphology and
effect on glycolytic enzymes, therefore, an alternative citric acid productivity in submerged fermentation has
oxidase to recycle NADH for further glycolysis would long been established. Despite disagreement over
seem to be an essential component for maintaining a whether the pelleted or filamentous form is more
high flux through the glycolytic pathway. desirable for citric acid production, in all cases, the
mycelium of acidogenic A. niger has been found to
2.1.5. Trace elements conform to the morphological type described by Snell
A. niger requires certain trace metals for growth and Schweiger (1951), that is, short, swollen hyphal
(Mattey, 1992). However, a limitation in other trace branches that may have swollen tips.
elements is necessary for production of citric acid (Shu In submerged culture, the morphology of filamentous
and Johnson, 1948a,b), especially during submerged fungi varies between pellets and free filaments, depending
fermentation. The metals that must be limiting include on culture conditions and the genotype of the strain.
Zn, Mn, Fe, Cu, heavy metals and alkaline metals. Discussion of the factors influencing A. niger morphology
Shu and Johnson (1948a) established the optimal in submerged culture needs to distinguish between macro-
levels of Zn and Fe at 0.3 and 1.3 ppm, respectively. and micro-morphology although a number of similarities
Addition of Mn at concentrations as low as 3 μg/l has been exist for both in relation to citric acid production and
shown to drastically reduce the yield of citric acid under responses to the environment. Snell and Schweiger (1951)
otherwise optimal conditions (Clark et al., 1966). Mattey reported the formation of compact aggregates or pellets
and Bowes (1978) reported that the addition of 10 mg (b 0.5 mm diameter) as being necessary for citric acid
Mn2+ per liter reduced accumulation of citric acid by 50% production. Takahashi et al. (1958) also found the pellet
relative to control culture. Investigations by Clark et al. growth to be superior to the diffuse filamentous form for
(1966) and Kisser et al. (1980) confirmed the key acid accumulation. In contrast, Moyer (1953) reported
regulatory role of Mn2+ ions. The influence of Mn2+ that pellet formation was undesirable for citric acid
ions on protein synthesis was considered to be of major production. Papagianni et al. (1994, 1998, 1999a,c) using
importance because cycloheximide, an inhibitor of de image analysis studies, reported a strong relationship
novo protein synthesis, was found to antagonize the effect between morphology and productivity in various bior-
of manganese addition (Kisser et al., 1980). Cellular eactors while the strain used grew in the form of “clumps”.
anabolism of A. niger is impaired under manganese Mycelial clumps are stable particles of filaments inter-
deficiency and/or nitrogen and phosphate limitation. The twined around a small core. They lack the characteristic
protein breakdown under Mn deficiency results in a high compact structure of pellets, but they represent the main
intracellular ammonium concentration which causes morphological type for many filamentous fungal fermen-
inhibition of the enzyme phosphofructokinase (PFK, an tations. Under appropriate conditions, citric acid yields
essential enzyme in the conversion of glucose and exceeded 85% with this particular macro-morphological
fructose to pyruvate) leading to a flux through glycolysis form, while micro-morphology was characterized by
pathway and the formation of citric acid. The combination short, swollen branches with swollen tips (Fig. 2).
of high glucose and ammonium concentrations, on the The main factors that affect A. niger morphology in
other hand, strongly repress the formation of 2-oxoglu- submerged culture and subsequently influence the
tarate dehydrogenase, thus inhibiting the catabolism of process outcome are the following: the levels of applied
M. Papagianni / Biotechnology Advances 25 (2007) 244–263 249
Fig. 1. Schematic representation of the metabolic reactions involved in citric acid production, the enzymes (italics), the known feedback loops (dashed
lines) and their locations within the cellular structure of A. niger.
