Citric Acid
Content
Experiment 14
Citric acid production from food
waste materials by submerged
fermentation
Groups 8 & 9
Po
Reyes, A.
Reyes, C.
Saguinsin
Sehawanlop
Suelo
Tabajonda
Tiongson
What is Citric acid?
• Citric acid (IUPAC name:2-hydroxypropane-1,2,3-tricarboxylic
acid) is a weak organic acid that is found naturally in citrus
fruits. Citric acid has a formula of C6H8O7 and a molecular
weight of 192.12 g/mol. Citric acid is mainly used as an
acidifier in food and pharmaceutical industries and as a
chelating agent.
Fig1.1 Citric acid
• In biological processes, Citrate (conjugate base
of Citric acid) is an intermediate in the Citric
acid cycle and therefore occurs in the
metabolism of almost all living things.
Fig 1.2 Citric acid cycle
• The 19th century citric acid was produced by extraction
from citrus fruits. By the 1890's demand for this versatile
material had increased to the extent that supply had
become a problem. In addition, its quality could not be
guaranteed.
• In 1892, C. Wehmer, reader in organic chemistry at the
University of Hannover, discovered that citric acid could be
produced by certain moulds. This paved the way for large
scale production of citric acid by microorganisms as World
War I disrupted Italian citrus exports and alternative
means to produce Citric acid are needed.
• It was not until the 1920's that researchers in the US and
Europe were able to perfect the large-scale, commercial
production of citric acid by the microorganism Aspergillus
niger, a mould belonging to the same family as the
penicillins.
Aspergillus niger
• Aspergillus niger or A. niger is a fungus and one of
the most common species of the genus Aspergillus.
• A. niger has been a very important microbe used in
the field of biotechnology. Many of the enzymes
produced by A. niger, such as citric acid, amylases,
lipases, cellulases and proteases, are essential
because of its importance for transformation to
food enzymes.
• Other properties of this species include pathogens
that cause the spoilage of food and production of
secondary metabolites, such as aflatoxin, that are
toxic. A. niger has also been used for waste
treatment and chemical modifications known as
biotransformations.
• About 99% of world production of citric acid
occurs via microbial processes, which can be
carried out using surface or submerged
cultures. The product is sold as an anhydrous
or monohydrate acid. Total production of citric
acid per year is 1.5 million tons.
• 70% of total Citric acid production is used in
food and beverage industry
– Used as acidifier or antioxidant
• 20% is used in the pharmaceutical industry
– Used as an antioxidant for vitamin preservation,
pH corrector, blood preservative
• 10% used in the chemical industry
– Used as a foaming agent, phosphate substitute for
detergents
Fig 1.3 Aspergillus niger culture
• A. niger can produce Citric acid from glucose through the
Citrate formation pathway.
• Following glycolysis, pyruvate undergoes one of two equally
important processes, one in the cytosol, and one in the
mitochondrion. Pyruvate entering the mitochondrion is
converted to acetyl coenzyme A, while pyruvate remaining in
the cytosol becomes converted to oxaloacetate in a
carboxylation reaction catalyzed by pyruvate carboxylase.
• Under the cytosolic redox conditions provided by glycolytic
NADH, oxaloacetate is then converted to malate by the
enzyme malate dehydrogenase. Mitochondrial membrane
transfer can now occur via a citrate/malate antiport referred
to as Tricarboxylate Transporter (TCT) (Kubicek and Karaffa
(2003)).
• TCT competes with aconitase for citrate, thus removing
significant TCA intermediates, and resulting in an influx of
malate to the mitochondria coupled with the equivalent
export of citrate. This system allows acetyl coenzyme A and
oxaloacetate to accumulate in near equal amounts at the end
of glycolysis. Citrate synthase compresses the two molecules
into citrate, which is then exported by TCT. This export of
citrate does not completely obstruct the TCA cycle.
Objectives
• Demonstrate bench-scale liquid fermentation
of food waste materials for citric acid
production that will help solve pollution
problems.
• Compare the effects of using chemicallydefined and complex media using food waste
materials.
• Compare fermentation efficiency between
spore inoculation and mycelium inoculation.
Methodology
• Inoculum preparation
.
.
.
• Prepare spore suspension using A. niger grown on PDA plates then
incubate for 5 days.
• After incubation, wash the plates with 0.1% Tween 80 solution to collect
spores. Collect in sterile tubes.
