APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2005, p. 7178–7186 Vol. 71, No.

11
0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.11.7178–7186.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Fate and Role of Ammonium Ions during Fermentation of

Citric Acid by Aspergillus niger
Maria Papagianni,1* Frank Wayman,2 and Michael Mattey2
Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary Medicine,
Aristotle University of Thessaloniki, Thessaloniki 54006, Greece,1 and Department of Bioscience,
University of Strathclyde, Glasgow G1 1XW, United Kingdom2
Received 19 April 2005/Accepted 16 June 2005

Stoichiometric modeling of the early stages of the citric acid fermentation process by Aspergillus niger
revealed that ammonium ions combine with a carbon-containing metabolite inside the cell, in a ratio 1:1, to
form a nitrogen compound which is then excreted by the mycelium. High-performance liquid chromatography
analysis identified glucosamine as the product of the relationship between glucose and ammonium during the
early stages of the citric acid fermentation process. Slightly acidic internal pHs, extremely low ammonium ion
concentrations inside the cell, and glucosamine synthesis come into direct contradiction with the earlier theory
of the ammonium pool inside the cell, regarded as responsible for inhibition of the enzyme phosphofructoki-
nase. At later fermentation stages, when the mycelium is involved in a process of fragmentation and regrowth,
the addition of ammonium sulfate leads to a series of events: the formation and secretion of glucosamine in
elevated amounts, the short inhibition of citrate synthesis, growth enhancement, the utilization of glucosamine,
and finally, the enhancement of citric acid production rates. Obviously, the enzymatic processes underlining
the phenomena need to be reexamined. As a by-product of the citric acid fermentation, glucosamine is reported
for the first time here. Suitable process manipulations of the system described in this work could lead to
successful glucosamine recovery at the point of its highest yield before degradation by the fungus occurs.

Much has been reported on the mechanism of citric acid and phosphate limitation. According to Habison et al. (4) and
accumulation by Aspergillus niger, but the majority of the stud- Röhr and Kubicek (17), the protein breakdown under manga-
ies were performed on the main production phase while vari- nese deficiency results in a high intracellular NH4⫹ concentra-
ous aspects of the early stages of the citric acid fermentation tion (the “ammonium pool”), which causes inhibition of the
still remain unclear today. Citric acid overflow requires a enzyme phosphofructokinase, an essential enzyme in the con-
unique combination of several unusual nutrient conditions, i.e., version of glucose and fructose to pyruvate, leading to a flux
excessive concentrations of carbon source, hydrogen ions, and through glycolysis and the formation of citric acid. The high
dissolved oxygen or suboptimal concentrations of certain trace glucose and NH4⫹ concentrations, on the other hand, strongly
metals and phosphate, which synergistically influence the

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repress the formation of 2-oxoglutarate dehydrogenase and
yield of citric acid (5). Citrate is one of the best known inhibi- thus inhibit the catabolism of citric acid within the tricarboxylic
tors of glycolysis, and the ability of A. niger to overproduce acid cycle (17).
citrate by an active glycolytic pathway has therefore attracted The concentrations of both nitrogen and phosphate are low
biochemical interest for a long time. Under appropriate nutri- in media designed for organic acid production by Aspergillus
ent conditions, this inhibition is more than counteracted by the species, but little is known about their mode of influence. In
accumulation of various positive effectors of the phosphofruc-
defined media, nitrogen is supplied as ammonium sulfate or
tokinase gene (pfk1), one of which is ammonium (NH4⫹), and
ammonium nitrate. The advantage of ammonium salts is that
hence this feedback does not occur (1, 3). Citrate inhibition of
they decrease the pH as they are consumed, which is a pre-
pfk1 seems to be antagonized in vivo by ammonium ions (4),
requisite of citric acid fermentation. Reports concerning the
and this antagonism is functionally linked to the well-known
limitation of nitrogen during the production phase of citric
effect of trace metal ions, particularly manganese ions, on citric
acid-producing A. niger cultures have been contradictory. An
acid accumulation (21). A critical role in the citric acid fer-
mentation process by A. niger has been attributed to manga appropriate balance of nitrogen, phosphate, and certain trace
nese ions, the influence of which on protein synthesis was metals appears to be important for the accumulation of citric
considered to be of major importance since cycloheximide, an acid in batch cultures (18). In continuous as well as fed-batch
inhibitor of de novo protein synthesis, was found to antagonize culture, nitrogen must be limiting to attain the highest yields
the effect of manganese addition (21). Cellular anabolism of A. (6). This would be consistent with a role of an inhibited glu-
niger is impaired under manganese deficiency and/or nitrogen tamine synthetase in citric acid production as proposed by
Punekar et al. (16). On the other hand, it has to be clarified
how this fits with the increased intracellular NH4⫹ levels
* Corresponding author. Mailing address: Department of Hygiene throughout citric acid fermentation (8) and the stimulation of
and Technology of Food of Animal Origin, School of Veterinary Med-
citric acid production by the addition of extra ammonium sul-
icine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece.
Phone: 30-2310-999804. Fax: 30-2310-999829. E-mail: mp2000@vet fate, which is also reported. Exogenous addition of NH4⫹
.auth.gr. during the citric acid fermentation has been found to stimulate