agitation; the pH of the broth; the growth rate of the Culture pH can have a profound effect on citric acid
microorganism; nutritional factors; and the type and production by A. niger because certain enzymes within
concentration of inoculum. the TCA cycle are pH sensitive. Maintenance of a low
Intensive agitation is associated with development of pH during fermentation is vital for a good yield of citric
short, thick, and highly branched filaments that acid and it is generally considered necessary for the pH
overproduce citric acid (Fig. 3). However, high shear to fall to around 2.0 within a few hours of the initiation
stress can cause breakage of filaments. A cycle of of the fermentation. Failing this, the yields are reduced
mycelial fragmentation and regrowth has been observed (Mattey, 1992). Quantitative data on the effect of pH on
in citric acid producing A. niger cultures. Fragmentation A. niger morphology and productivity has been reported
and regrowth are beneficial to the citric acid production (Papagianni, 1995; Papagianni et al., 1999a). The
because the mycelia that are most susceptible to preferred morphology (i.e. small aggregates and short
fragmentation are the old and heavily vacuolated parts filaments) associated with increased citric acid produc-
of the filaments that are metabolically inactive; the new tion was obtained at pH values around 2.0 ± 0.2. At pH
tips generated from fragmentation give rise to new 1.6, morphological development was abnormal (bul-
filaments (Papagianni et al., 1999c). bous hyphae) and citric acid production was reduced
250 M. Papagianni / Biotechnology Advances 25 (2007) 244–263
dramatically. At pH 3.0, the aggregates had longer Citric acid overproduction requires a unique combi-
perimeters and oxalic acid formation was evident. nation of several unusual nutrient conditions, i.e.
Of the trace metals that greatly affect the citric acid excessive concentrations of carbon source, hydrogen
fermentation, only manganese concentration has been ions, and dissolved oxygen, and suboptimal concentra-
shown to influence A. niger morphology. Kisser et al. tions of certain trace metals and phosphate, that
(1980) studied morphology and cell wall composition of synergistically influence the yield of citric acid (Kristian-
A. niger under conditions of manganese sufficient and sen and Sinclair, 1978). Citrate is one of the best-known
deficient cultivation in an otherwise citric acid produc- inhibitors of glycolysis, and the ability of A. niger to
ing medium. Omission of manganese ions (less than overproduce citrate by an active glycolytic pathway has
10− 7 M) from the nutrient medium resulted in abnormal therefore attracted substantial interest. Under particular
morphological development that was characterized by nutrient conditions citrate inhibition is counteracted
increased spore swelling and squat, bulbous hyphae. because of the accumulation of various positive effectors
Manganese ions influenced the frequency of mycelial of the phosphofructokinase gene (pfk 1) (Habison et al.,
branching and chitin synthesis (Kisser et al., 1980).
Morphological changes, that included prevention of
clumping, absence of swollen cells and reduced
diameters of filaments, accompanied by a 20% reduc-
tion in citric acid yield, have been reported following
addition of 30 mg/l Mn to an Mn-free medium
(Papagianni, 1995; Papagianni et al., 1999a).
Spore inoculum level is another parameter that
influences citric acid fermentation. Papagianni and
Mattey (2006) studied the development of germinating
spores and the morphology of A. niger in a stirred tank
bioreactor under citric acid production conditions, as a
function of the spore inoculum level that ranged from
104 to 109 spores per ml. Morphological features,
evaluated by digital image analysis, were classified
using an artificial neural network (ANN). The input
layer consisted of the morphological features and
cultivation time, while the output layer consisted of
four object types: globular and elongated pellets, clumps Fig. 3. The effect of agitation: the relationship between circulation time
and free mycelial trees. The significance of the (tc) and citric acid production by A. niger in a tubular loop bioreactor.
M. Papagianni / Biotechnology Advances 25 (2007) 244–263 251
1979; Arts et al., 1987). One of the positive effectors is invertase hydrolyzing sucrose to fructose and glucose
ammonium (NH4+). Citrate inhibition of pfk 1 seems in for transport into the cell (Boddy et al., 1993; Rubio and
vivo to be antagonized by ammonium ions (Habison et al., Maldonado, 1995). High temperatures of the steriliza-
1983) and this antagonism is functionally linked to the tion process also breakdown sucrose in the medium into
well-known effect of trace metal ions, particularly its component sugars. The availability of glucose from
manganese ions, on citric acid accumulation (Wolschek sucrose is not thought to have any influence upon the
and Kubicek, 1999). A critical role in citric acid fer- rate of citric acid production.