• Count the number of spores and use 0.1 mL of this suspension as
inoculum.
• Prepare intact mycelium.
• From the agar plates, inoculate the discs in an Erlenmeyer flask containing
PDA broth.
• Incubate for 72 hours at 30OC untile the mycelia mat develops.
• Substrate preparation
Prepare
saccharification and
fermentation
medium ( coconut
water, banana peel,
orange peel, orange
pulp, calamansi peel,
calamansi pulp) using
distilled water and
5% substrate (pulp).
Prepare six
Erlenmeyer flasks
contaning 5g/100mL
of the respective
substrates.
• Simultaneous Saccharification and Fermentation
Autoclave the substrate
solutions in a 250 mL
erlenmeyer flask
Estimate the initial
amount of sugar by
using Somogyi-Nelson’s
test
Check the efficiency of
intact mycelium and
spores of A. niger in the
fermentation of Citric
acid (titration)
Every 48 hours, collect
the flasks and estimate
citric acid concentration
by titration with 0.1M
NaOH
Incubate 30oC for 6
days
Inoculate 1mL of spore
suspension to all
substrates
Also, estimate the left
over reducing sugar
concentration by
Somogyi-Nelson’s test
Inoculate 1 intact
mycelia (7 cm in
diameter) as the
inoculum to a new set
of substrates and
repeat the procedure.
Somogyi-Nelson’s test (quantitative
determination of reducing sugars)
Prepare Nelson's
reagent. (12.5 mL
Nelson's A with 0.5 mL
Nelson's B)
Label 7 test tubes and
transfer measured
amounts of standard
glucose solution into
the test tubes.
Add 1.0 mL Nelson's
reagent to each tube
and shake well.
Read the absorbance of
the standards and
unknown against a
reagent blank at 480
nm.
Add 1.0 mL of
arsenomolybdate
reagent to ech tube
and shake occasionally.
Heat tubes in boiling
water for 20 minutes
then cool in a beaker of
water.
Construct a glucose
standard curve by
plotting absrorbance
readings against
concentrations of
standard solutions.
Determine the
concentration of the
unkonwn in mg/tube
and mg/mL.
Results and Discussion
1.2
Glucose standard curve
1
0.8
0.6
Glucose standard curve
0.4
Linear (Glucose standard curve)
y = 10.632x
R² = 0.8311
0.2
0
0
0.02
0.04
0.06
0.08
0.1
0.12
• Figure 3.1 Glucose standard curve
Table 3.1 Concentration of reducing sugars
(spore inoculum=9.0x10-6/mL, inoculum size:9.0x10-6
spores)
Substrate
Day 0
Day 2
Day 4
Day 6
Coconut water
1.68 mg/mL
1.12 mg/mL
0.591 mg/mL 0.319 mg/mL
Banana peel
1.45 mg/mL
0.964 mg/mL
1.29 mg/mL 0.536 mg/mL
Calamansi Peel
1.63 mg/mL
1.09 mg/mL
1.42 mg/mL 0.799 mg/mL
Calamansi Pulp
1.61 mg/mL
1.08 mg/mL
1.53 mg/mL 0.460 mg/mL
Orange Peel
1.70 mg/mL
1.13 mg/mL
1.59 mg/mL 0.902 mg/mL
Orange Pulp
1.722 mg/mL
1.14 mg/mL
1.55 mg/mL 0.686 mg/mL
Fig 3.2 Concentration of reducing
sugar over time (spore inoculum)
2
1.8
1.6
Concentration (mg/mL)
1.4
Coconut juice
1.2
Banana peel
1
Calamansi peel
Calamansi pulp
0.8
Orange peel
Orange pulp
0.6
0.4
0.2
0
0
1
2
3
Days
4
5
6
7
Table 3.2 Concentration of reducing sugars
(mycelium inoculum)
Substrate
Coconut water
Banana peel
Day 0
Day 2
Day 4
Day 6
1.805 mg/mL 1.382 mg/mL 1.091 mg/mL 0.479 mg/mL
1.401mg/mL
1.439 mg/mL 0.921 mg/mL 0.451 mg/mL
Calamansi Peel
1.344 mg/mL 2.097 mg/mL 1.344 mg/mL 0.592 mg/mL
Calamansi Pulp
1.260 mg/mL 2.069 mg/mL 1.269 mg/mL 0.573 mg/mL
Orange Peel
0.150 mg/mL 2.793 mg/mL 1.457 mg/mL 0.677 mg/mL
