Wignyanto Dept. Agro-Industrial Technology Brawijaya University.
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Transcript of Wignyanto Dept. Agro-Industrial Technology Brawijaya University.
Citric Acid Fermentati
onWignyanto
Dept. Agro-Industrial TechnologyBrawijaya 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
• 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?
Produced by several molds and bacteria froma
variety of substrates
2-hydroxy-1, 2, 3-propane tricarboxylic
acid
first isolated from lemon
juice
Natural intermediate in Krebs cycle
Naturally non-toxic due to its widespread
presence
Pleasant taste and property of enhancing existing flavours have
ensured its dominance in industry
produced in anhydrous or monohydrate
270,00 tonnes worldwide/year=$
1.4 billion
Andhydrous form is obtained by crystallization from hot aqueous solutions.Monohydrate is obtained by crystallization at temperature below 36.6°C
food, confectionery and beverages (75%)pharmaceutical (10%)industrial (15%)
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
Citric Acid Function
Methods for Citric Acid Production• Submerged or surface fermentation process, batch fermentation
• 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 PROCESSES-FERMENTATION
• A. niger– (i)Surface fermentation using beet molasses– (ii) submerged fermentation using beet or cane
molasses or glucose syrup. Submerged processes using sucrose as carbohydrate source are also belived to be running in areas where sugar is cheap
• Yeast– Submerged fermentation using beet molasses or
glucose syrup
Over production of citric acid in A. niger requires several pathways and pathway modifications:
• High flux of metabolites through glycolysis, glucose transport
• Block of TCA cycle reactions that degrade citrate
Schematic: Citric Acid Fermentation
Continuous Production of Citric Acid from Dairy Wastewater
Using Immobilized Aspergillus niger ATCC 9142
Se-Kwon KimPyo-Jam ParkHee-Guk Byun
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.
Immobilized Biocatalyst
Technology
a means to utilize as efficient and heterogeneous catalyst for a multitude of industrial and medical applications
Enzymes Whole cells&
found to be more advantageous than
enzymes
as in situ regeneration of activity is merely by supplementing suitable
nutrients
produceModern DairiesVery high quantities
waste(thousand cubic
meters / day)which is High concentration in
organic matter
Problem on sewage treatment systems
Main organic load contributor of
lactose, fats, and proteins originated
from milk
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.
• 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
total working volume was 250 mL (300 mm
length and 25 mm diameter)
operation temperature by pumping
temperature-controlled water through a jacket
a positive displacement
peristaltic pump was used to vary the
liquid feed flow rate
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
• The pH of the substrate is an important factor that affects the performance of dairy wastewater fermentation
• Citric acid concentration and yield were highest at pH 3.0
• consumption of sugar was highest at pH 2.0
pH did not directly influence the citric acid production mechanism
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
In general, a low pH is essential for achieving the maximum production of
citric acid
Low initial pH has the advantage of checking contamination and inhibiting
oxalic acid formation.
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
• 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
Major ObjectiveOf Cell
Immobilization Technology
achieve a reactor system with a high productivity that can be maintained for
extended time periods
Material should be inexpensive
Actual immobilization procedure and subsequent reactor operation should be
simple
Comparison of Citric Acid Production between Shake-Flask and Continuous
Fermentation
• 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.
• 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
• 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
• Tambahkan masalah blocking after citrate?• Why??• How??• Sumber??
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