mentation has been attributed to manganese ions. In- A. niger has several hexokinases. One of the
fluence of manganese ions on protein synthesis was hexokinases has an affinity for glucose that is 1000-
considered to be of major importance because cyclohex- fold higher than its affinity for fructose. This glucoki-
imide, an inhibitor of de novo protein synthesis, was nase has been found to be inhibited non-competitively
found to antagonize the effect of manganese addition by citrate (Steinböck et al., 1994). Experiments with
(Wolschek and Kubicek, 1999). Cellular anabolism of A. niger mutants indicate that the level of trehalose-6-
A. niger is impaired under manganese deficiency and/or phosphate is important and regulates the flux from
nitrogen and phosphate limitation. According to Habison glucose into glycolysis (Arisan-Atac et al., 1996). The
et al. (1983) and Röhr and Kubicek (1981), the protein citrate inhibition of glucokinase was found to be due to
breakdown under manganese deficiency results in a high chelation of the Mg2+ that is required to bind ATP, and is
intracellular NH4+ concentration (the “ammonium pool”), most probably irrelevant under physiological conditions
that causes inhibition of the enzyme phosphofructokinase, where Mg2+ is present in excess.
an essential enzyme in the conversion of glucose and Schreferl-Kunar et al. (1989) produced A. niger
fructose to pyruvate. This leads to a flux through mutants with a reduced lag time for growth on high
glycolysis and the formation of citric acid. The high sucrose concentrations. These mutants were found to
glucose and NH4+ concentrations, strongly repress the possess enhanced hexokinase and phosphofructokinase
formation of 2-oxoglutarate dehydrogenase and thus activities. Another class of A. niger mutants that were
inhibit the catabolism of citric acid within the tricarbox- resistant to 2-deoxyglucose and had an increased
ylic acid cycle (Röhr and Kubicek, 1981). glucose metabolizing ability, produced citric acid at a
Recent studies on the early stages of citric acid accu- high rate (Kirimura et al., 1992), but the precise stage of
mulation by A. niger (Papagianni et al., 2005) contradict glucose metabolism (probably likely to be at the start of
the existence of an intracellular ammonium pool that has glycolysis) was only poorly defined.
been claimed to be responsible for inhibition of the en- A. niger also possesses the ability to oxidize glucose
zyme phosphofructokinase. Investigating the fate and role using glucose oxidase (Hayashi and Nakamura, 1981).
of ammonium ions in the early phase of the citric acid The enzyme is induced under conditions that are
fermentation process, Papagianni et al. (2005) came to the otherwise typical for the citric acid fermentation, i.e.
conclusion that ammonium ions are not simply deposited by high concentrations of glucose and strong aeration in
inside the cell to make an ammonium pool but they enter the presence of low concentrations of other nutrients
the cell to combine with glucose and form glucosamine. (Mischak et al., 1985; Rogalski et al., 1988; Dronawat
Glucosamine is immediately released in the fermentation et al., 1995). Glucose oxidase is formed early in
broth. The slightly acidic intracellular pH, the extremely fermentation and converts a significant amount of
low ammonium ion concentrations inside the cell (about glucose into gluconic acid. However, its effect is
1% of the external concentration), and the synthesis and limited, as the enzyme is inactivated when the
release of glucosamine show that the inhibition of phos- accumulation of protons in the broth causes the pH to
phofructokinase is certainly not due to an increased con- fall below 3.5 (Mischak et al., 1985; Roukas and
centrations of ammonium ions. The long-held relationship Harvey, 1988). It is not known whether A. niger can
between high glucose and ammonium ion concentrations utilize gluconic acid to make citric acid later in the
and the enzymes phosphofructokinase, 2-oxoglutarate fermentation.