7178
VOL. 71, 2005 AMMONIUM IONS AND CITRIC ACID FERMENTATION BY A. NIGER 7179

the rate of citrate production (2, 22), and this is consistent with resuspended in a small quantity of buffer, and the cell membranes were disrupted
the effect of NH4⫹ on pfk1. by further addition of methanol to make a suspending solution of approximately
50% strength. After standing for 24 h and removal of the solids, the ions were
Although several aspects of the role of NH4⫹ in the regula- measured with the electrode.
tion of citrate overproduction have been investigated, no in- Glucosamine determination was done by high-performance liquid chromatog-
formation seems to exist in the literature concerning the rela- raphy (HPLC) analysis (using a mass detector). The concentration range of
tionship between ammonium ion concentration in the medium standards [D(⫹)-glucosamine hydrochloride, C6H13NO5-HCl, molecular weight
of 215.6; Sigma] was 30, 15, 7.5, and 3.75 mM, and they were assayed using a
and ammonium ion uptake by the mycelium during the first
Redex RNM carbohydrate column (300 by 7.8 mm; Phenomenex) and a mass
hours of fermentation, nor that between ammonium ion con- detector (Sedex 55). The standards used for glucose were 54, 27, 13.5, and 5.4
centration and proton release. The present work aimed to mM. The mobile phase was water and the flow rate 0.4 ml/min (0.6 ml/min for
investigate the particular phase of fermentation and follow glucosamine). The procedure was performed at 85°C and a pressure of 1.9 bars
stoichiometrically the processes of ammonium ion and glucose and repeated three times. The software Gilson 715 HPLC was used.
Fungal morphology was characterized by using an automatic image analysis
uptake. Modeling and subsequent analysis revealed the release system consisting of an Olympus microscope (Olympus, New Hyde Park, NY)
of an aminated compound in the fermentation medium at operated as phase contrast, a charge-coupled-device camera (Sony, Cambridge,
elevated concentrations. This compound, namely, glu- United Kingdom), a PC with a frame-grabber, and image analysis software (SIS,
cosamine, was traced throughout the process of citric acid Olympus, Germany). The inoculum consisted of free mycelial trees that, within
24 h from inoculation, formed microscopic clumps. Mycelial ‘clumps’ are stable
production, and factors affecting its formation were investi-
particles of intertwined filaments around a small core. They lack the character-
gated. Since mycelial fermentations carried out in bioreactors istic compact structure of pellets, while they represent the main morphological
represent morphologically dynamic systems in multifactorial type for many filamentous fungal fermentations. The preparation of the samples
environments, it was considered absolutely necessary to exam- and the measurements were as described in earlier publications (13, 14, 15). A
ine the morphology of the fungus and quantify certain mor- magnification of ⫻100 was applied for measurements of mean perimeters of
clumps (morphology parameter P, ␮m) and mean lengths of filaments protruding
phological parameters with the use of an automated image from the cores of mycelial clumps (morphology parameter L, ␮m). For the
analysis system. Therefore, the whole system, performed under detection and characterization of vacuoles, the image was processed, at a mag-
production conditions, was viewed with respect to the relation- nification of ⫻400, by means of adjustment of grayness levels, detection of the
ship between glucose and ammonium, the biosynthesis of glu- objects of interest, and binary image processing. The exact method and the
quantification of vacuolated areas were described in detail in an earlier publica-
cosamine, and the dynamics of the morphology of the fungus.
tion on the hyphal vacuolation and fragmentation of A. niger (15).
Fermentations were carried out in triplicate. All data presented are averages
of results obtained in three or more independent measurements.
MATERIALS AND METHODS
Microorganism, media, and inoculum preparation. The organism used in this
work was the industrial strain A. niger PM1 (University of Strathclyde, Glasgow, RESULTS
Scotland). This was maintained on molasses agar, which contained 300 g/liter
cane molasses (pH adjusted to 6.8) and 18 g/liter agar (technical, grade 3; Oxoid,
Ammonium ion concentration and citric acid fermentation.
Basingstoke, United Kingdom). The plates were incubated at 30°C for 7 days. Using the standard medium composition described in Materi-
The inoculum was prepared by washing spores from a mature culture plate with als and Methods, the final citric acid concentration (168 h of
20 ml of sterile distilled water and transferring them aseptically to 50 ml of fermentation) reached 110 g/liter (Fig. 1) while mycelium dry
medium in 500-ml Erlenmayer flasks. The spore content was approximately 107
weight reached 7.3 g/liter (not shown). Citric acid production
spores/ml. The shake flasks were incubated at 30°C and 200 rpm in an orbital
rate increased as the NH4⫹ concentration in the fermentation

