1. CITRIC ACID
PRODUCTIO
N

2. JYOTHI R
LAKSHMI
KANTAMMA KAVYA N
LAKSHMI BN RAJESHWARI K SAHANA K
MONIKA VERMA
KAVYA KS
MONIKA.M
L JAHNAVI REDDY
TEAM
MEMBERS

3. WHICH YIELDS MORE?
SUGARCANE OR BEET…
SOLID STATE V/S SHAKE
FLASK TECHNIQUE

4. 1.INTRODUCTION
2.REVIEW OF LITERATUE
3.OBJECTIVE AND MATERIALS
4.METHODOLOGY
5.EXPECTED OUTCOME
6.REFERENCES

5. CITRIC ACID
• Citric acid (C₆H₈O₇) (2-hydroxy-1,2,3-propane
tricarboxylic acid) is a weak organic tricarboxylic
acid found in citrus fruits
• . Citric acid is found naturally in citrus fruits,
especially lemons and limes. It’s what gives
them their tart, sour taste.
• It has a monoclinic crystal structure.
• It is odourless and colourless compound.

7. The industrial citric acid production can be
carried in three different ways:
• Surface fermentation
• Submerged fermentation
• Solid-state fermentation

8. BACTERIA:
Bacillus licheniformis
Arthrobacter paraffinens
Corynebacterium sps
FUNGI:
Aspergillus niger
A.aculeatus
A.Awamori
Penicillium janthinelum

9. The most common species involved
extensively in citric acid production in labs
is Aspergillus niger

10. Uses of Citric Acid
• It is used as an antioxidant
• It is used as a cleaning agent – as an ingredient in
kitchen and bathroom cleaning solution
• It is used as an emulsifying agent in ice creams
• It is used to add a sour taste to soft drinks and
other food items
• It used in shampoo
• It is used in sucrose crystallization in caramel
• It is used in food colouring
• It is used as a natural preservative
• It is used to remove the chalky deposit from
evaporators, kettles, boilers etc.

11. ARTICLE 01
Solid-state fermentation for
the synthesis of citric acid
by Aspergillus niger
Luciana P.S. Vandenberghe a,b, Carlos R. Soccola, Ashok
Pandeya, J.-M. Lebeault

12. • Sugarcane formation through solid state
method is carried out through three different
agro industrial wastes viz., sugarcane bagasse,
coffee rust and cassava bagasse.
• The main thing that they focussed on were
sugarcane bagasse efficiency of citric acid
production.
• Solid state method is one such method that
requires lower energy and produces much less
waste water and environmental concerns
because of the disposal of the solid substrate.
• SSF is by development of microorganisms in low
water environment.

13. • The results showed that citric acid production showed
more sugar consumption in Aspergillus niger : Coffee
rust(97%)>Sugarcane bagasse(90%)> Cassava
bagasse(87%).
• Citric acid production was found high in cassava with a
difference of 88g/kg by sugarcane.

14. • In sugarcane when the strain is mutant the
production of citric acid was 120g/l, while
parental produced 80g/l. (1.5 fold
improvement from 150g/l of molasses sugar).
• Period of citric acid production with wild type-
10days, while in mutant strain: 6-7days.

15. ARTICLE 02
Citric Acid Fermentation
by Aspergillus niger NG-
110 in Shake Flask
Rubina Mazhar, Sikander Ali, Abdul Waheed
Biotechnology Laboratory Government college
University, Lahore,Pakistan

16. • The aim of the experiment was to conduct a study on the
culture conditions for the production of citric acid by
mutant strain of Aspergillus niger NG-110 using shake
flask technique.
• Enhanced citric biosynthesis optimized by alcohol and
CaCl₂ was carried out. On comparison on kinetic
parameters the mutant strain of A.niger was a fast
growing organism whereas NG-110 had the ability to
hyper produce the citric acid
• The utilisation of cane molasses as a cheap and
abundantly available source was also taken into account.
• Conidia inoculum was carried out with sterilisation with
Monoxal OT solution and clumps were aseptically
separated and then counted with haemocytometer.