dehydrogenase, and the synthase of glucosamine within
the TCA cycle certainly needs further investigation. 3.2. Phosphofructokinases
3.1. Invertase, hexokinases, and glucose oxidase The first step in the pathway is the phosphorylation
of fructose-6-phosphate by one of two phosphofructo-
The pathway of reactions leading from sucrose to kinases (PFK1 and PFK2). PFK1 is the best known of
citric acid starts outside the cell, with a membrane bound these enzymes. It phosphorylates the C1 position to
252 M. Papagianni / Biotechnology Advances 25 (2007) 244–263
make fructose-1:6-bisphosphate. PFK1 is known to be acid producing strain were not increased. This supported
inhibited by high concentrations of ATP, manganese and calculations of Torres (1994a,b) who concluded that it
citrate and is activated by NH4+, Zn2+, Mg2+ and would be necessary to increase the enzyme activities at
fructose-2:6-bisphosphate, the product of the PFK2 seven different glycolytic steps to increase the effective
reaction (Kubicek-Pranz et al., 1990). A study by Legisa flux through the pathway. A 17-base pair sequence was
and Bencina (1994) showed that A. niger possesses a found upstream of the A. niger pkiA genes that may act
cAMP dependent protein kinase that activates PFK1 as upstream regulating sequence (De Graaff et al.,
and, possibly, also PFK2. PFK1 is not regulated at the 1992). It has also been proposed that a cis-acting
transcription stage as inactive PFK1 has been isolated element initiates transcription of pyruvate kinase during
from mycelia early in the batch fermentation process growth on glycolytic carbon sources (De Graaff et al.,
(Legisa and Bencina, 1994). 1988).
Schreferl et al. (1986) isolated a mutant with a citrate
insensitive PFK2 enzyme. This strain was more tolerant 3.5. Fixation of carbon dioxide
of manganese when accumulating citric acid, showing
that the inhibitory effect of Mn2+ on PFK1 is antagonized The catabolism of glucose via glycolysis leads to two
by fructose-2:6-bisphosphate. Citrate inhibition of PFK1 moles of pyruvate, that are subsequently converted by
is antagonized by NH4+ ions. Several researchers have two separate reactions into the precursors of citrate (i.e.
shown that citrate accumulation does not occur unless a oxaloacetate and acetylCoA). Cleland and Johnson
certain amount of NH4+ions is taken up at the start of the (1954) were the first to show that in these reactions,
batch fermentation. The overall effect of activators and A. niger uses a quantity of carbon dioxide for the for-
inhibitors on PFK is small. Torres (1994a,b) has shown mation of oxaloacetate that equals the quantity of CO2
that in steady-stage fermentation, PFK enzyme step has released during acetylCoA formation. This is important
no great effect on the flux through glycolysis. The for producing high citric acid yields because the only
combination of the levels of inhibitors and activators in alternative way of producing oxaloacetate would be a
productive cytoplasm (Habison et al., 1983; Jernejc et al., complete turn of the tricarboxylic acid cycle, with the
1992; Mattey, 1992) simply permit the accumulation of associated loss of two moles of CO2. If that were to
citric acid through glycolysis. happen, only two-thirds of carbon source would ac-
cumulate as citric acid and at least a third would be
3.3. Glycolytic and the pentose phosphate pathway wasted.
Fixation of carbon dioxide does not seem to occur
The well-known tracer studies by Cleland and during the early phases of fermentation (Kubicek et al.,
Johnson (1954) and Martin and Wilson (1951) showed 1979). Continuous analysis of CO2 and oxygen in the
that citric acid is mainly formed via the reactions of the exit air of a pilot plant citric acid fermentation showed
glycolytic pathway, as shown in Fig. 1. Aspergillus sp. that during the first 70 h of the batch fermentation the
use glucose and other carbohydrates for biosynthesis respiratory coefficient (i.e. CO2 released/O2 taken up)
and maintenance by channeling glucose into the was close to unity. Respiratory quotient then began to
reactions of the glycolytic and pentose phosphate decrease and reached the level predicted from the
pathways. The pentose phosphate pathway accounts operation of the pyruvate carboxylase reaction (i.e.