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shaker incubator for 40 h to produce the fermenter inoculum. The contents of six
flasks were used to inoculate the bioreactor. broth lowered to very low levels after the 24th h from inocu-
The composition of the standard fermentation medium was the following, in lation (Fig. 1 and 2). The effect of different concentrations of
g/liter: D-glucose, 150; (NH4)2SO4, 2.5; MgSO4 · 7H2O, 0.5; KH2PO4, 2.0; Fe3⫹ (NH4)2SO4 in the medium on the rate of NH4⫹ uptake (rN,
[as Fe2(SO)4 · 24H2O], 0.1 ⫻ 10⫺3; Zn2⫹ (as ZnSO4 · 7H2O), 0.1 ⫻ 10⫺3; Cu2⫹
(as CuSO4 · 5H2O), 0.06 ⫻ 10⫺3.
mM/h) was investigated using media containing various frac-
Culture conditions. The stirred tank bioreactor used in this work was a New tions (100, 80, 60, 40, 20, and 0%) of the optimal amount of 2.5
Brunswick Scientific BIOFLO 410 model with a working volume of 10 liters. The g/liter (NH4)2SO4. As Fig. 2 shows, all cultures appeared to
agitation system consisted of three disk turbine impellers, 8 cm in diameter, with have a slow initial uptake, which has no clear relationship with
six flat blades, operating at a stirrer speed of 600 rpm.
the number of initial ammonium ions. Between approximately
Fed-batch experiments were carried out with a 150-g/liter initial glucose con-
centration and ammonium sulfate added at three concentrations, 1, 0.5, and 2 20 and 25 h, the bulk of ammonium was removed from the
g/liter, at a single pulse, at 75 h into the process. Process temperature was medium. The rate of uptake does not appear to have any
maintained at 28°C and the airflow rate at 1 vol/vol/min air/medium (vvm). pH relationship with the amount of biomass present in this stage,
was controlled at 2.1 by the automatic addition of titrants (2 M NaOH and 20% as all cultures had identical start points. The rate of uptake
H2SO4 solutions).
Analytical methods. Dry weights were determined by filtering 20 ml of broth
does appear to be related to the initial ammonium ion con-
through preweighed glass fiber filters (grade GF/C, 4.25 cm; Whatman Interna- centration. Figure 3 shows a very clear linear relationship be-
tional, Maidstone, United Kingdom), washing and drying them in a microwave tween initial ammonium ion concentration and the rate of the
oven (15 min at low power), and leaving them in a desiccator for 24 h before bulk uptake (20 to 25 h). The concentration of protons ex-
reweighing. Citric acid was determined by the method of Marier and Boulet (11).
ported from the biomass appears to be directly related to the
Glucose was determined by using a glucose oxidase-peroxidase method as de-
scribed by Kunst et al. (9). Proton concentration was calculated from pH mea- initial ammonia concentration for the fermentations per-
surements. formed with up to and including 60% of the optimal amount
The concentration of ammonium ions in solution was calculated using an (Fig. 4). Dry weight and ammonia measurements in fermenta-
ammonium electrode (Asea Brown Boveri/Kent Taylor 8002-8). A protocol was tions performed with complete medium (Fig. 5) showed that
developed (20) to allow for good reproducibility over a large volume of samples
and an extended period of time. Concentrations of ammonium ions within the
the uptake of ammonia equivalents from the broth is very
mycelium were calculated by filtering the broth and washing the filter cake with similar to the increase in biomass in the early phase of fermen-
tap water (buffered to the same pH as the broth with HCl). The cells were then tation (20 to 25 h from inoculation).
7180 PAPAGIANNI ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 3. Relationship between initial ammonium ion concentration

and uptake rate between 20 and 24 h. The trend line is a least-squares
fit through the origin. Correlation coefficient r2 ⫽ 98.6%.

