Biological Products Engineering

Semiannual Project: Production of itaconic acid by fermentation

Granados Gonzlez Jos Guadalupe, Negrete Bosques Nora Liliana, Oate Robledo Hugo
Instituto Politcnico Nacional. Unidad Profesional Interdisciplinaria de Ingeniera Campus
Guanajuato
Biological Products Laboratory
8FM1
Summary
In this project, itaconic acid was obtained from Aspergillus niger fungus in liquid medium. the methodology
was adapted for peniclilina, demonstrating the effectiveness of this culture medium for growing A. niger.
sensidiscs tests were performed to verify the inhibitory potency of itaconic acid in 3 bacteria and results were
compared to Ampicillin.
Abstract
Obtained from the fungus Aspergillus niger as antibiotic itaconic acid, being the product of a fermentation
process in a bioreactor, in order to determine the fermentation process of a biopharmaceutical product.
Emphasis
Develop an experimental protocol for itaconic acid as the fermentation product from the fungus
Aspergillus niger growth.
Determine the parameters of growth of the fungus Aspergillus niger necessary to, through a
fermentation process to produce itaconic acid as an antibiotic.
Quantify the minimum inhibitory concentration of itaconic acid obtained from the fungus Aspergillus
niger by an airlift bioreactor fermentation (or the simulation in a flask) using an antibiogram, to
verify their antibiotic effectiveness.
Recognize the importance of biological products and to design an appropriate methodology for
collection.
Introduction. The first system was the penicillin
production method known as surface where the
fungus grew on the surface of a layer of culture
medium in trays. But after 1944, the development
of submerged fermentation method helped to
reduce space requirements and consequently
production costs (ArgenBio, 2011). Then
processes for penicillin production are described.
Surface Process (surface method). In this
process the growth of the microorganism is
carried out as a pad on the surface of a liquid
medium, for it trays or flasks are used, on the
surface of a finely fragmented wet solid substrate,
e.g., wood chips or wheat bran.
Process immersion (submerged fermentation
method). The culture medium for fermentation is

basically composed of a corn broth with the

addition of lactose and inorganic compounds.
After adjusting the pH in a range of 4.5-5.0, the
culture medium is passed to the fermentor
equipped with an overhead stirrer and an injection
system of sterile air and streamers to maintain the
temperature between 23 and 25 C. The fungus
enters sterile and fermentation begins, during
which the sterile air allows the growth of the
fungus and agitation facilitates its distribution in
the fermenter. After about 50 to 90 hours the rate
of growth of the fungus decreases, the fermenter is
cooled to 5 C to prevent destabilization of the
antibiotic and the fungus is filtered. Penicillin
subsequently
extracted
using
solvents,

Biological Products Engineering

concentrated, sterilized by filtration and then the

product is crystallized and packed.
Acid production "alkylitaconic" by Aspergillus
niger. During the investigation of new antibiotics,
6 new compounds known as acids "tensyuic",
isolated from the culture broth of Aspergillus
niger, the structures of these acids from A to F,
known as the family of itaconic acid were
detected.
Itaconic acid (IA) is a promising organic acid. It is
a crystalline unsaturated dicarbonic acid white in
which a carboxyl group is conjugated to the
methylene group. IA is used worldwide in the
industrial synthesis of resins such as polyesters,
plastics and synthetic glass and in the preparation
of bioactive compounds in agriculture, pharmacy
and medicine, Matsumaru et al, (2008).
In recent years it has been reported antimicrobial
activity and anticancer by "tensyuic" acids,
identified as antibiotics against Bacillus subtilis
with moderate concentration.
In general, the production of itaconic acid by
Aspergillus terreus depends on the temperature
(30-40 C), continuous aeration, starting low pH
(3-5), lower operating pH (2.2 to 3.8), glucose
concentrations (10- 20%), sufficient nitrogen, high
concentration of magnesium sulphate (0.5%),
phosphate low to limit the growth of mycelium
and adequate levels of trace metals, zinc, copper
and iron, Jury (2010).

