Transcript Citric Acid Fermentation

Wignyanto
Dept. Agro-Industrial Technology
Brawijaya University
Industrial Fermentation
• Batch-fed and continuous fermentation processes are
common
• In the batch-fed process, sterilized nutrients are added to
the fermenter during the growth of the biomass.
• In the continuous process, sterilized liquid nutrients are fed
into the fermenter at the same flow rate as the
fermentation broth leaving the system.
• This will achieve a steady-state production.
• Parameters like temperature, pH, oxygen consumption and
carbon dioxide formation are measured and controlled to
optimize the fermentation process.
Major Industrial Fermentation
Products
Non-Food Applications
• Antibiotics
• Vitamins
• Amino Acids
Food Applications
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Organic Acids
Enzymes
Polysaccharides
Oils and Fatty Acids
Colors
Flavors
Organic acids
• Used as food acidulants
• Most versatile ingredients in industry because they are:
– Soluble and hydroscopic
– Buffers and chelators
• Organic acids produced by fermentation and
commonly used as food acidulants include:
–Citric
– Lactic
– Gluconic
– Propionic
What is Citric acid?
first isolated
from lemon
juice
produced in
anhydrous or
monohydrate
270,00 tonnes
worldwide/year=$
1.4 billion
Natural
intermediate
in Krebs cycle
2-hydroxy-1,
2, 3-propane
tricarboxylic
acid
Andhydrous
form is obtained
by
food, confectionery
and beverages
(75%)
crystallization
from
hot
aqueous
solutions.
pharmaceutical (10%)
Monohydrate is obtained by crystallization
industrial (15%)
at temperature below 36.6°C
Naturally non-toxic
due to its widespread
presence
Produced by
several molds
and bacteria
froma variety
of substrates
Pleasant taste and
property of enhancing
existing flavours have
ensured its dominance in
industry
Citric Acid Function
Stabilizer of oils and fats, ascorbic acid.
Emulsifire in processed foodstuffs, e.g. cheese.
Mixtures of citric acid and its salts have good buffering capacity
Monostearyl citrate as an antioxidant in oils and fats
Citric acid esters as non-toxic plasticizers in plastic films used to
protect foodstuffs
Methods for Citric Acid Production
• Submerged or surface fermentation process, batch fermentation
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Production media:
Beet molasses or glucose syrup as raw materials
Nitrogen added as ammonium nitrate or sulphate
Metals are often removed from raw material as high levelsof iron
appear to inhibit citrate production
• Fermentation:
• Inhibition of formation of long hyphae (which would resultin
dramatic increase of viscosity of fermentation medium)
• Low pH (pH is controlled at 2.2 –2.6 by addition of NH3), dissolved
oxygen concentrations, and temperature controlare important
PRODUCTION PROCESSESFERMENTATION
• A. niger
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Over production of citric
acid in A. niger
(i)Surface fermentation using beet molasses
requires several
pathways
pathway
(ii) submerged fermentation using beet
or and
cane
modifications:
molasses or glucose syrup. Submerged
processes
using sucrose as carbohydrate source
areflux
also
• High
of belived
to be running in areas where sugar is cheap
metabolites through
• Yeast
– Submerged fermentation using
glucose syrup
glycolysis, glucose
transport
• Block of TCA cycle
reactions that degrade
citrate
beet molasses
or
Schematic: Citric Acid Fermentation
Continuous Production of Citric
Acid from Dairy Wastewater
Using Immobilized Aspergillus niger
ATCC 9142
Se-Kwon Kim
Pyo-Jam Park
Hee-Guk Byun
Immobilized
Biocatalyst
Technology
Enzymes
a means to utilize 
as efficient and
heterogeneous catalyst for a
multitude of industrial and
medical applications
as in situ regeneration of activity
is merely by supplementing
suitable nutrients
&
Whole cells
found to be more
advantageous than
enzymes
In addition, whole metabolic pathway of
microbe or part of it required for the
production of a particular compound, can
be efficiently used over a longer period
under immobilized state.
Modern Dairies
produce
Very high quantities
waste
(thousand cubic
meters / day)
Main organic load
contributor of
lactose, fats, and
proteins originated
from milk
which is
High concentration in
organic matter
Problem on sewage
treatment systems
PURPOSE:
Examine the potential of dairy wastewater as
“a source for citric acid production by Aspergillus niger via a continuous
reactor as well as to study the effect of various fermentation
parameters, such as the dilution rate, pH, and temperature”
MATERIALS AND METHODS
• Microorganism and Inoculum Preparation
– Aspergillus niger ATCC 9142 (American Type Culture Collection, Rockville, MA,
USA)
• Medium
– The dairy wastewater contained about 2.5 % reducing sugar mostly as lactose
that had moisture rate of 97%.
– The total reducing sugar was concentrated up to 5 % for the experiment, and
sterilized at 121oC for 20 min.