for only a small percentage of the carbon metabolized respiratory quotient = 0.66) only at stages where citrate
during citric acid production and this decreases further accumulation was already taking place at a constant rate
during prolonged cultivation (Legisa and Mattey, 1986). (e.g. b 120 h into fermentation). The Cleland and
Johnson reaction may therefore be important only at
3.4. Pyruvate kinase later stages of fermentation, with the initial phase of
citric acid accumulation taking place without any
Pyruvate kinase was considered to be an important overall carbon dioxide uptake. The enzyme catalyzing
regulatory stage in the synthesis of citric acid until Cleland and Johnson reaction was shown to be pyruvate
Meixner-Monori et al. (1984) showed that the purified carboxylase (Woronick and Johnson, 1960; Bloom and
enzyme was only slightly affected by metabolic levels of Johnson, 1962), and has been characterized by Feir and
inhibitors. When Ruijter et al. (1996) amplified the Suzuki (1969) and Wongchai and Jefferson (1974).
genes encoding phosphofructokinase 1 (pfkA) and Unlike the enzyme from several other eukaryotes, the
pyruvate kinase (pkiA) individually and in combination, pyruvate carboxylase of A. niger is located in the
the rates of citrate accumulation by a moderately citric cytosol (Bercovitz et al., 1990; Jaklitsch et al., 1991).
M. Papagianni / Biotechnology Advances 25 (2007) 244–263 253
as NAD+-dependent and NADP+-dependent isocitrate necessary for citric acid overproduction (Clark and
dehydrogenases. The NAD+-dependent form exists only Lentz, 1961; Kubicek et al., 1980; Dawson et al.,
in mitochondria. One of the two NADP+-dependent forms 1988a). Sudden interruptions in aeration cause an
exists in mitochondria and the other exists in the cytoplasm. irreversible impairment of citric acid production without
The NADP+-dependent form is present in greater quantities affecting growth (Kubicek et al., 1980; Dawson et al.,
compared with the NAD+-dependent enzyme (LaNauze, 1988b). The biochemical basis for this observation lies
1966; Kubicek and Röhr, 1977). in the induction of an alternative respiratory pathway
The isocitrate to α-ketoglutarate is a two step process. that is required for re-oxidation of the glycolytic NADH
Protons are removed first and CO2 is produced in the (Zehentgruber et al., 1980; Kubicek et al., 1980;
second step as shown in Fig. 4. Divalent metal ions such as Kirimura et al., 1987, 1996).
Mg2+ and Mn2+ are required for the reaction, and are The activity of the proton pumping NADH:ubiqui-
bound by the enzyme at the active site. The metal ion has none oxidoreductase (complex I) in the mycelium was
two functions: it is involved in the binding of the substrate found to be proportional to the levels of Mn2+ in the
to the enzyme and has an important role in stabilizing the growth medium by Wallrath et al. (1992) further
reaction intermediate, oxalosuccinate. Mg2+ and Mn2+ can suggested that complex I is at least partly inactive in
be used interchangeably in the enzyme without influenc- citric acid producing conditions. A high yielding
ing enzyme activity. Citrate and α-ketoglutarate have both producer of citric acid (A. niger strain B60) was
been shown to act as inhibitors of the NADP+-dependent observed to lose its entire complex I activity at the
form of the enzyme (Mattey, 1977; Kubicek and Röhr, onset of citric acid production (Wallrath et al., 1991).
1977) and this helps to perpetuate the accumulation of Strain B60 has the normal peptide sequence in complex
citric acid once the process has started. I, but the assembly of the complex appears to be
different (Schmidt et al., 1992). In citric acid production
3.10. Glutamine synthase conditions, excess NADH is oxidized by an alternative
oxidase that does not pump protons. This enzyme is
Punekar et al. (1984) have suggested that the roles of produced constitutively, but does not normally have any
ammonia, pH and manganese ions in citric acid effect on metabolism as its affinity for NADH is only
accumulation are linked with glutamine synthase. Gluta- 1% of the affinity of complex I for NADH (Prömper
mine synthase has two forms, depending on whether the et al., 1993; Schmidt et al., 1992).