⫺ rx
r N共x兲 ⫽ (2)
YN

r N ⫺ r N共x兲
r s共P兲 ⫽ (3)
YP/N
FIG. 1. Time course of citric acid production in standard condi- The calculated value for rs was compared with the measured
tions and with the addition of 0.5-, 1-, and 2-g/liter pulses of ammo-
nium sulfate. values of rs obtained from the original data. Biomass was as-
sumed to have the generalized empirical formula CH1.8O0.5
N0.2. The fate of ammonium ions not incorporated into bio-
Stoichiometric modeling. A simple stoichiometric model mass could be altered by changing the yield of hypothetical
was constructed by calculating the material balances for bio- product from nitrogen and carbon sources. The values for yield
mass, glucose, and ammonium ions throughout the first 30 h of coefficients were as follows: 65% for biomass from glucose
fermentation. Raw fermentation data were placed into a (12), 500% for biomass from ammonium ions (theoretical mo-
spreadsheet (Microsoft Excel), and for each data point, the lar yield), and 100% for a simple hypothetical compound con-
expected glucose uptake rate was calculated from rN (rate of taining glucose and ammonium ions in equal molar amounts.
ammonium ion uptake), rx (rate of biomass formation), and The calculated glucose uptake was plotted alongside the ex-
yield coefficients (Y) using the following equations (19): perimental values in Fig. 6. The close correlation between

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calculated and experimental profiles indicates that ammonium
⫺ rx
r s共x兲 ⫽ (1)
Ys

FIG. 2. Ammonium ion levels in the broth of fermentations per-

formed with different initial amounts of ammonium ions correspond- FIG. 4. The effect of initial ammonium ion concentration upon
ing to 0, 20, 40, 60, 80, and 100% of 2.5 g/liter ammonium sulfate. proton excretion.
VOL. 71, 2005 AMMONIUM IONS AND CITRIC ACID FERMENTATION BY A. NIGER 7181

FIG. 5. Batch profile of biomass concentration and mass of ammo- FIG. 7. HPLC chromatogram for broth samples. The solid line
nia equivalents from the broth. represents the standard solutions used: glucosamine at 15 mM and
glucose at 13.5 mM. The short-dashed line represents a broth sample
(1:10 dilution) taken at 24 h of fermentation (50 g/liter initial glucose
concentration). The long-dashed line represents a broth sample (1:100
ions combine with a carbon-containing metabolite inside the dilution) taken at 48 h of fermentation (150 g/liter initial glucose
cell. It also shows that the most likely ratio for this combination concentration). The peaks obtained at about 8 min retention time
correspond to glucosamine, while those at 12.5 min correspond to
is equivalent to 1 mole of glucose per mole of ammonia.
glucose.
Following modeling work which showed that glucose and
ammonium uptake are stoichiometrically linked with a ratio of
1:1, glucosamine was identified as a potential nitrogen storage identify glucosamine, HPLC analysis was set up as described in
compound. As a component of cell walls, glucosamine is po- Materials and Methods. Broth samples were taken in 24-h
lymerized and removed from the cell. Shorter polymer chains intervals from fermentations with 50- and 150-g/liter initial
are also produced to form a protective and adhesive “bioslime” glucose concentrations (all other medium constituents’ con-
layer outside the cell wall. The presence of loosely attached centrations remained unchanged). The results of HPLC anal-
compounds has been noted in the literature (7). In order to ysis were very satisfactory, as glucosamine was identified in
broth samples. Figure 7 shows the chromatogram of analysis

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for standards (glucosamine, 15 mM; glucose, 13.5 mM) and
broth samples (24-h culture, 50-g/liter initial glucose concen-
tration, 1:10 dilution, and 48-h culture; 150-g/liter initial glucose
concentration, 1:100 dilution). In Fig. 7, the peaks obtained at
about 8 min retention time correspond to glucosamine while
those at 12.5 min correspond to glucose.
Formation and release of glucosamine during fermentation.
To investigate the conditions of formation and release of glu-
cosamine into the broth, fermentations were carried out with
initial glucose concentrations of 150 and 50 g/liter, performed
with the optimal concentration of ammonium ions [2.5 g/liter
(NH4)2SO4] and a 60% fraction of the optimal concentration.
According to Fig. 8, no glucosamine was detected until about
16 h following inoculation. The maximum concentration of 48
g/liter was detected when both glucose and NH4⫹ concentra-
tions were optimal (150 g/liter and 100%, respectively). This
was noticed at 48 h of fermentation and remained almost
stable up to 85 h until it dropped sharply afterwards to level
zero at about 126 h. With suboptimal concentrations of glucose
and ammonium ions, glucosamine formation commenced at
the same time, while its maximum concentration in the broth
was detected earlier and in lower levels compared to the stan-
FIG. 6. Comparison of calculated and experimental cumulative up- dard medium conditions. Results of the same trend were ob-
take of glucose during fermentation. tained in all cases, as glucosamine concentration in the fer-
7182 PAPAGIANNI ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 8. Time courses of glucosamine concentrations in fermentation broth. Fermentations carried out with optimal and suboptimal initial
concentrations of glucose and ammonium ions and with ammonium added as a single pulse.