Orange Pulp
1.486 mg/mL 3.395 mg/mL 2.323 mg/mL 0.846 mg/mL
Fig 3.3 Concentration of reducing
sugar over time (mycelium inoculum)
Concentration of reducing sugar over time
4
Concentration (mg/mL)
3.5
3
2.5
Coconut juice
Banana peel
2
Calamansi peel
1.5
Calamansi pulp
Orange peel
1
Orange pulp
0.5
0
0
1
2
3
Days
4
5
6
7
Table 3.3 Concentration of Citric
acid over time (spore inoculum)
Substrate
Day 2
Day 4
Day 6
Coconut water
0.128 mg/mL
0.154 mg/mL
0.256 mg/mL
Banana peel
0.256 mg/mL
0.320 mg/mL
0.384 mg/mL
Calamansi Peel
0.320 mg/mL
0.512 mg/mL
0.589 mg/mL
Calamansi Pulp
0.518 mg/mL
0.538 mg/mL
0.569 mg/mL
Orange Peel
0.269 mg/mL
0.487 mg/mL
0.653 mg/mL
Orange Pulp
0.640 mg/mL
0.833 mg/mL
0.884 mg/mL
Fig 3.4 Concentration of Citric acid over
time (spore inoculum)
1
0.9
0.8
0.7
mg/mL Citric acid
0.6
Coconut juice
Banana peel
0.5
Calamansi Peel
Calamansi Pulp
0.4
Orange Peel
0.3
Orange Pulp
0.2
0.1
0
0
1
2
3
4
Days
5
6
7
Table 3.4 Concentration of Citric
acid over time (mycelium inoculum)
Substrate
Day 2
Day 4
Day 6
Coconut juice
0.077 mg/mL
0.102 mg/mL
0,167 mg/mL
Banana peel
0.043 mg/mL
0.107 mg/mL
0.162 mg/mL
Calamansi Peel
0.085 mg/mL
0.171 mg/mL
0.243 mg/mL
Calamansi Pulp
0. 99 mg/mL
0.153 mg/mL
0.177 mg/mL
Orange Peel
0.043 mg/mL
0.162 mg/mL
0.243 mg/mL
Orange Pulp
0.085 mg/mL
0.278 mg/mL
0.346 mg/mL
Fig 3.5 Concentration of Citric acid
over time (mycelium inoculum)
0.4
0.35
0.3
0.25
mg/mL Citric acid
Coconut juice
Banana peel
0.2
Calamansi Peel
Calamansi Pulp
0.15
Orange Peel
Orange Pulp
0.1
0.05
0
0
1
2
3
4
Days
5
6
7
Table 3.5 Comparison of Citric
acid yields
Substrate
% Yield spore % Yield mycelium
at day 6
at day 6
Coconut juice
1.53%
3.00%
Banana peel
2.30%
2.92%
Calamansi Peel
3.54%
4.38%
Calamansi Pulp
3.41%
3.18%
Orange Peel
3.92%
4.38%
Orange Pulp
5.30%
6.22%
Conclusions
The concentration of reducing sugars increases at
day 2 to day 4 for spore inoculated substrates and
day 0 to day 2 for mycelium inoculated substrates.
This is can be attributed to the mycelium breaking
down the complex carbohydrates of the substrates
into simple sugars.
Conclusions
• The amount of sugar in Coconut water kept on
decreasing unlike the other media because its
main composition is mostly simple sugars, trace
amounts of fats and protein and water unlike the
other
substrates
which
has
complex
carbohydrates that can be degraded into sugars.
Conclusions
• Mycelium inoculated substrates have a higher
yield of Citric acid at day 6 than spore inoculated
substrate. This is because the biochemical
processes for Citric acid synthesis occurs at the
mycelium. Synthesis and accumulation of Citric
acid in spore inoculated substrates is generally
slower than the mycelium inoculated substrates
as it requires some time to develop its mycelium.
References
• Kubicek and Karaffa (2003), Aspergillus niger
citric acid accumulation: do we understand this
well working black box?, Appl Microbial
Biotechnol 61:189-196
• N.Torres and D.Guebel (2001), Optimization of
the citric acid production by Aspergillus niger
through a metabolic flux balance model,
Electronic Journal of Biotechnology 4: No.1, April
15, 2001
Citric acid production from food
waste materials by submerged
fermentation
Groups 8 & 9
Po
Reyes, A.