enzyme has Mg2+ or Mn2+ ion in its structure. Only the Mutant strains of A. niger that lack complex I activity
Mg2+ form can exist during growth on Mn2+-free medium have been prepared by Schmidt et al. (1992) and
used for citric acid accumulation. As the pH in the Prömper et al. (1993). Deletion of complex I in A. niger
medium falls to low levels, the intracellular pH also falls B60 (Schmidt et al., 1992) reduced this strain's ability to
to between 6.0 and 6.5. The activity of the Mg2+ form of form ATP via oxidative phosphorylation to 60% of that
glutamine synthase is greatly reduced under these of the wild type. The mutant strain accumulated a high
conditions, leading to the accumulation of free glutamic concentration of intracellular citrate, but appeared
acid, TCA cycle intermediates and NH4+ ions. unable to excrete significant amounts of it into the
medium. The mutant actually secreted less citrate than
3.11. NADH regeneration and oxidative phosphorylation the wild type strain. Schmidt et al. (1992) postulated that
this could be due to a higher alternative oxidase activity
Strong aeration and dissolved oxygen tensions higher in the high yielding strain, which oxidized all of the
than those required for vegetative growth of A. niger are excess NADH that could not be used by complex I. As
the mutant strain possessed only the wild type
alternative oxidase, it was unable to recycle enough
NADH to maintain the high glycolytic flux that is
essential for citric acid production.
During citric acid accumulation, the quantitative
conversion of citric acid to glucose yields 1 molecule of
ATP and 3 molecules of NADH. Unless a significant
proportion of the NADH pool can be re-oxidized without
the formation of ATP by the alternative respiratory
pathway described above, the yield of ATP will probably
Fig. 4. The conversion of isocitrate to α-ketoglutarate. exceed the cell's maintenance requirement. Wolschek and
M. Papagianni / Biotechnology Advances 25 (2007) 244–263 255
Kubicek (1999) have suggested that the ATP could be during the production phase of the A. niger citric acid
consumed by the membrane bound ATPase which process. Models for the known glucose transporters in
maintains the pH gradient (Mattey et al., 1988), and that A. niger and for simple diffusion of glucose through
the inhibition of the alternative pathway would therefore the hyphal membrane were prepared and the simulations
inhibit glycolysis by causing an accumulation of NADH. from these models were compared with published
fermentation data. The purely physical and uncontrolled
4. Membrane transport aspects in A. niger process of diffusion was found to satisfactorily explain
the specific rate of glucose uptake observed during the
Both the transport of sugars and ammonia into the cell production phase in several different types of fermenta-
and the export of citrate ions from the cell are crucial to an tions (Papagianni and Mattey, 2004).
understanding of the overproduction of citric acid by The only publications on uptake of ammonium ions
A. niger. Membrane transport is also the subject of much by A. niger, are those of Papagianni et al. (2005) (see
disagreement. Theoretical calculations by Torres (1994a,b) Section 3) and Mattey et al. (1988). The effect of
suggest that a major part of the control of citric acid metabolic inhibitors on the rate of uptake implies that
production must occur at hexose uptake and/or phosphor- the ammonium uptake requires energy either directly or
ylation. That is, if the rate of glucose uptake can be indirectly. Published studies of ammonium uptake in
increased, the rate of citrate production will increase. Gupta other microorganisms all focus on active uptake when
and Sharma (1995) demonstrated this without influencing ammonium ions are present in low concentrations.
the overall yield. Gupta and Sharma (1995) showed that an Mattey and co-workers (Mattey, 1992; Kontopidis
increase in the rate of glucose transport reduced the lag time et al., 1995) explained the export of citrate through the
between ammonia uptake and citrate production. However, plasma membrane in terms of the large pH gradient
in fed-batch experiments by Papagianni (1995), maintain- between the cytosol and the extracellular medium. Citrate
ing the concentration of glucose at specific levels during exists in different ionic states in the cell, which has a pH
production of citric acid, did not affect the yield of citric between 6.0–7.0, and the medium that is at pH 2.0 or
acid, but its rate of production strongly correlated with lower. The cell membrane has been proposed to be
glucose concentration. permeable to citrate2− (Mattey, 1992; Kontopidis et al.,
At least two glucose transporters have been identified 1995), the prevalent form of citrate at the intracellular pH.