mentation broth remained rather unchanged for a period had a more moderate effect. Mean filament lengths, as shown
following the maximum concentration point. In fermentations in Fig. 10 for the standard run and the 2.0-g/liter (NH4)2SO4
carried out with the 50-g/liter initial glucose concentration, pulse run, increased following (NH4)2SO4 addition, while vacuo-
glucosamine levels remained almost stable in the period be- lation levels were significantly reduced. Obviously, (NH4)2SO4
tween 22 h and 48 h of fermentation. When ammonium was

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supplied in the optimal concentration, no glucosamine was
detected beyond 72 h, while with 60% of optimal ammonium
ion concentration, no glucosamine was detected beyond 65 h of
fermentation. In these two runs, glucose was depleted by
around 70 h of fermentation. It appears from Fig. 8 that glu-
cosamine, a nitrogen storage compound, is synthesized early in
fermentation in amounts dependent on the initial concentra-
tions of glucose and ammonium, to be utilized by the fungus in
later stages. In a typical citric acid fermentation (standard
medium conditions), glucosamine is fully degraded by the fun-
gus in the period between 85 and 126 h.
Fed-batch cultures. Three runs were carried out in which
2.0, 1.0, and 0.50 g/liter (NH4)2SO4 were added at 75 h of
fermentation. Immediately after addition, production of citric
acid was much reduced, the effect being more pronounced with
the addition of 2.0 g/liter (NH4)2SO4 (Fig. 1), which almost
inhibited citrate synthesis until around 120 h. From that time
and for the rest of fermentation, citric acid production rates
appeared enhanced, as Fig. 1 shows, and final concentration
reached 90 g/liter. The microorganism’s very rapid response to
the addition of a supplementary nitrogen source is mirrored in
the specific growth rate time courses (Fig. 9). Immediately
FIG. 9. Time courses of the specific growth rate of Aspergillus niger
after the addition, specific growth rates increased sharply in the in standard run and in fermentations in which a single ammonium
case of 2.0 g/liter (NH4)2SO4 while lower levels of (NH4)2SO4 pulse was added.
VOL. 71, 2005 AMMONIUM IONS AND CITRIC ACID FERMENTATION BY A. NIGER 7183

FIG. 10. The morphological profile of Aspergillus niger mycelium in the standard run and with the addition of a 2-g/liter ammonium sulfate
pulse. Time courses of mean filament length and percentage of vacuolated filament volume are shown.

addition enhanced the formation of new cells from the tips of DISCUSSION
fragmented hyphae. (NH4)2SO4 addition at 75 h led to glu-
cosamine accumulation, as shown in Fig. 8, in amounts de- A successful citric acid fermentation with A. niger, apart
pending on the (NH4)2SO4 concentration of the pulse. Follow- from the suitable production medium, requires intensive mix-
ing ammonium addition, glucosamine concentration in the ing and aeration conditions. At 600 rpm applied in the present
broth remained at high and stable levels until 120 h, after case and an aeration rate of 1 vvm, after a lag period of some
which it degraded sharply. These results indicate that the newly 40 to 50 h, citric acid formation commenced (Fig. 1). The onset
formed mycelium utilizes glucosamine. of citrate accumulation in the broth was associated with a
Fungal morphology. A. niger grew in the form of clumps. It greatly increased glucose consumption rate (Fig. 6). At the end

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is the characteristic morphology of the particular strain under of the process, carried out with an initial glucose concentration
citric acid-producing conditions (12, 14). Morphological mea- of 150 g/liter, citric acid reached the high concentration of 110
surements were done by means of image analysis, and the time g/liter while biomass did not exceed 7.3 g/liter. Therefore, the
courses of morphology parameters, like mean perimeters of present system represents a successful citric acid-producing
clumps and mean lengths of filaments, were obtained. Mean system.
perimeters of mycelial clumps ranged between 800 ␮m and 500 It is well known that nitrogen in the medium must be lim-
␮m and declined steadily during the first half of fermentation iting in order to attain increased citric acid yields (5). However,
while appearing almost stable during the second half (results the process of ammonium ion uptake in A. niger has never been
not shown). Mean lengths of filament (L, ␮m) measurements closely studied, despite the importance of these ions to the
are presented in Fig. 10. According to Fig. 10, a decline in industrial production of citric acid using this fungus. Our study
mean filament length occurs at the same time when specific focused on the early fermentation stages and attempted an
growth rate increases. The volumes of vacuolated filaments investigation on the fate and role of these ions and the overall
were also recorded throughout fermentation, and the percent- dynamics of the system under production conditions. Results
age of the total vacuolated volume of the mycelium was cal- presented in Fig. 1 and 2 show that, by applying the optimum
culated and presented in Fig. 10. Also, 3.5% of the total vol- initial ammonium ion concentration in the medium, this has to
ume of the mycelium appears to be vacuolated at the end of be depleted before citric acid production establishes. The bulk
the process in the standard run, while the 2-g/liter pulse of of ammonium is removed from the broth between 20 and 25 h,
ammonium sulfate kept vacuolation at the very low levels of and it is almost depleted by 36 h. At that time, biomass is still
approximately 1.2%. The effect of the 2-g/liter pulse of ammo- less than 2 g/liter. Using a range of different concentrations of
nium sulfate on the mean length of filaments is shown in Fig. ammonium ions in the medium, we investigated the relation-
10. Along with lower vacuolation levels, an increase in mean ship between concentration and rate of uptake. It appeared
filament length immediately after ammonium addition was re- that the two are related and the relationship between the initial
corded, a situation reflected in the specific growth rate mea- ammonium ion concentration and the bulk uptake (20 to 25 h)
surements of Fig. 9. is linear (Fig. 3).
7184 PAPAGIANNI ET AL. APPL. ENVIRON. MICROBIOL.