Reyes, C.
Saguinsin
Sehawanlop
Suelo
Tabajonda
Tiongson
What is Citric acid?
• Citric acid (IUPAC name:2-hydroxypropane-1,2,3-tricarboxylic
acid) is a weak organic acid that is found naturally in citrus
fruits. Citric acid has a formula of C6H8O7 and a molecular
weight of 192.12 g/mol. Citric acid is mainly used as an
acidifier in food and pharmaceutical industries and as a
chelating agent.
Fig1.1 Citric acid
• In biological processes, Citrate (conjugate base
of Citric acid) is an intermediate in the Citric
acid cycle and therefore occurs in the
metabolism of almost all living things.
Fig 1.2 Citric acid cycle
• The 19th century citric acid was produced by extraction
from citrus fruits. By the 1890's demand for this versatile
material had increased to the extent that supply had
become a problem. In addition, its quality could not be
guaranteed.
• In 1892, C. Wehmer, reader in organic chemistry at the
University of Hannover, discovered that citric acid could be
produced by certain moulds. This paved the way for large
scale production of citric acid by microorganisms as World
War I disrupted Italian citrus exports and alternative
means to produce Citric acid are needed.
• It was not until the 1920's that researchers in the US and
Europe were able to perfect the large-scale, commercial
production of citric acid by the microorganism Aspergillus
niger, a mould belonging to the same family as the
penicillins.
Aspergillus niger
• Aspergillus niger or A. niger is a fungus and one of
the most common species of the genus Aspergillus.
• A. niger has been a very important microbe used in
the field of biotechnology. Many of the enzymes
produced by A. niger, such as citric acid, amylases,
lipases, cellulases and proteases, are essential
because of its importance for transformation to
food enzymes.
• Other properties of this species include pathogens
that cause the spoilage of food and production of
secondary metabolites, such as aflatoxin, that are
toxic. A. niger has also been used for waste
treatment and chemical modifications known as
biotransformations.
• About 99% of world production of citric acid
occurs via microbial processes, which can be
carried out using surface or submerged
cultures. The product is sold as an anhydrous
or monohydrate acid. Total production of citric
acid per year is 1.5 million tons.
• 70% of total Citric acid production is used in
food and beverage industry
– Used as acidifier or antioxidant
• 20% is used in the pharmaceutical industry
– Used as an antioxidant for vitamin preservation,
pH corrector, blood preservative
• 10% used in the chemical industry
– Used as a foaming agent, phosphate substitute for
detergents
Fig 1.3 Aspergillus niger culture
• A. niger can produce Citric acid from glucose through the
Citrate formation pathway.
• Following glycolysis, pyruvate undergoes one of two equally
important processes, one in the cytosol, and one in the
mitochondrion. Pyruvate entering the mitochondrion is
converted to acetyl coenzyme A, while pyruvate remaining in
the cytosol becomes converted to oxaloacetate in a
carboxylation reaction catalyzed by pyruvate carboxylase.
• Under the cytosolic redox conditions provided by glycolytic
NADH, oxaloacetate is then converted to malate by the
enzyme malate dehydrogenase. Mitochondrial membrane
transfer can now occur via a citrate/malate antiport referred
to as Tricarboxylate Transporter (TCT) (Kubicek and Karaffa
(2003)).
• TCT competes with aconitase for citrate, thus removing
significant TCA intermediates, and resulting in an influx of
malate to the mitochondria coupled with the equivalent
export of citrate. This system allows acetyl coenzyme A and
oxaloacetate to accumulate in near equal amounts at the end
of glycolysis. Citrate synthase compresses the two molecules
into citrate, which is then exported by TCT. This export of
citrate does not completely obstruct the TCA cycle.
Objectives
• Demonstrate bench-scale liquid fermentation
of food waste materials for citric acid
production that will help solve pollution
problems.
• Compare the effects of using chemicallydefined and complex media using food waste
materials.
• Compare fermentation efficiency between
spore inoculation and mycelium inoculation.
Methodology
• Inoculum preparation
.
.
.
• Prepare spore suspension using A. niger grown on PDA plates then
incubate for 5 days.
• After incubation, wash the plates with 0.1% Tween 80 solution to collect
spores. Collect in sterile tubes.