17. • The supernatant was diluted to 15%sugar levels
and 2ml of molasses. After series of steps the
solution was used for the estimation of citric acid
and sugar levels.
• Assay method was carried out by DNS and
pyridine acetic an-hydric method.
• In the result it was seen that the rate of citric acid
fermentation by a strain of Aspergillus was 24-
242h. After initial incubation for 24 hours the
citric acid produced was 10.50g/l, increase in
incubation time lead to increase in yield.
• On the basics of sugar the citric acid production
was 60.96% but further increase in incubation
period did not show any enhancement.

19. ARTICLE 3
Citric Acid Production from Non
treated Beet Molasses by a
Novel Aspergillus niger Strain:
Effects of pH, Sugar and
Ingredients
Seda Guc and Osman Erkmen; Department of of
Food Engineering, Faculty of Engineering,
University of Gaziantep, 27310 Gaziantep, Turkey

20. • Effects of factors on the citric acid production from
non-treated beet molasses were studied in
Aspergillus niger OE55. Here Maximum amount of
citric acid (19.13 and 34.62 g/L) was achieved when
the initial pH of fermentation medium was 6.0 from
200 g/L and 150 sugar respectively.
• Citric acid production and biomass formation
continuously increased during fermentation period
in the media.
• A. niger is very sensitive to trace metal ions in the
beet molasses. Despite treatment with chelating
agents (such as sodium or potassium ferro-
cyanide), molasses give low yields of citric acid .
Therefore, beet molasses requires treatment before
use in the production of citric acid. There is a
requirement to find the mutant.

22. • In this study, a novel A. niger OE55 mutant strain has
been tested to able to produce citric acid from non-
treated beet molasses under different fermentation
media. Since, this type of strain necessary to reduce
cost of citric acid production.
• The fermentation media were prepared from non-
treated beet molasses together with different
ingredients and used in shake flask studies. pH of the
fermentation media is adjusted to 6.0.
• Inoculum culture were used to inoculate the
fermentation flasks which were then incubated at 30
± 1°C under shaking conditions at 220 rpm in the
shaker for 8 days. About 10 ml of samples (cultures)
were removed at each sampling time (0, 2, 4, 6 and 8
days of fermentation) under aseptic conditions and
used in analysis.

23. ARTICLE 04
SOME FACTORS AFFECTING CITRIC
ACID PRODUCTION FROM SUGAR
CANE MOLASSSES BY
ASPERGILLUS NIGER
Hauka,F. I. A. ;M. M. A. Ell-Sawah; M. M Kassem
and Sh. M. El-Kady

24. • The production or formation of citric acid is carried out from
molasses by Aspergillus niger in submerged culture.
• The main thing that they focused on the different factors were
affected on the citric acid such as concentration ,volume,
maximum productivity obtained, effect of metal ions
,treatment ,pH, high inoculum size, yield ,etc.
• Citric acid production is one such method that is used for the
food and pharmaceutical industries and in using agricultural
products and their wastes such as molasses and sugar beet
molasses.
• Pre treatment of molasses is carried out in order to eliminate
or remove heavy metals or inhibitory substrate.
• The fermentation method using in Aspergillus niger provides
ease of separation for the product which has been produced by
conventional submerged culture in which biomass or spores
are suspended in medium and can be readily achieved

25. • Incubation period of citric acid production takes place within 6
days and performed under haemocytometer
• The result of the effect of concentration was increased with the
increasing of heavy metals which gives the highest value of citric
acid concentration.
• The pH decreased during fermentation because the total untreated
molasses remained constant and was low in comparison during
fermentation time through which immobilized spores are present.
• The maxim citric acid concentration was obtained after 6 days of
incubation period.
• The sugarcane molasses in Aspergillus niger reached its maximum
without fungal effect and be treated in free environment.
• The biomass of or total yield spores was counted under
haemocytometer.