The uptake of ammonium ions is followed by a release of tal data. To investigate the formation of the hypothetical prod-
protons. The protons released from the mycelium appear to be uct from nitrogen and carbon sources, it was decided that the
directly related to the initial ammonium ion concentration in fate of ammonium ions, not incorporated into biomass, could
fermentations carried out with up to and including 60% of the be altered by changing the yield of that product. The value for
optimal number of ammonium ions (Fig. 4). The relationship the yield coefficient for a simple hypothetical compound con-
appears to break down at higher initial concentrations, but this taining glucose and ammonium ions in equal molar amounts
is because the ammonium ions were not totally removed from was 100%. Figure 6 shows the plot of the calculated and ex-
the broth in the 80 and 100% fermentations until about 40 h. perimental glucose uptake rates, which appear in close corre-
Figure 5 does not show this extra time, as it is at approximately lation. Obviously, the results of the model indicate that am-
this time that acid production starts in fermentations with a monium ions combine with a carbon-containing metabolite
lower initial concentration of ammonium ions, confusing the inside the cell and that the most likely ratio of this combination
overall picture. Detailed time studies on the relationship be- is equivalent to 1 mol of glucose to one more mol of ammonia.
tween ammonium ion uptake and proton release carried out by The observed close correlation between the calculated and
Wayman (20) showed that the two are linked but only indi- experimental profiles of the glucose uptake rate indicates also
rectly. The release of protons into the broth does not coincide that the metabolic process combining the carbon and nitrogen
precisely with the uptake rate of ammonium ions but lags by a sources must be rapid and therefore must take place before the
couple of hours. This delay precludes a proton/ammonium carbon structure of the glucose has been greatly altered by
antiport as the means of ammonium ion uptake. Coincidently, glycolysis or the pentose phosphate pathway. There are no
peaks in the rate of ammonium uptake follow peaks in the reports in the literature on any kind of product of the described
growth rate by about 4 h. It would seem that a chain of events relationship between glucose and ammonium. Weight analysis
where growth leads to ammonium uptake leads to proton re- indicates that the product almost certainly is not being stored
lease is established in this early phase. However, one phenom- inside the cell and must be exported, possibly as an extracel-
enon that does coincide precisely with ammonium uptake is lular aminated polysaccharide. Following the modeling work,
glucose uptake, which is at such a high level that it must be which showed that glucose and ammonium are stoichiometri-
protein mediated (Fig. 6). cally linked with a ratio of 1:1, glucosamine was identified as a
If the ammonia were stored inside the biomass at the bulk potential nitrogen storage compound. Following synthesis in-
uptake period (20 to 25 h), just under 40% of the biomass side the cell, this must be removed from the cell. There are no
would be pure ammonia. Measurements of the pH inside the reports in the literature on the presence of glucosamine in
mycelium (unpublished data) (10) show that the internal pH is citric acid fermentation broths, but there is work that reported
slightly acidic under these conditions and so cannot contain the presence of loosely attached compounds (7). HPLC anal-
this amount of pure ammonia, which would also be highly ysis was set up in order to identify glucosamine, and it proved
toxic. This was confirmed by direct measurement of the inter- to be very satisfactory, as the compound was indeed identified
nal concentration, which showed that the internal concentra- to be glucosamine. The peaks appearing at about 8 min reten-
tion of ammonium ions was below detectable levels (less than tion time corresponded to glucosamine, while those obtained
1 mM/g dry weight) at 25 h. Other published work indicates at 12.5 min retention time were the glucose peaks (Fig. 7).
that the level of ammonium ions later in the fermentation is in Following detection of the aminated compound, the forma-