• Count the number of spores and use 0.1 mL of this suspension as
inoculum.
• Prepare intact mycelium.
• From the agar plates, inoculate the discs in an Erlenmeyer flask containing
PDA broth.
• Incubate for 72 hours at 30OC untile the mycelia mat develops.
• Substrate preparation
Prepare
saccharification and
fermentation
medium ( coconut
water, banana peel,
orange peel, orange
pulp, calamansi peel,
calamansi pulp) using
distilled water and
5% substrate (pulp).
Prepare six
Erlenmeyer flasks
contaning 5g/100mL
of the respective
substrates.
• Simultaneous Saccharification and Fermentation
Autoclave the substrate
solutions in a 250 mL
erlenmeyer flask
Estimate the initial
amount of sugar by
using Somogyi-Nelson’s
test
Check the efficiency of
intact mycelium and
spores of A. niger in the
fermentation of Citric
acid (titration)
Every 48 hours, collect
the flasks and estimate
citric acid concentration
by titration with 0.1M
NaOH
Incubate 30oC for 6
days
Inoculate 1mL of spore
suspension to all
substrates
Also, estimate the left
over reducing sugar
concentration by
Somogyi-Nelson’s test
Inoculate 1 intact
mycelia (7 cm in
diameter) as the
inoculum to a new set
of substrates and
repeat the procedure.
Somogyi-Nelson’s test (quantitative
determination of reducing sugars)
Prepare Nelson's
reagent. (12.5 mL
Nelson's A with 0.5 mL
Nelson's B)
Label 7 test tubes and
transfer measured
amounts of standard
glucose solution into
the test tubes.
Add 1.0 mL Nelson's
reagent to each tube
and shake well.
Read the absorbance of
the standards and
unknown against a
reagent blank at 480
nm.
Add 1.0 mL of
arsenomolybdate
reagent to ech tube
and shake occasionally.
Heat tubes in boiling
water for 20 minutes
then cool in a beaker of
water.
Construct a glucose
standard curve by
plotting absrorbance
readings against
concentrations of
standard solutions.
Determine the
concentration of the
unkonwn in mg/tube
and mg/mL.
Results and Discussion
1.2
Glucose standard curve
1
0.8
0.6
Glucose standard curve
0.4
Linear (Glucose standard curve)
y = 10.632x
R² = 0.8311
0.2
0
0
0.02
0.04
0.06
0.08
0.1
0.12
• Figure 3.1 Glucose standard curve
Table 3.1 Concentration of reducing sugars
(spore inoculum=9.0x10-6/mL, inoculum size:9.0x10-6
spores)
Substrate
Day 0
Day 2
Day 4
Day 6
Coconut water
1.68 mg/mL
1.12 mg/mL
0.591 mg/mL 0.319 mg/mL
Banana peel
1.45 mg/mL
0.964 mg/mL
1.29 mg/mL 0.536 mg/mL
Calamansi Peel
1.63 mg/mL
1.09 mg/mL
1.42 mg/mL 0.799 mg/mL
Calamansi Pulp
1.61 mg/mL
1.08 mg/mL
1.53 mg/mL 0.460 mg/mL
Orange Peel
1.70 mg/mL
1.13 mg/mL
1.59 mg/mL 0.902 mg/mL
Orange Pulp
1.722 mg/mL
1.14 mg/mL
1.55 mg/mL 0.686 mg/mL
Fig 3.2 Concentration of reducing
sugar over time (spore inoculum)
2
1.8
1.6
Concentration (mg/mL)
1.4
Coconut juice
1.2
Banana peel
1
Calamansi peel
Calamansi pulp
0.8
Orange peel
Orange pulp
0.6
0.4
0.2
0
0
1
2
3
Days
4
5
6
7
Table 3.2 Concentration of reducing sugars
(mycelium inoculum)
Substrate
Coconut water
Banana peel
Day 0
Day 2
Day 4
Day 6
1.805 mg/mL 1.382 mg/mL 1.091 mg/mL 0.479 mg/mL
1.401mg/mL
1.439 mg/mL 0.921 mg/mL 0.451 mg/mL
Calamansi Peel
1.344 mg/mL 2.097 mg/mL 1.344 mg/mL 0.592 mg/mL
Calamansi Pulp
1.260 mg/mL 2.069 mg/mL 1.269 mg/mL 0.573 mg/mL