– To remove protein and lipid, the initial pH was identified as 4.3 by 4 N of HCl,
and carried out the activating charcoal treatment.
• Cell Immobilization
– The cell immobilization for the continuous flask culture was achieved by
putting 4 % sodium alginate (cell : alginate = 1 : 10, w/w) into ATCC 9142 strain
growing in a 500 mL flask at 30oC for 72 h, then the cell-alginate mixture was
extruded into a 2% CaCl2 solution through a needle. The Ca-alginate beads
with entrapped cells of Aspergillus niger exhibited a typical shrinking
behavior, and the sizes of the beads were from 2.5 mm to 3.5 mm particle
diameter.
a positive
displacement
peristaltic pump was
used to vary the
liquid feed flow rate
total working volume
was 250 mL (300 mm
length and 25 mm
diameter)
operation temperature
by pumping
temperature-controlled
water through a jacket
• Bioreactor and Fermentation
• The Ca-alginate beads entrapped with immobilized cells were packed up to
30% in the column.
• The shake-flask experiments were performed in 500 mL Erlenmeyer flasks
containing 150 mL of the medium mixture (bead volume : medium volume =
1 : 2).
• The flasks were incubated at 30oC on a rotary shaking incubator 200 rpm
Analytical Techniques
• The citric acid  colorimetric method of
Marrier and Boulet
• The reduced sugar  colorimetric method
with 3,5-dinitrosalicylic acid (DNS) as the color
reagent
• All analyses were performed in duplicate.
Effect of pH on Citric
Acid Production
general,
a low
• The In
pH of
the substrate
is an pH is essential for
important
factor the
that affects
achieving
maximum
production
of
the performance of dairy
citric acid
wastewater fermentation
• Citric acid concentration and
yield were highest at pH 3.0
• consumption of sugar was
highest at pH 2.0
Low initial pH has the advantage of
pH did
not directly influence
the citric
checking
contamination
and inhibiting
acid production mechanism
oxalic acid formation.
but rather affected the enzymes which
were active in degrading the substrate
and/or the permeability of the cell
membrane of the substrate and product
Effect of Temperature
on Citric Acid Production
• Temperature is also an
important factor for citric acid
production.
• The maximal citric acid
production was obtained at
30oC
• Higher temperatures  very
rapid process & abundant
mycelial growth = consuming
large amounts of sugar thus
lowering the yield of citric acid
• Lower temperatures  higher
yields of citric acid are possible,
by prolonging the fermentation
process
Effect of Dilution Rate
on Citric Acid Production
• The dilution rate is the medium
flow rate per effective reactor
volume
• The optimum citric acid
productivity and yield were
160mg L-1 h-1 and 70.3% at a
dilution rate of 0.025 h-1
achieve a reactor system with a high
productivity that can be maintained for
extended time periods
Major Objective
Of Cell
Immobilization
Technology
Material should be inexpensive
Actual immobilization procedure and
subsequent reactor operation should be
simple
• The current study attempted to improve the citric
acid productivity by a continuous fermentation
process using calcium-alginate immobilized cells
of Aspergillus niger in a bioreactor
Comparison of Citric Acid Production
between Shake-Flask and
Continuous Fermentation
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In a batch shake-flask fermentation, the
productivity (63.3 mg L-1 h-1) and yield
(51.4%) of citric acid reached a maximum
after 3 days, and the citric acid concentration
and residual sugar concentration after 20
days were 1.38g/L and 8.85 g/L, respectively.
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Meanwhile, the yield (70.3 %) of continuous
citric acid production when using
immobilized Aspergillus niger reached a
maximum level after 9 days (Fig. 5). In
addition, the productivity value (160.0 mg L-1
h-1) was twice that of the batch shake-flask
culture (Table 3)
Conclusion
• important aspects of citric acid production from dairy waste-water
by immobilized Aspergillus niger :
– The optimum conditions were pH, 3.0
– temperature, 30oC
– dilution rate, 0.025 h-1.
• In addition, the productivity of citric acid by the immobilized
Aspergillus niger in a continuous reactor was more than two-fold
higher than that in a the shake-flask culture.
• The results suggest that the bioreactor used in the current study
could be potentially used for continuous citric acid production from
dairy wastewater by calcium-alginate immobilized Aspergillus niger.
Citric Acid: Downstream Processing
• Three separation methods
– Direct Crystallization
• Most successful with highly refined raw materials
– Precipitation as calcium citrate tetrahydrate
• Dominant process
• Calcium hydroxide is added to filtrate
– Liquid extraction
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Suspended particles are removed under vacuum
Precipitate is washed to remove impurities
Solution is concentrated using evaporators
Fed to a crystallizer, Crystals arecentrifuged, dehydrated,
and ground
Citric Acid Plants
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Tambahkan masalah blocking after citrate?
Why??
How??
Sumber??
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