26. • The objective of our project is to estimate
the yield of citric acid produced by
sugarcane and beet molasses.
• To observe which technique produces
more citric acid , whether it’s solid state
fermentation or shake flask fermentation.

27. MATERIALS REQUIRED
BASIC REQUIREMENTS
ASPERGILLUS CULTURE
BEET MOLASSES
CANE MOLASSES
GLASSWARES
MRBA MEDIA

28. Shake flask fermentation: satellite glassware's,
inoculation loop, mechanical shaker
Estimation: 0.1 NaOH solution, phenolphthalein
indicator ,burette apparatus.
Solid state fermentation: micropipette,
inoculation loop, hay, cotton plugs.
Estimation: 0.1 NaOH solution, phenolphthalein
indicator ,burette apparatus.

29. SHAKE FLASK
FERMENTATION
1000ML of sterile MRBA broth was taken and spore inoculation was carried
out for A.niger.
It was then incubated at 25-30⁰C for 3 days in a mechanical shaker. And
then estimation was carried out by titration method
The cultured medium was filtered and collected. Pipette out 10ml of
filtrate and add phenolphthalein indicator.
Titrate against 0.1N NaOH until the pale pink colour persists and collect
concordant readings.
Estimation of citric acid was carried out after required calculations.

30. The solid substrate is soaked with water up to 65-70% of water content. After the
removal of excess water, the mass undergoes a steaming process.
Sterile starch paste is inoculated by spreading Aspergillus niger conidia in the
form of aerosol as a liquid conidia suspension on the substrate surface
The pH of the substrate is about 5.5 and incubated at 28-30⁰C .Growth can be
accelerated by adding ᾳ-amylase , although the fungus can hydrolyse starch on its
own too. During citric acid production the pH is dropped due to acid formation.
The solid state process takes about 5 to 8 days at the end of which citric acid is
extracted using hot water. In other cases, mechanical passes are used to produce
more citric acid.
SOLID STATE
FERMENTAION

31. EXPECTED OUTCOME
• Sugarcane molasses will have to yield more citric
acid compared to beet because of its impurities
content being more in beet than in cane.
• Shake flask is considered more feasible to
perform compared to solid state because of easy
inoculation process.
• Solid state still needs new designs and
technologies for developing industrially on large
scale.
• The amount was yield would be directly
proportional to incubation period but only up to
a certain limit after which no changes would be
observed in the yield.

32. CONCLUSION
• Cane and beet molasses are suitable for citric acid
production. However, beet molasses is
preferred due to its lower content of trace metals.
• Generally, cane molasses contains calcium,
magnesium, manganese, iron and zinc, which have a
retarding effect on the synthesis of citric acid.
• But industrially sugarcane molasses are preferred
because of low impurities content compared to beet
molasses.

33. REFERENCES
• Bayraktar, E. and U. Mehmetoglu (2000). Production of citric acid using
immobilized conidia of Aspergillus niger. Appl. Bioch. And Biotechnol. Part A,
Enzyme Engin. and Biotechnol. 87 (2): 117-125.
• Benuzzi, D. A. and R. F. Segovia (1995). Effect of the copper concentration on
citric acid productivity by an Aspergillus niger strain. Appl.Biochem. and
Biotechnol. 61: 393–397.
• Show PL, Oladele KO, Siew QY, Zakry FAA, Lan JCW, et al. (2015) Overview of
citric acid production from Aspergillus niger . Fron Life Sci 8: 271-283.
• Lotfy WA, Ghanem KM, El-Helow ER (2007) Citric acid production by a novel
Aspergillus niger isolate: II. Optimization of process parameters through
statistical experimental designs. Bioresour Technol 98:3470-3477
• Vervack, W., 1973. Analyse des aliments. Methodes courantes d'analyses,
Laboratoire de Biochimie de la Nutrition, Luvain-la Neuve, U.C.I.