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the range of 10 to 30 mM/g dry weight (8). Dry weight and tion, release, and fate of glucosamine throughout fermentation
ammonia measurements in fermentations performed with were investigated in fermentations carried out with 150- and
complete medium show that the uptake of ammonia equiva- 50-g/liter initial concentrations of glucose and with the optimal
lents from the broth is very similar to the increase in biomass concentration of ammonium ions [production medium, 2.5 g/li-
in the 20- to 25-h period of fermentation (Fig. 5). As the ter (NH4)2SO4] and 60% of the optimum concentration. The
increase in biomass cannot be due to the accumulation of highest detected concentration of glucosamine was obtained
ammonia inside the biomass, a nitrogen compound must be when both glucose and ammonium ions were supplied at con-
produced and excreted by the mycelium. Urea is a simple centrations regarded as optimal for a citric acid production
molecule with a high carbon-to-nitrogen ratio, and it is the only medium: 150 g/liter glucose and 2.5 g/liter (NH4)2SO4. This
compound that could be used to store nitrogen within the was estimated to be 48 g/liter and noticed in samples taken at
biomass without greatly increasing its mass. It is produced 48 h (Fig. 8). From that point until 85 h, glucosamine appeared
during the deamination of amino acids by the ornithine cycle. quite stable in the broth but it reduced to zero levels at about
Although no reports have been found to indicate that A. niger 126 h of fermentation. When the concentrations of glucose and
possesses the ability to make urea, fermentations carried out ammonium were suboptimal, glucosamine formation was de-
with full medium and ammonium ion concentration were mea- tected at the same time; however, maximum concentrations
sured in the presence and absence of jack-bean urease. The obtained earlier compared with the standard run and these
measurements of ammonium ions did not vary with the treat- were significantly lower. Figure 8 presents the time courses of
ment of the samples with urease, and this demonstrates that glucosamine concentrations (g/liter) in the broths of all the
urea is not used by A. niger as a nitrogen storage compound. combinations between glucose and ammonium in the fermen-
Calculating the material balances for biomass, glucose, and tation medium. The conclusion drawn from these experiments
ammonium ions throughout the first 30 h of fermentation, we is that glucosamine is synthesized early in fermentation in
constructed a simple stoichiometric model. Using equations 1, amounts which depend on the availability of glucose and am-
2, and 3 as described in Results, the calculated values were monium in the medium. These amounts can be remarkably
compared with the measured values obtained from experimen- high, such as 48 g/liter, and they can be detected in the broth
VOL. 71, 2005 AMMONIUM IONS AND CITRIC ACID FERMENTATION BY A. NIGER 7185

for a period of approximately 40 h (between 48 and 85 h in productivity increased, along with an increase in the maximum
fermentation). Afterwards, glucosamine concentrations biomass concentration. These works did not include any mor-
sharply decrease to no detectable levels beyond 126 h. phological observations in the course of batch fermentation;
We showed in a number of previously published reports (12, however, the timing suggested for nitrogen supplementation is
13, 14, 15) that fungal morphology cannot be neglected in noteworthy, since the fermentation was carried out in a 12-liter
studies dealing with the biochemistry of a system. Instead, it is stirred tank reactor at 400 rpm with an initial sucrose concen-
related to fermentation rates and it affects overall productivi- tration of 140 g/liter, conditions which strongly indicate that a
ties directly or indirectly, through mass transfer phenomena process of mycelial fragmentation and regrowth took place. It
and perhaps other processes yet not fully understood. From would be interesting therefore to investigate the result and the
the morphology point of view, a fungal fermentation repre- final outcome of a single ammonium sulfate pulse upon glu-
sents a dynamic system with an ever-changing behavior. De- cosamine formation and the overall progress of a batch citric
tailed morphological studies of this particular system, using the acid fermentation.
standard medium with a 150-g/liter initial glucose concentra- Three runs were performed (standard medium) in which
tion, as published earlier (12, 13), revealed that in a range of ammonium sulfate was added as a single pulse at 75 h at
stirrer speeds between 400 and 600 rpm, the mycelium forms concentrations of 2.0, 1.0, and 0.50 g/liter. Immediately after
microscopic clumps, the mean perimeter of which reduces with addition, Aspergillus response was rapid: specific growth rates
increasing stirrer speeds, with the mean filament length fol- enhanced, while the synthesis of citrate was affected. The most
lowing the same trend. It has also been observed and described pronounced effects were noted with the 2-g/liter pulse, where
in detail (15) that under intensive agitation conditions, as in citrate synthesis was inhibited until around 120 h. From that
the present case, the mycelium undergoes a cycle of fragmen- time and for the rest of the run, production rates appeared
tation and regrowth at later stages. Mycelial fragmentation enhanced, as Fig. 1 shows, and final concentration reached 90
results from increased vacuolation, a natural process that g/liter. The effect was more moderate with the addition of
weakens the filaments and predisposes them to damage and lower ammonium sulfate concentrations. Mean filament
fragmentation due to mechanical stress. A combination of ob- lengths increased following the 2-g/liter pulse, and the vacuo-
servations that take place in the period between 72 and 126 h, lation levels were reduced significantly (Fig. 10). Obviously,
e.g., a drop of the mean diameter of vacuoles and a significant ammonium sulfate addition resulted in growth enhancement
drop in the percentage of vacuolated volume of filaments, and with the formation of new cells from the tips of fragmented
a reduction of the mean filament length along with an increase hyphae. Figure 8 shows the pulse results on glucosamine ac-
in the specific growth rate of the organism suggests that new cumulation and release. Glucosamine was accumulated in
mycelium is formed from the new tips resulting from fragmen- amounts depending on the (NH4)2SO4 of the pulse, the highest
tation (15). The degradation of glucosamine in the period concentration achieved being almost 53 g/liter in the case of
between 85 and 126 h strongly indicates that the mycelium the 2-g/liter pulse. This was detected at 102 h and remained at
utilizes it in building the cell walls of the newly formed hyphae. high levels until 120 h, to degrade sharply afterwards. The
Specific growth rate values, mean filament lengths, and the pulse experiments show that glucosamine can be synthesized
vacuolation profile, as presented in Fig. 9 and 10, show the and released to the broth when nitrogen is supplied in excess.
state of the mycelium and the time courses of a series of Investigating the fate and role of ammonium ions in the