Suplies and Substances.

Suplies
Bacteriological loop
250 mL Erlenmeyer
flask
Beaker 500 mL
Glass funnel
Glass pipette 5 mL
Latex hoses
1000 mL flask
Kitazato
Bunsen burner
Glass petri dishes
Micropipettes 1001000 L and 20-200
L
Tips micropipette
100-1000 L and 20200 L
Dissecting forceps
Gauze pad
Tape witness
Cotton
Parafilm
Filter paper
500 mL Erlenmeyer
flask
Beaker 250 mL
Foil

Equipment
Autoclave
Ice machine
Grill heating with
stirring
Incubator
-

Substances

NH

K 2 HPO 4
Figure 1. Family of itaconic acid

NaH 2 PO 4
MgSO47 H 2 O
KCl

CaCl2
ZnSO 47 H 2 O

Biological Products Engineering

MnSO44 H 2 O
CuSO 45 H 2 O

according to the nutritional requirements of the

fungus occurs.
Table 1. Defined medium in which A. niger can grow.

FeSO 47 H 2 O
Na 2 SO 4
Glucose
Distilled wter
Cultures of different bacteria
Aspergillus niger

Procedure. For the production of itaconic acid as

commercially speaking (Najafpour, 2007) must
take into account a number of steps:
1. Preparation of inoculum.
2. Preparation and sterilization of the medium.
3. Inoculation medium in the fermenter.
4. forced aeration with sterile air during
fermentation.
5. Elimination of the mycelium from the culture
medium after fermentation.
6. Purification of itaconic acid.
To produce appropriately itaconic acid is
necessary to use a bioreactor, it should contain the
culture medium with the requirements for the
growth of A. niger. Optimal conditions for fungal
growth are: temperature 30-35 C, in a range vvm
and pH 0.5-1 3.0. (Najafpour, 2007).
Culture medium. Filamentous fungi as is A.
niger are able to grow in simple defined media.
Microorganisms of this type used for developing a
wide range of compounds such as carbon,
nitrogen and energy sources, so using various
sugars such as polysaccharides, amino acids and
organic acids lipids. They can use amino acids as
the sole source of carbon and nitrogen. Amino
acid metabolism intermediates to carbon are
degraded by one or two steps are the best sources
that require multiple steps. (Nielsen, 1997)
Nielsen shows the following table in which a list
of components that can be included in the culture
medium for development of A. niger I press,

For optimal development of A. niger (Gonzalez,

2010) proposes the culture medium Czapek (Cz),
which is composed of Sucrose, NaNO3, K2HPO4,
MgSO4 x 7 H2O, FeSO4 x 7 H2O and distilled
water, the glucose the sole carbon source and
ammonium sulfate who provides the means of
nitrogen; given the requirements of the
microorganism such means can be modified
according to the arrangements of the components
supporting table 2 serves as support for select
components of the culture medium proposed for
the fermentation process.
This medium is recommended for the
development and isolation of fungi such as A.
niger growth in this medium is excellent so it was
a suitable option.
For medium preparation follows the following
methodology:
1. Weigh each one of the components according to
the amount to be prepared following the ratio of
the amount of each component per liter of
medium (Table 1)
2. Mix components.
3. Add ingredients to an Erlenmeyer flask with the
necessary amount of water, heated with stirring to
boiling for 1 min
4. Autoclave at 121 C, 15 lb pressure for 15
minutes
5. Cool.
Propagation of A. niger. First we need to know if
the medium described in Table 1 is capable of
growing A. niger therefore must prepare two petri
dishes with glass this culture medium and some
nutrient agar to make it grow.

Biological Products Engineering

Preparation and mounted of the biorreactor.