in A. niger. Torres et al. (1996a) showed that a high Thus, citrate2− is postulated to diffuse out of the cell
glucose concentration (N 50 g/l) was a prerequisite for rapidly. If this assumption is correct, a low pH would be
the formation of an alternative low-affinity glucose necessary to keep the external concentration of citrate2−
transporter that was capable of providing the high flux negligible. The rate of citrate export at different external
of glucose required for citrate production. With such pH levels would also be related to the ratio of the different
high levels of glucose in the medium, there is a potential ionic forms of citrate at the external pH.
for their diffusive transport through the membrane at According to Wolschek and Kubicek (1999), intracel-
significant rates. This idea is supported by Mischak et al. lular citrate is excreted from the cell by an active process
(1984), but it appears to have been mostly overlooked. that requires ATP to pump citrate into the medium. The
Mischak et al. (1984) clearly showed that small quan- authors showed that citrate export would not occur at pH
tities of extracellular citric acid inhibited the uptake of 5.0 unless the mycelium was Mn2+ deficient. This
glucose (at 0.5 mM concentration) in mycelium that had particular type of transport was also sensitive to metabolic
been grown under citric acid producing conditions. inhibitors, implying an active process. At a pH of 5.0, the
Inhibition of the glucose transporter was not affected by rate of citrate export was reduced to 50% of the rate at pH
metal ions (Mischak et al., 1984), indicating that citrate 2. In a different study (Papagianni, 1995), the export of
affected the carrier directly, and not by chelating citrate in producing mycelium at pH 4.0 was reduced to
essential divalent metal ions. Under these conditions, 42% of the rate at pH 2.0. The transporter implicated by
the low affinity glucose transporter should theoretically Wolschek and Kubicek (1999), continued to export citrate
be present (Torres et al., 1996a), and so it must be at pH 7.0 at 25% of the rate at pH 2.0, but at pH 7 citric
concluded that the high flux transport mechanism is acid export generally does not occur. This suggests that
inhibited under producing conditions and that some the transporter system investigated by Wolschek and
other mechanism is responsible for the transport of Kubicek (1999) is not the main means of citrate export in
glucose into the cell. A. niger under producing conditions.
Wayman and Mattey (2000) identified simple Unpublished studies referenced by Mischak et al.
diffusion as the primary mechanism for glucose uptake (1984), suggest that citrate is not taken up at pH 2.0 by
256 M. Papagianni / Biotechnology Advances 25 (2007) 244–263
5.1. Kinetic modeling by Röhr, Zehntgruber and In these equations, Xt is the biomass concentration at
Kubicek (1981) time t, X0 is the initial biomass concentration, μ is the
specific growth rate, and Kc and Kt are constants.
Kinetics of citric acid production by A. niger growing Product formation was related to the growth rate by a
on sucrose in a pilot plant were investigated by Röhr modified Luedeking–Piret equation, which relates the
et al. (1981). A typical growth curve for A. niger under formation of products (rp) to either the biomass
citric acid producing conditions shows an initial rapid concentration (x) or the rate of biomass accumulation
growth phase followed by a phase of slow growth. Röhr (rx):
et al. (1981) subdivided cell growth and product
formation into several phases, each described by a rp ¼ ðad rx Þ þ ðbd xÞ ð4Þ
simple deterministic model (Fig. 5). The best fitting
model equations in the various phases were identified.