Orange Peel
0.150 mg/mL 2.793 mg/mL 1.457 mg/mL 0.677 mg/mL
Orange Pulp
1.486 mg/mL 3.395 mg/mL 2.323 mg/mL 0.846 mg/mL
Fig 3.3 Concentration of reducing
sugar over time (mycelium inoculum)
Concentration of reducing sugar over time
4
Concentration (mg/mL)
3.5
3
2.5
Coconut juice
Banana peel
2
Calamansi peel
1.5
Calamansi pulp
Orange peel
1
Orange pulp
0.5
0
0
1
2
3
Days
4
5
6
7
Table 3.3 Concentration of Citric
acid over time (spore inoculum)
Substrate
Day 2
Day 4
Day 6
Coconut water
0.128 mg/mL
0.154 mg/mL
0.256 mg/mL
Banana peel
0.256 mg/mL
0.320 mg/mL
0.384 mg/mL
Calamansi Peel
0.320 mg/mL
0.512 mg/mL
0.589 mg/mL
Calamansi Pulp
0.518 mg/mL
0.538 mg/mL
0.569 mg/mL
Orange Peel
0.269 mg/mL
0.487 mg/mL
0.653 mg/mL
Orange Pulp
0.640 mg/mL
0.833 mg/mL
0.884 mg/mL
Fig 3.4 Concentration of Citric acid over
time (spore inoculum)
1
0.9
0.8
0.7
mg/mL Citric acid
0.6
Coconut juice
Banana peel
0.5
Calamansi Peel
Calamansi Pulp
0.4
Orange Peel
0.3
Orange Pulp
0.2
0.1
0
0
1
2
3
4
Days
5
6
7
Table 3.4 Concentration of Citric
acid over time (mycelium inoculum)
Substrate
Day 2
Day 4
Day 6
Coconut juice
0.077 mg/mL
0.102 mg/mL
0,167 mg/mL
Banana peel
0.043 mg/mL
0.107 mg/mL
0.162 mg/mL
Calamansi Peel
0.085 mg/mL
0.171 mg/mL
0.243 mg/mL
Calamansi Pulp
0. 99 mg/mL
0.153 mg/mL
0.177 mg/mL
Orange Peel
0.043 mg/mL
0.162 mg/mL
0.243 mg/mL
Orange Pulp
0.085 mg/mL
0.278 mg/mL
0.346 mg/mL
Fig 3.5 Concentration of Citric acid
over time (mycelium inoculum)
0.4
0.35
0.3
0.25
mg/mL Citric acid
Coconut juice
Banana peel
0.2
Calamansi Peel
Calamansi Pulp
0.15
Orange Peel
Orange Pulp
0.1
0.05
0
0
1
2
3
4
Days
5
6
7
Table 3.5 Comparison of Citric
acid yields
Substrate
% Yield spore % Yield mycelium
at day 6
at day 6
Coconut juice
1.53%
3.00%
Banana peel
2.30%
2.92%
Calamansi Peel
3.54%
4.38%
Calamansi Pulp
3.41%
3.18%
Orange Peel
3.92%
4.38%
Orange Pulp
5.30%
6.22%
Conclusions
The concentration of reducing sugars increases at
day 2 to day 4 for spore inoculated substrates and
day 0 to day 2 for mycelium inoculated substrates.
This is can be attributed to the mycelium breaking
down the complex carbohydrates of the substrates
into simple sugars.
Conclusions
• The amount of sugar in Coconut water kept on
decreasing unlike the other media because its
main composition is mostly simple sugars, trace
amounts of fats and protein and water unlike the
other
substrates
which
has
complex
carbohydrates that can be degraded into sugars.
Conclusions
• Mycelium inoculated substrates have a higher
yield of Citric acid at day 6 than spore inoculated
substrate. This is because the biochemical
processes for Citric acid synthesis occurs at the
mycelium. Synthesis and accumulation of Citric
acid in spore inoculated substrates is generally
slower than the mycelium inoculated substrates
as it requires some time to develop its mycelium.
References
• Kubicek and Karaffa (2003), Aspergillus niger
citric acid accumulation: do we understand this
well working black box?, Appl Microbial
Biotechnol 61:189-196
• N.Torres and D.Guebel (2001), Optimization of
the citric acid production by Aspergillus niger
through a metabolic flux balance model,
Electronic Journal of Biotechnology 4: No.1, April
15, 2001