The bioreactor is a stirred tank used because it
allows the optimal development of S. niger
because the proper growth conditions for the
fungus are set forth therein; 30 C- 35 C, pH
3.0-3.5 and 0.5-1 vvm of (Atkinson, 1986) in
addition to these conditions can be monitored
throughout the process. This type of bioreactor
provides the necessary agitation in order to
solubilize the amount of oxygen required by the
fungus for optimal growth. Also the mixed liquid
is produced by the liquid flow due to the rise of air
bubbles. (Gurrola Vazquez et al, 2007)
For the preparation and mounted equipment
should follow the following process:
1. Remove and wash.
2. Sterilize:
a) Through a chemical process
b) Using hot steam
c) In an autoclave, for it after disassembling the
bioreactor and then the plastic craft paper wrapped
pieces are placed in an autoclave at 121 C, 15 lb
for 15 minutes and then allowed to cool.
3. Put together properly.
4. Connect to a power source and turn
5. To establish conditions for microorganism
growth
6. Add culture medium and inoculate, this requires
emptying A. niger in the middle taking care cause
environmental pollution.
7. Keep the growing conditions for the necessary
time.
Method for testing the antibiotic activity of
itaconic acid. Perform filter paper discs with a
hole punch, placing 15 discs filter paper in a glass
petri dish, in total do 5 petri dishes with their
respective disks (one box per team), wrap in foil
box. Sterilize at 121 C for 15 min. After
sterilizing dry boxes in an oven at 80-90 C.
Stored until use in a dry place.
1. Preparation of the test plates
Spending 0.1 ml of bacterial culture LB agar
plate, spread over the entire surface with the
extension triangle, let stand inoculated plates at
room temperature.
2. Preparation of disks.
Place 5 discs in a sterile Petri dish. It impregnate
the filter paper discs 5 with the extract obtained.

Let dry the discs on every lap to place the next

volume of extract. At the end you will have 5
disks with different amount of concentrated
extract. Let dry discs.
3. Checking the antibiotic action itaconic acid
extract.
4. Place the discs prepared in the previous step on
the test plates made in step 1, using sterilized
tweezers flame, place 5 discs as shown in the
following figure.
5. Label plates perfectly with the microorganism,
date, group and project. Incubate assay plates at
37 C for 24 hrs.
6. Control of the plates. itaconic acid activity is
evidenced by the appearance of halos of inhibition
around the discs.

Results. 2nd May session. In the first session, the

culture medium in which we would grow to A.
niger was prepared. The culture medium was
prepared as described in Table 1. 800 mL of liquid
culture medium were prepared and also two glass
petri dishes with the same medium but were added
nutrient agar.
one Kitazato 1000 mL flask was mounted with a
sterile surgical hose to the air inlet, and was added
800 mL of liquid culture medium. At the entrance
of the hose stopper he was placed sterile cotton to
avoid environmental contamination. Figure 2
shows the Kitazato flask containing the medium
of sterile liquid culture.

Figure 2. Kitazato flask with liquid culture mdium

(bioreactor).

Biological Products Engineering

May 4th session. In the second session, Professor

Garibay provided a strain of A. niger dated 2014,
which was inoculated in Petri dishes with
medium prepared in the previous session (2 May
2016). It was grown strain in the boxes inoculated
for 5 days at 25 C. The results of inoculation are
shown in Figure 3.

Session May 16. A. niger fungus grew without

pollution Kitazato 1000 mL flask. It was decided
not to use aeration because could pollute the
environment. The results of inoculation can be
seen in Figures 5 and 6.

Figure 5. Kitazato flask with A. niger (biorreactor).

Figure 3. Propagated A. niger.

Session May 9. In the third session of project,

Kitazato 1000 mL flask containing the sterile
liquid medium with the culture of the petri dish
shown in Figure 3. In addition to this was
inoculated culture medium (nutrient agar) was
prepared and they conducted the sensidiscs. The
test material was sterilized sensidiscs. Medium
composition for sensidiscs can be seen in Figure
4.

Figure 6. Kitazato flask with A. niger (biorreactor).