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changes, the most important of which take place at the same early phase of the citric acid fermentation process, we come to
period when glucosamine depletion was observed. the conclusion that ammonium ions are not simply deposited
The natural process of vacuolation, and subsequent frag- inside the cell to make an ammonium pool but that they enter
mentation, is induced by low glucose levels, as experiments in the cell to combine with glucose and form glucosamine, which
batch and fed-batch cultures have shown (14, 15). This can is immediately released in the fermentation broth. Concerning
explain the earlier degradation of glucosamine as it appears in the biochemistry of citric acid accumulation by A. niger, the
runs starting with 50 g/liter glucose (Fig. 8). However, a clear situation described by Habison et al. (4) and Röhr and Kubicek
effect of the concentration of ammonium ions on the time (17) is generally accepted and no report has appeared so far to
course of glucosamine concentrations during fermentation can put it in question. According to them, a high intracellular
be observed in Fig. 8, comparing the plots obtained from fer- ammonium concentration, resulting from protein breakdown
mentations carried out with optimal and suboptimal concen- in manganese-deficient media, causes inhibition of the phos-
trations of ammonium ions. It appears so far that the fungus phofructokinase enzyme and leads to a flux through glycolysis
reacts in excess ammonium by converting it to glucosamine, a and the formation of citric acid. The slightly acidic intracellular
compound that will be utilized later in a regeneration process pH, the very low internal concentration of ammonium ions
depending always on the culture conditions. Citrate accumu- (about 1% of the external concentration), and the synthesis
lation commences with the exhaustion of nitrogen in the liquid and release of glucosamine described in the present case show
medium, and it is widely accepted that nitrogen limitation is a that the inhibition of phosphofructokinase is certainly not due
prerequisite for a successful citric acid process. However, a to increased concentrations of ammonium ions, and to this
very limited number of reports (2, 22) indicate that yields of point, the enzymatic profile may need to be realigned. The
citric acid in batch culture may be increased by the addition of existing relationship between high glucose and ammonium ion
nitrogen after the mycelial growth stage. Yigitoglou and Mc- concentrations from one side and the enzymes of phosphofruc-
Neil (22) reported as optimum addition time for nitrogen sup- tokinase, 2-oxoglutarate dehydrogenase, and the synthase of
plementation the range between 40 and 75 h: by supplement- glucosamine from the other side within the citric acid cycle
ing the culture with 0.5 g/liter (NH4)2SO4, fermentation certainly needs further investigation.
7186 PAPAGIANNI ET AL. APPL. ENVIRON. MICROBIOL.

Following the fate of ammonium ions in the citric acid fer- synthesis and citric acid accumulation by Aspergillus niger. Eur. J. Appl.
Microbiol. 4:167–175.
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ously reported as a by-product of the citric acid fermentation. studying the fine control of citric acid accumulation by Aspergillus niger.
Glucosamine synthesis and release into the broth can take Biotechnol. Lett. 1:47–52.
9. Kunst, A., B. Draeger, and J. Ziegenhom. 1986. Colorimetric methods with
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