Although the similar relationships applied to both
The growth phases identified were: the hyphal growth
growth and acid formation (Fig. 5), the acid formation
phase (Bx), pellet growth phase (Cx), restricted growth
kinetics were usually different from the growth kinetics
phase (Dx), transition period between trophophase and
by a term that represented the lag time. This lag time,
idiophase (Ex) and idiophase growth (Fx). The logarith-
also known as the maturation time, is the period during
mic, cube root and linear equations described by Trinci
which the culture taken up all the ammonium ions but
(1970) were used to describe the growth in each phase,
has not yet started producing citric acid. The rate of
as follows:
product formation was then said to be proportional to the
Log of growth linear with time; i:e: rate at which the cells entered the maturation state. The
ð1Þ rate of product formation was expressed as follows:
ln Xt ¼ ln X0 þ lt
Cube root of growth linear with time; or rPt ¼ krX ðt−tm Þ ð5Þ
ð2Þ
1=3 1=3
Xt ¼ X0 þ Kc t
where rPt is the product formation rate at time t, rX(t−tm)
Growth linear with time; i:e: Xt ¼ X0 þ Kt t ð3Þ is the rate of biomass growth at time t beyond the
M. Papagianni / Biotechnology Advances 25 (2007) 244–263 257
Table 1
Calculated values of the growth-associated and non-growth associated The permissible values below the diagonal line
constants k1 and k2 (Röhr et al., 1981) depended on the value of the ratio YEX /YC, that
Parameter Microorganism suggest that energy limits the cell growth. Y E
A. niger B60/B3 A. niger B60/B5b (dimensionless) is the upper limit of the yield factor
tm (h) 15 24 Yij for a compound j on a compound i based on energy
k1 1.9 2.0 availability. Similarly YC (dimensionless) is the upper
k2 0.090 0.038
limit of Yij based on carbon availability. The highest
258 M. Papagianni / Biotechnology Advances 25 (2007) 244–263
model and experimental data obtained under various the medium, but also later if glucose is available and
process conditions in two different reactors. further nitrogen is added. Glucosamine acts as a storage
compound and it is used by the fungus during the course
5.7. Stoichiometric modeling by Wayman (2001) and of the fermentation.
Papagianni, Wayman and Mattey (2005)
6. Concluding remarks
Wayman (2001) made a stoichiometric model that
involved mass balances for biomass, glucose and This review focused on citric acid fermentation by
ammonium ions during the first 30 h of the fermenta- A. niger. Fermentation biochemistry, membrane trans-
tion. For each measured data point, the expected glucose port in the producing microorganism and fermentation
uptake rate was calculated from the rate of ammonium modeling were discussed. Findings concerning the role
ion uptake rN, the rate of biomass formation rx, and the of ammonium ions (Papagianni et al., 2005) during the
relevant yield coefficients (Y ). The following equations early stages of the citric acid fermentation lead to ques-
were used: tions concerning the long-held theory of an “ammoni-
um” pool within the cells. This pool has been regarded
−rx as the cause of inhibition of the enzyme phosphofruc-
rsðxÞ ¼ ð18Þ tokinase, a consequent flux through glycolysis and for-
Ys
mation of the acid. Clearly, the relationship between
high concentrations of glucose and ammonium ion
−rx and the impact of this relationship on the various meta-
rN ðxÞ ¼ ; ð19Þ
YN bolic enzymes (phosphofructokinase, 2-oxoglutarate
dehydrogenase, glucosamine synthase) deserves further
rN −rN ðxÞ investigation.
rsðPÞ ¼ ð20Þ Concerning membrane transport in A. niger, the
YP=N
uptake of glucose under the high glucose conditions that
saturate the known transporters, appears to be driven by
The calculated value for rs was compared with the pure diffusion that does not depend on any transporter
measured values of rs obtained from the measured data. proteins (Wayman and Mattey, 2000). The mediated
Biomass was assumed to have the generalized empirical transport systems previously described in the literature
formula CH1.8O0.5N0.2. The fate of ammonium ions not do not fit the observations reported in the literature. A
incorporated into biomass could be altered by changing fermentation model based on diffusive transport of
the yield of hypothetical product from nitrogen and glucose does explain well many of the previously un-
carbon sources. The values for yield coefficients were as explained observations.
follows: 65% for biomass from glucose; 500% for Advances in image analysis technologies and their
biomass from ammonium ions (theoretical molar yield); application to fungal fermentations have made available
and 100% for a simple hypothetical compound contain- quantitative morphological data on A. niger for various
ing glucose and ammonium ions in equal molar amounts. stages of the fermentation. These developments have
The experimental and calculated fermentation pro- enabled quantitative investigation of the long-known
files agreed closely, indicating that ammonium ions empirical relationship between morphology and citric
combine with a carbon containing metabolite inside the acid productivity. Fermentation models that are mor-
cell. This good agreement further showed that the most phologically structured have been developed to better
likely ratio for this combination was one mole of describe the behavior of this fermentation.
glucose per mole of ammonia. Based on this stoichi-
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