As we can observe in Figures 5 and 6, the fungus

grew and formed a white precipitated. Probably in
this precipitated itaconic acid could be.
We proceeded to remove with a sterile syringe 12
mL of white precipitate produced by the fungus.
The withdrawn extract was filtered with filter
paper 3 to reduce the load of A. niger. You can not
esterilicar the precipitate balnco because the
itaconic acid be degraded. In Figure 7 white
precipitate extraction shown.
Figure 4. Composition of mdium for sensidiscs
test.

Biological Products Engineering

Figure 7. Itaconic acid extraction of the culture

medium.

After extraction of itaconic acid from the culture

medium and filtration, the sensidiscs for
antibiogram test were impregnated. The procedure
is shown in Figure 8.

Figure 9. Antibiotic drug used as a target in tests

sensidiscs (Ampicillin).

The inoculated sensidiscs Petri dishes shown in

Figures 10, 11 and 12.

Figure 10. Sensidiscs test Bacillus subtillis.

Figure 8. Preparation sensidiscs.

The sensidiscs were impregnated with white

precipitate. In addition to this, sensidicos were
prepared as antibiotic Ampicillin. 3 petri dishes
were inoculated with the following organisms:
Bacillus subtillis, E. coli and Streptomyces.
The antibiotic used as blank shown in Figure 9.

Biological Products Engineering

Figure 13. Test results for Bacillus subtilis sensidiscs.

Figure 11. Sensidiscs test E. coli.

Figure 14. Test results for E. coli sensidiscs.

Figure 12. Sensidiscs test Streptomyces.

Sensidiscs tests allowed incubated at 37 C for 3

days. The results obtained are shown in Figures
13, 14 and 15 for each microorganism.

Figure 15. Test results for Streptomyces sensidiscs.

As shown in Figures 13, 14 and 15, partial

inhibition shown by the extract of A. niger.
Discussion

Biological Products Engineering

In the beginning we made the selection of this

experiment because we had already expected the
production of itaconic acid would be performed
by Aspergillus niger, this is because A. niger has a
versatile and tolerant character in various growth
environments, so, it was not going to be difficult
to generate the strain and we were sure about A.
niger will grow in the selected media, it suppose
that the selected media has the needed
components to cause stress in the fungal strain, so,
in those conditions the fungal cells will be in the
maximum level of itaconic acid production, in
agreement with Li (2012) some A. niger strains
are good itaconic acid producers, this function
was studied in recent works.
But in our results we growth A. niger in a
Kitasato flask with no forced aeration for 8 days,
and we saw that we were in the good way to
produce something in the flask, in fact, the fungal
growth was going fast, because on the surface of
the substrate we got a plate of biomass, it was
sporulating already and at the time that we took
from the broth some samples we noticed that in
the bottom of the flask, there was a fungal plate,
and we can say that it was a A. niger plate because
in agreement with Li (2012) the better growth for
A. niger and A. terreus is on submerged fungal
fermentation, so that is the reason of why the
fungal cells were growing in the bottom of the
flask.
To know if we got some itaconic acid on the
fermented, we impregnated several paper discs
with enough amount of fermented broth.
Testing the inhibition of bacterial growth in the
results we saw that for tha cases of Basillus
subtilis that there was not growth inhibition, but in
the plate where we sow Streptomices, we can
notice that the bacterial growth in some parts of
the plate is slightly inhibited, because in most of
the paper discs that were impregnated with
fermented broth solution, we do not see saturated
growth, so, we must speculate that this strain of
A.niger produces some fraction of itaconic acid,
but this amount is not strong enough to inhibit all
the bacterial samples that were tested. In
agreement with Juy (2010), the A. niger strains
that have been proved to produce the itaconic acid
,they were genetically modified, are implanted

with several genes from Aspergillus terreus strain

that produces high amounts of acid per requires
more specific growth conditions.
We did not know from where the strain used, so if
acid production was not expected, it is because it
lacks these genetic attributes to the strain used to
produce more acid.
Conclusions.
It was performed a simple bioreactor design,
although it was a very simple design, this reactor
gave the needed conditions for the growth of A,
niger, the strain that we used did not produce the
enough amount of the acid. The reason is that is
not the best specimen to do the job.

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